An antisense oligodeoxynucleotide approach to investigate the function of the nuclear-encoded subunits of human cytochrome c oxidase.
On treatment of human liver Hep G2 cells with thiamphenicol, intramitochondrial levels of cytochrome c oxidase (COX) nuclear gene products were shown to decrease in tandem with the mitochondrially-encoded subunits except, however, for subunit IV which proved to be remarkably stable. This nonspecific decrease in subunit level was utilized to assess the function of subunit VIIa-L. Thiamphenicol-pretreated cells were bathed in anti-VIIa-L oligonucleotides and the recovery rate of COX activity compared to control oligomer-untreated cells or cells with a similar concentration of a randomized oligomer. No difference in recovery rate was noted for the first two days post oligomer addition, after which a sharp decrease in recovery was noted for the anti-VIIa-L treated cells only. We conclude: (i) subunit IV is stable in the absence of other COX components, (ii) subunit VIIa-L is required for maintaining normal levels of COX activity, (iii) an antisense oligodeoxynucleotide approach for assessing the function of COX nuclear gene products is made feasible by first reducing levels of the complex and then assaying its recovery rate in the presence of transcript-specific antisense oligonucleotides.
Chrzanowska-Lightowlers
Z M
ZM
Division of Clinical Neuroscience, University of Newcastle upon Tyne, U.K.
Turnbull
D M
DM
Bindoff
L A
LA
Lightowlers
R N
RN
D001483:Base Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008928:Mitochondria / Q000187:drug effects; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000235:genetics; D013839:Thiamphenicol / Q000494:pharmacology
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D003576:Electron Transport Complex IV; D013839:Thiamphenicol