This dissertation reports the discovery of gene amplification in the soil bacterium Acinetobacter sp. strain ADP1. Amplification mutants were selected from a strain that lacks two transcriptional activators. Without these regulators, it does not express two cat-gene transcriptional units at levels high enough to grow on benzoate. Mutant colonies were selected on benzoate medium at a frequency of 10-8 within a three-week period. Of 105 independently isolated mutants, 104 carried tandem head-to-tail repeats of a chromosomal segment, or amplicon, containing the cat genes. The amplicons ranged in size from approximately 12 to 290 kbp and varied in copy number from 3 to more than 30. Gene amplification occurred via a twostep process whereby an initial duplication event was followed by the generation of higher amplification. Recombinational sites involved in the initial duplication event were isolated using a transformation assay that takes advantage of the natural transformability of these Acinetobacter strains. Analysis of sequences from 72 mutants revealed that most of the initial duplications were generated by illegitimate recombination. In some cases, the recombination events were site specific in a fashion that was independent of short homologous repeats. These duplications occurred independently of recA and spontaneously in the absence of selection. In contrast, the second amplification step required RecA and was only apparent after selection. Additional experiments addressed the issue of the delay in amplification mutant colony formation during the original selection period. These studies tested the possibility of adaptive amplification. According to this controversial theory, amplification rates can increase in response to selective conditions via inducible mechanisms for surviving stress. However, our results indicated that this delay in colony formation resulted from a natural selection process whereby higher amplification from preexisting duplications was slowly generated through a series of homologous recombination events. Thus, these mutants did not arise adaptively. Furthermore, this system presented new evidence that supports the theory that the co-amplification of functionally related genes is a driving force in the evolution of higher-order gene clusters. This new model system for studying gene amplification illuminates a process with important implications for evolution, drug resistance, increased virulence, bioremediation, and cancer.
