This chapter describes the analysis and manipulation of yeast mitochondrial genes. In assessing the effects of mitochondrial (or nuclear) mutations on the expression of individual mitochondrial genes and the nature of their products, it is useful to examine mitochondrially encoded proteins directly. Mitochondrial translation products can be selectively 35S-labeled in cycloheximide- treated cells because cytoplasmic ribosomes are sensitive to this drug but mitochondrial ribosomes are not. A crude preparation of mitochondria can then be prepared, subjected to denaturing gel electrophoresis, and autoradiographed to reveal the handful of major proteins encoded on the mitochondrial genome. Restriction fragments of purified mtDNA can be end-labeled and sequenced directly by chemical degradation. Recently, it is found that mtDNA present in crude preparations of total yeast DNA 4° serves well as a template for dideoxynucleotide chain-termination reactions, using 5'-end-labeled oligonucleotide primers instead of incorporating labeled nucleotides during polymerization. The Sequenase kit is used modified for the omission of labeled nucleotides. This allows rapid analysis of mitochondrial mutations. For mitochondrial genetic transformation and gene replacement, it is found that high-velocity bombardment of yeast with tungsten microprojectiles carrying mtDNA sequences can result in delivery of the DNA to the mitochondrial matrix. Furthermore, rho– strains can be converted to stable synthetic rho– strains by transformation with bacterial plasmids carrying mitochondrial genes.
|Original language||English (US)|
|Number of pages||17|
|Journal||Methods in enzymology|
|State||Published - Jan 1991|
All Science Journal Classification (ASJC) codes
- Molecular Biology