Different genetic approaches to mutate the mitochondrial ribosomal protein S12

• Over the last decades, an ever-growing number of tools became available to manipulate the genome of the model organism Saccharomyces cerevisiae. The most common approach to study a mutation in a protein is to first replace the native gene with a selection cassette via homologous recombination. In a second step, the mutated gene can be expressed from a plasmid. For certain applications, however, it is necessary to integrate the mutation in the genome. Here we introduced a mutated variant of the mitochondrial ribosomal protein S12 (Mrps12), a protein of the highly conserved accuracy center of the mitochondrial ribosome, using an integrative plasmid. First, we attempted to use a counter-selectable strategy by employing the uracil selection cassette (URA3) in combination with 5-fluoroorotic acid (5-FOA). We observed that this approach is not ideal for mutating certain crucial mitochondrial proteins. In our hands, this method only gave false-positive results. Most likely, deletion of MRPS12 and subsequent loss of mitochondrial DNA caused genome instability. This gave rise to mutated versions of URA3 which could no longer be used for counter selection. Therefore, we eventually introduced the MRPS12* under control of its endogenous promotor and terminator via an integrative plasmid in the deletion strain.

Subject headings

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Keyword

mitochondrial ribosome

yeast genetics

counter selection

Biochemistry

biokemi

Publication and Content Type

vet (subject category)

ovr (subject category)