Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae

Abstract

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8(m)) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

Figure 1

Reporter genes for monitoring mitochondrial (a) or nuclear (b) microsatellite stability in yeast. (a) Reporter gene for monitoring mitochondrial microsatellite stability (cox3∷arg8^m). ARG8^m is a derivative of the yeast ARG8 gene in which the codon usage has been altered to be consistent with mitochondrial expression (16). This gene is expressed by using the COX3 transcriptional and translational signals in mtDNA (thin line). COX3 protein coding sequences are represented by the solid box. The fusion protein is processed posttranslationally at the site indicated by the arrow. Microsatellite sequences [in-frame or out-of-frame insertions of poly(AT) or poly(GT)] were inserted at the designated AccI site. (b) Reporter gene for monitoring nuclear microsatellite stability (20). The components of this fusion gene encoding a protein with wild-type Ura3p activity are GAL1,10 promoter (shaded box), N-terminal amino acids derived from LYS2 (solid box), HIS4-derived amino acids (striped box), and URA3-derived sequences (open box). Microsatellite sequences [in-frame insertions of poly(AT) or poly(GT)] were inserted at the designated BglII site. These plasmids were integrated into the LEU2 locus in a strain with a ura3 mutation.