Franklin Quan

Mitochondrial Encephalomyopathy and Complex III Deficiency Associated with a Stop-Codon Mutation in the Cytochrome b Gene

We have reinvestigated a young woman, originally reported by us in 1983, who presented with exercise intolerance and lactic acidosis associated with severe deficiency of complex III and who responded to therapy with menadione and ascorbate. Gradually, she developed symptoms of a mitochondrial encephalomyopathy. Immunocytochemistry of serial sections of muscle showed a mosaic of fibers that reacted poorly with antibodies to subunits of complex III but reacted normally with antibodies to subunits of complexes I, II, or IV, suggesting a mutation of mtDNA. These findings demonstrate the diagnostic value of immunocytochemistry in identifying specific respiratory-chain deficiencies and, potentially, distinguishing between nuclear- or mtDNA-encoded defects. Sequence analysis revealed a stop-codon mutation (G15242A) in the mtDNA-encoded cytochrome b gene, resulting in loss of the last 215 amino acids of cytochrome b. PCR-RFLP analysis indicated that the G15242A mutation was heteroplasmic and was present in a high percentage (87%) of affected tissue (skeletal muscle) and a low percentage (0.7%) of unaffected tissue (blood) but was not detected in controls. Analysis of microdissected muscle fibers showed a significant correlation between the immunoreactivity toward the Rieske protein of complex III and the percentage of mutant mtDNA: immunopositive fibers had a median value of 33% of the G15242A mutation, whereas immunonegative, ragged-red fibers had a median value of 89%, indicating that the stop-codon mutation was pathogenic in this patient. The G15242A mutation was also present in several other tissues, including hair roots, indicating that it must have arisen either very early in embryogenesis, before separation of the primary germ layers, or in the maternal germ line. The findings in this patient are contrasted with other recently described patients who have mutations in the cytochrome b gene.

Human cytochrome c oxidase subunit VIb: characterization and mapping of a multigene family

We report the isolation and sequence of a human heart cDNA coding for cytochrome c oxidase (COX) subunit VIb (COX VIb). This cDNA extends 50 bp upstream from the region coding for the mature peptide. By Northern analysis, a single transcript of approx. 550 nucleotides (nt) has been identified in six human tissues. Southern analysis of human genomic DNA demonstrates the presence of multiple loci that show high homology to the cDNA. These loci cosegregate with either five or six different human chromosomes in human-rodent somatic cell hybrids. Using the COX6b cDNA, genomic sequences representing two of these loci have been isolated and characterized. The nt sequence analysis suggests that both loci represent COX6b pseudogenes.

[33] Vaccinia virus systems for expression of Gα genes in S49 cells

In this chapter, we describe the use of recombinant VV to express G alpha subunits efficiently in S49 cyc- cells, a murine lymphoma cell line deficient in endogenous Gs alpha activity. Additional studies have shown that VV infection does not interfere with G-protein-coupled signal transduction events. In particular, the VV-expressed rat Gs alpha subunit is able to mediate efficient receptor-dependent and receptor-independent activation of adenylyl cyclase in cyc- cells. This system should therefore be useful for rapidly assessing the activity of modified or chimeric Gs alpha subunits.