Characterization of a novel open reading frame, urf a, in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Abstract

Between the genes for tRNA(gin) and tRNA(ile) an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the "mitochondrial" genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the "standard" genetic code (UGG for tryptophan). A previous paper has demonstrated that "mutator" strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (anar-6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.

Mitochondrial Genome Rearrangements in Glomus Species Triggered by Homologous Recombination between Distinct mtDNA Haplotypes

“…In the absence of external factors, heteroplasmy should be the default state for mtDNA under a simple mutation-drift scenario (White et al 2008). In yeasts, the presence of a heteroplasmic state was shown to be transient due to the presence of segregation mechanisms, such as nucleoid formation, and mtDNA molecular repair pathways which maintain mitochondrial genome integrity (Zimmer et al 1991; Hu et al 1995; Yasuhira and Yasui 2000). The presence of nucleoids, similar mtDNAs linked by Holliday junctions, induces a genetic bottleneck responsible for a fast segregation (Lockshon et al 1995; White and Lilley 1997; MacAlpine et al 1998).…”

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

“…Sequencing of 12 cloned cDNA fragments from three independent ampli®cation experiments revealed the identity of the transcript with the cloned DNA sequence, and thus the absence of introns (see Zimmer et al 1991;Fig. 4).…”

“…These isolates were tested to ensure that they showed the same phenotypic properties as the original mutant ana r -6. As described by Zimmer et al (1991), the isolates were grown in static cultures in YED10 medium to late-logarithmic phase. The cells were plated out onto glucose complete medium and colonies were replica plated onto glycerol medium.…”

“…This does not lead to complete respiration de-®ciency, but a culture of ana r -6 continuously segregates subclones that dier in their ability to grow on glycerol (see Fig. 2 in Zimmer et al 1991); after replica plating, Mol Gen Genet (1998) 258: 389±396 a proportion of the colonies shows wild-type growth on glycerol, whereas others display reduced growth on glycerol. Up to 8% of the colonies fail to grow on glycerol.…”

“…Up to 8% of the colonies fail to grow on glycerol. The appearance of colonies with a graded respiratory impairment may be a consequence of translational frameshifting by mitochondrial ribosomes at a run of A residues in urf a, as discussed by Zimmer et al (1991).…”