Restoration of the yeast LEU2 gene by transcriptionally controlled recombination between tandem repeats

Abstract

The LEU2 gene of a his3 strain was inactivated by inserting the HIS3 gene between two overlapping inactive leu2 gene fragments, and mitotic stability of the resulting leu2:HIS3::leu2 sequence was measured under leucine repression and derepression. Both inactive leu2 regions were transcribed under derepressing conditions (growth in low leucine), and the LEU2 gene was completely restored by illegitimate recombination between the overlapping tandem repeats, leading to the loss of the intervening HIS3 gene. In contrast, only the downstream leu2 fragment was transcribed upon leucine repression, and the HIS3 insert in the leu2 region remained intact. The reciprocal experiment (inactivation of the HIS3 gene by inserting the marker gene LEU2) revealed a moderate rate of HIS3 restoration and LEU2 excision, reflecting transcriptional activity of the HIS3 region intermediate between that of LEU2 transcription in the repressed and derepressed state.