Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae. Application to the selection of strains containing novel forms of cytochrome-c oxidase.
A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain. Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.
Glynn Research Institute, Bodmin, England.
S
S
Brown
A M
AM
Colson
B
B
Meunier
P R
PR
Rich
D003580:Cytochromes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D009154:Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification
D003580:Cytochromes; D003576:Electron Transport Complex IV