Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination

Abstract

Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mitochondrial deoxyribonucleic acid (mtDNA). DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. Although transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology.

Fig. 1. Nuclear transformants and mitochondrial co-transformants obtained by bombardment of different yeast strains

The nuclear LEU2 plasmid Yep351 (8) and the COX2 plasmid pNB69 (2) were precipitated together onto tungsten particles and bombarded on lawns of the rho⁰ strains W303-1B/A/50 (MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, can1-100 [rho⁰]) (a rho⁰ derivative of W303-1B, (13)) and DFS160 (MATa ade2-101, leu2D, ura3-52, arg8D::URA3, kar1-1, [rho⁰]) (6), or on lawns of the rho⁺ strain NB104. NB104 rho⁺ mtDNA carries a 129 bp deletion, cox2-60, located around COX2 first codon (2), and is isonuclear to DFS160. The top plates correspond to minimal medium supplemented with sorbitol and lacking leucine. Typical plates showing about 3000 nuclear transformants for each strain have been presented. Nuclear transformants were crossed by replica plating to the nonrespiring tester strain (NB160), carrying a mutation of COX2 initiation codon (2), and mitochondrial transformants (bottom plates) were detected by replica-plating the mated cells onto non-fermentable medium. Reprinted from (7) with permission.

Fig. 2. Schematic diagram of recombination events that allow identification of nonrespiring recombinant cytoductants by marker rescue

Thick lines represent mtDNA sequences, thin lines represent vector DNA. The box represents a gene under study. (A) A karyogamy defective (kar1-1) synthetic rho⁻ donor containing an experimentally induced mutation “e” is mated to a rho⁺ recipient strain with a deletion in the region of interest. Among the cells present in the mixture after mating, are the desired rho⁺ recombinant cytoductants. (B) To distinguish the desired rho⁺ recombinant cytoductants from the unaltered recipient cells and other cell types present, clones derived from the mating mixture are mated to a rho⁺ tester strain bearing the marker mutation “m.” The desired rho⁺ recombinant cytoductants can yield respiring recombinants when mated to this tester by a crossover between “e” and “m” (and a second resolving crossover anywhere else). The ability to produce respiring recombinants identifies the desired cytoductant clones. Unaltered recipient clones, and other cells types present, cannot yield such respiring recombinants. Reprinted from (7) with permission.

Fig. 3. Selection of rho⁺ mitochondrial transformants directly after bombardment

The nuclear shuttle vector Yep351 (LEU2) and plasmid pNB69 (COX2) were bombarded together onto lawns of the rho⁺ cox2-60 strain NB104 (see legend of Fig. 1). The bombarded lawns had been spread either on minimal medium supplemented with sorbitol but lacking leucine (top right), or on non-fermentable YPEG medium supplemented with sorbitol and 0.1% glucose (top left). Leu⁺ transformants were replica plated to non-fermentable medium (bottom plate) to select for mitochondrial transformants. Reprinted from (7) with permission.