Reintroduction of a characterized Mit tRNA glycine mutation into yeast mitochondria provides a new tool for the study of human neurodegenerative diseases.
We report the identification and characterization of a new mutation (ts9) in the Saccharomyces cerevisiae mitochondrial genome, which was first genetically mapped in the tRNAgly region and further identified by means of sequencing as consisting of a G to A transition at position 30 in the tRNA. The mutation causes an almost complete disappearance of mature tRNAgly, while a second mitochondrial mutation with a compensatory C to T change restores it in normal quantities; this points to the importance of the strong bond between bases 30 and 40 of the anticodon stem in the stabilization of the tRNA. In addition to resulting in a clear-cut heat-sensitive phenotype, the ts9 mutation creates a new EcoRV restriction site. Both properties were used as markers to monitor the successful (re) introduction of the mutated allele into a wild-type mitochondrial genome through biolistic transformation. The mutant frequency in the progeny as well as the correct integration of the mutated allele at its proper site demonstrate the feasibility of this method for creating and investigating specific mitochondrial tRNA mutations. The method will provide important applications for the use of yeast as a model system of human mitochondrial pathologies.
Rohou
H
H
Laboratoire de Génétique Moléculaire, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Université Paris-Sud, Bat. 400, 91405 Orsay Cedex, France.
Francisci
S
S
Rinaldi
T
T
Frontali
L
L
Bolotin-Fukuhara
M
M
D001426:Bacterial Proteins; D001483:Base Sequence; D019470:Biolistics; D015152:Blotting, Northern; D015139:Blotting, Southern; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D016681:Genome, Fungal; D006358:Hot Temperature; D006801:Humans; D008928:Mitochondria / Q000235:genetics; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000235:genetics; D010444:Peptide Elongation Factor Tu; D017354:Point Mutation / Q000502:physiology; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics*; D000077278:RNA, Mitochondrial; D012353:RNA, Transfer, Gly / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004272:DNA, Mitochondrial; C034477:RNA primers; D012331:RNA, Fungal; D000077278:RNA, Mitochondrial; D012353:RNA, Transfer, Gly; D012313:RNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D010444:Peptide Elongation Factor Tu