Expression of the Saccharomyces cerevisiae mitochondrial COX1 locus, which contains several introns and is co‐transcribed with the downstream genes AAP1, OLI2 and ENS2, is controlled by at least 18 nuclear‐encoded proteins. The PET309 gene, encoding one of these proteins, was cloned, sequenced and shown to contain an open reading frame of 965 codons. Isonuclear PET309+ and delta pet309::URA3 strains carrying mitochondrial genomes that differ in the number of COX1 introns, were generated. Analysis of RNA species from these strains demonstrated an inverse relationship between the number of introns present in the precursor RNA and the amount of COX1 and AAP1/OLI2/ENS2 RNAs accumulated in a pet309 mutant. Hence, PET309 plays a role either in transcription of intron‐containing primary transcripts from the COX1‐AAP1‐OLI2‐ENS2 transcription unit or in stabilization of primary transcripts. PET309 is also required in translation of COX1 mRNA. A mitochondrial bypass suppressor of the pet309 deletion mutation was isolated, and shown to consist of a DNA rearrangement at the COX1 locus, such that the 5′ untranslated leader region (UTR) of the COB gene was fused to COX1 at nucleotide −174 of its 5′ UTR. This result suggests that Pet309p acts through the COX1 5′ UTR to activate initiation of translation of the COX1 coding region.

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of doublestrand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly-efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300 bp and 60 bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails.

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031Ϫ4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309Ϫc-myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309Ϫc-myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.Keywords : Saccharomyces cerevisiae; yeast; mitochondria; translation ; RNA processing.A novel aspect of mitochondrial gene expression is the con-[1, 4], may substitute for the function performed by IF3 in other prokaryotic-like translation systems. trol of mitochondrial RNA translation by gene-specific factors encoded by nuclear genes (for review, see [1Ϫ3]). In SaccharoMitochondrial RNA processing is also controlled by genespecific factors encoded by nuclear genes (for review, see [2, myces cerevisiae, it is likely that each mitochondrial mRNA employs specific factors required for translation initiation. These 3]). Several mitochondrial genes in S. cerevisiae contain introns may act in addition to general translation initiation factors, or which are thought to be spliced by ribozyme mechanisms faciliinstead of them. A nuclear gene for mitochondrial translation tated by protein factors (RNA chaperones) that facilitate proper initiation factor 2 (IF2) has been found, but a clear homologue folding of the intron ribozymes. Both mitochondrial-encoded of prokaryotic translation initiation factor 3 (IF3) has not been maturases and nuclear-encoded proteins required for mitofound, despite the availability of the complete DNA sequence chondrial RNA splicing have been identified. Additionally, of the S. cerevisiae genome. The function of yeast gene-specific nuclear-encoded trans-acting proteins required for mitochondrial mitochondrial translation factors such as Pet122p or Cbs2p, RNA 5′ or 3′ end processing or mRNA stability...