cDNA sequence of three cysteine‐rich clusters in the iron‐sulfur subunit of complex II (succinate‐ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer

Abstract

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

“…A number of nuclear genes for components of complex II have been identified and characterized (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), as have the homologous genes in both eubacteria (31)(32)(33)(34)(35)(36)(37)(38) and archaebacteria (39). In Escherichia coli, the genes sdhA (32) and sdhB (31) encode the two subunits of SDH (the flavin protein and the iron-sulfur protein, respectively), whereas sdhC and sdhD (32) specify apocytochrome b558 and a hydrophobic 13-kDa polypeptide, the two integral membrane components of complex II.…”