The MRS1 gene of S. douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes

Abstract

We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes. This methodology has been used to study a nuclear gene from the yeast S. douglasii (a close relative of S. cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S. douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S. cerevisiae. We have shown that this gene is the homologue of the S. cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S. cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S. douglasii. The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions. At the protein level there are 86% identical residues and 7% conservative substitutions. The divergence of the MRS1 genes of S. cerevisiae and S. douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.

Figure 1

Schema showing the strategy used to locate the complementing gene within the cloned insert and to sequence the site of insertion.

Figure 2

Partial restriction map of the cloned insert showing the positions of the URA3 insertions (▽). Insertions have been located to the specific restriction fragments shown, but their positions within the fragments are not known. Insertions that inactivate complementation are marked below the line. The deduced position of the gene is shown by a hatched bar. H: Hind III; E: EcoR I; ΔH: Hind III site deleted; ΔE: EcoR I site deleted.

Figure 3

Nucleotide sequence of the S. douglasii MRS1 gene and the deduced protein sequence. The Sau3A I sites where the fragment encoding the URA3 gene is inserted in the plasmids YCpCSI020 (751) and YCpCSI054 (1095) are underlined.

Figure 4

Comparison of the S. douglasii (above) and S. cerevisiae (below) MRS1 protein sequences. The two sequences show 86% identical residues and 7% similar residues. Identical residues are shown with a vertical bar.

Figure 5

Growth on glycerol after 3 days incubation at 28°C of S. douglasii strains with a wild-type (MRS1) or inactivated (mrs1::URA3) MRS1 gene, in the presence of the S. douglasii wild-type intron plus mitochondria (4707-22D and C0701) or an intronless mitochondrial genome (4707-22D/Δ and C0701/Δ). In the presence of the intronless mitochondrial genome the mrs1::URA3 strain shows a wild-type growth on glycerol.

Figure 6

MRS1 genes from both strains are iso-functional with S. cerevisiae mitochondria. Growth on glycerol after 3 days incubation at 28°C of a strain containing the S. cerevisiae nuclear and mitochondrial genomes, with an inactivated MRS1 gene (mrs1::URA3) transformed by the S. cerevisiae MRS1 gene on a multicopy plasmid (YEpIB00l), the S. douglasii MRS1 gene on a centromeric plasmid (YCpCSI007), and the vector controls. Both genes are able to efficiently complement the chromosomal disruption of the S. cerevisiae MRS1 gene.

Figure 7

The MRS1 genes of S. cerevisiae and S. douglasii display quantitative differences in the presence of the S. douglasii mitochondrial genome. A. The left-hand side shows the growth on a complete glycerol medium after 5 days incubation at 28° C of W303/SD (S. cerevisiae nuclear genome with the S. douglasii mitochondrial genome) transformed by the S. cerevisiae MRS1 gene on a multicopy plasmid (YEpIB00l), the S. douglasii MRS1 gene on a centromeric plasmid (YCpCSI007), and the vector controls, showing that on a multicopy vector the S. cerevisiae MRS1 gene can restore some growth on glycerol. The right-hand side shows a Northern blot of mitochondrial RNA from the same strains revealed with a coxI exonic probe, and shows that the S. douglasii MRS1 gene promotes an efficient excision of the S. douglasii intron all, while the S. cerevisiae MRS1 gene on a multicopy plasmid promotes a poor excision of this intron. B. Growth kinetics of the strains used in A on a synthetic ethanol/glycerol liquid medium.