Intrachromosomal recombination after targeted monocopy integration in Penicillium chrysogenum: stabilization of the direct repeats to prevent loss of the inserted gene.

Abstract

Monocopy systems obtained by targeted integration at the pyrG locus of P. chrysogenum led to the formation of unstable direct repeats in the genome. A previously isolated pyrG mutant was sequenced and the mutation was found to be located at nucleotide position 665 of the pyrG gene. A different pyrG mutation was introduced in vitro at the BamHI site of this gene. Recombination products arising from monocopy systems using the bleomycin/phleomycin resistance gene (ble) as a model were studied to elucidate the intrachromosomal recombination mechanisms. Experimental results showed that both gene conversion and deletion events occurred spontaneously at the integration site. Gene conversion products were obtained at a frequency of one in 3.4x10(4) viable transformant spores. When gene conversion occurred, the inserted exogenous gene was retained and was flanked by rearranged direct repeats of the pyrG gene, each containing at least one pyrG mutation. Deletion events resulted in the loss at high frequency of the inserted exogenous gene. Genetic stabilization of a monocopy system was obtained when both pyrG repeats (formed at the targeted integration site) contained at least one identical mutation, since in this case further recombinations can be easily counter-selected.