Tissue-specific expression and chromosome assignment of genes specifying two isoforms of subunit VIIa of human cytochrome c oxidase.

Subunit VIIa of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) exists in at least two isoforms, one present in all tissue types ('liver' isoform; COX VIIa-L) and the other specific for cardiac and skeletal muscle (COX VIIa-M). We have isolated a full-length cDNA encoding human COX VIIa-M. The deduced polypeptide represents the human ortholog of COX VIIa-M, as it shares 78% identity with bovine COX VIIa-M, but only 63% identity with human COX VIIa-L. Northern-blot analysis of primate tissues demonstrated that COXVIIa-M mRNA is present only in muscle tissues; in contrast, the COXVIIa-L mRNA is present in both muscle and nonmuscle tissues. Southern-blot hybridization of human-rodent cell hybrid genomic DNA indicates that the COXVIIa-M gene maps to a single locus on chromosome 19, designated COX7AM. In contrast, COXVIIa-L cDNA probes hybridized to fragments from two COX7AL loci, on chromosomes 4 and 14.     

Molecular characterization and chromosome assignment of the porcine gene COX7A1 coding for the muscle specific cytochrome c oxidase subunit VIIa-M

The COX7A1 gene encodes a heart- and muscle-specific isoform of the subunit VIIA of cytochrome c oxidase, which is the last component of the mitochondrial electron transfer chain. Cloning and characterization of the porcine COX7A1 gene revealed a highly conserved organization with respect to other mammalian COX7A1 orthologs. The porcine gene consists of four exons spanning approximately 1.5 kb and codes for a peptide of 80 amino acids. The COX7A1 gene showed no variation between pigs from different breeds. The gene was assigned by FISH and RH-mapping to SSC 6q1.1-->q1.2 which is in agreement with previously established comparative maps.     

Evolution of the primate cytochrome c oxidase subunit II gene

We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. Correspondence to: R.L. Honeycutt     

Structure and evolution of primate cytochrome c oxidase subunit II gene

The sequence of cytochrome oxidase subunit II (COII) mRNA from the cynomolgus macaque has been determined. Availability of the sequence from a non-human primate has allowed examination of the evolution of the COII gene and protein along the primate lineage. Comparison with existing protein and DNA sequences, combined with estimates of divergence derived from calculations designed to compensate for multiple mutation and reversion events, indicates that although the rate of fixation of nucleotide substitutions at silent sites is somewhat lower in primates than non-primates, the rate of fixation at replacement sites is 4-5-fold higher. The data also suggest that the rate of divergence at replacement sites along the primate lineage has not been uniform, but has decreased 2-2.5-fold since the higher primate branch point, in the absence of a comparable change in the rate substitution at silent sites. Both primate mRNAs differ from their non-primate homologues in having 3'-untranslated regions of 20-25 nucleotides. Examination of the monkey and human untranslated sequences suggests that these regions have evolved by duplication events occurring in both cases within 2-3 nucleotides following the translational stop codon. The primate mRNAs are also exceptional in that both can form stable stem and loop structures immediately preceding the postulated duplication site that may have played a role in facilitating the mutational events involved. Comparison of the human and monkey protein sequences has revealed regions conserved in primates that are significantly more hydrophobic than their non-primate counterparts. The possible effects of these alterations on the interaction between COII and cytochrome c are discussed.     

Subunit III of cytochrome c oxidase is expressed in Paracoccus denitrificans

Polyclonal antibodies have been obtained against a synthetic dodecapeptide identical to the aminoacid sequence 120-131 DSPIKDGVWPPE (inferred from its DNA sequence) of Paracoccus denitrificans cytochrome c oxidase subunit III. The antibodies had a titer higher than 1:10000 when tested against the antigen. These antibodies have been used to produce immunological evidence that, despite the fact that subunit III is not isolated with cytochrome c oxidase, it exists in Paracoccus denitrificans lysates. The antibodies did not show reactivity with bovine heart cytochrome c oxidase either by ELISA or immunoblotting. It was also shown that the antibodies react with a single polypeptide present in Paracoccus denitrificans cell lysates, having an apparent molecular weight close to that of subunit III of bovine heart oxidase.     

Two bovine genes for cytochrome c oxidase subunit IV: a processed pseudogene and an expressed gene

We have isolated and analyzed 17 clones from a bovine genomic library in phage lambda Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.     

Reproductive function for a C-terminus extended, male-transmitted cytochrome c oxidase subunit II protein expressed in both spermatozoa and eggs

Our previous study documented expression of a male-transmitted cytochrome c oxidase subunit II protein (MCOX2), with a C-terminus extension (MCOX2e), in unionoidean bivalve testes and sperm mitochondria. Here, we present evidence demonstrating that MCOX2 is seasonally expressed in testis, with a peak shortly before fertilization that is independent of sperm density. MCOX2 is localized to the inner and outer sperm mitochondrial membranes and the MCOX2 antibody's epitope is conserved across >65 million years of evolution. We also demonstrate the presence of male-transmitted mtDNA and season-specific MCOX2 spatial variation in ovaries. We hypothesize that MCOX2 plays a role in reproduction through gamete maturation, fertilization and/or embryogenesis.     

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.     

Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues.

Cytochrome c oxidase (EC 1.9.3.1) is an enzyme which is composed of subunits derived from both the mitochondrial and the nuclear genomes. To determine whether or not the expression of these two genomes is co-ordinated at the mRNA level, we have examined the steady-state levels of mRNAs coding for cytochrome c oxidase subunit III (mitochondrially encoded) and subunit VIc (nuclear-encoded) in rat tissues. This was compared with the tissue concentration of the holoenzyme, which was estimated by measuring cytochrome c oxidase enzyme activity. The tissues (heart, brain, liver, kidney, soleus muscle and superficial white vastus muscle) possessed a 13-fold range of enzyme activity, which was highest in heart and lowest in the superficial vastus muscle. Specific subunit mRNA levels were quantified by using slot-blot hybridization of cDNA probes to total tissue RNA. The highest values for subunit III and Vlc mRNA tissue contents were found in kidney, followed by liver and heart (40-60% of that of kidney). The white vastus muscle contained the lowest subunit mRNA level (15% of that of kidney). Although some variability was apparent within each tissue, a parallel pattern of mRNA expression of the nuclear- and mitochondrially encoded subunits was observed. Differences between muscle (heart, vastus and soleus) and non-muscle tissues were noted in the relationship between mRNA and protein levels of expression. Thus, although this suggests that tissue-specific regulatory processes operate, the steady-state expression of subunit III and subunit Vlc mRNAs appears to be co-ordinately regulated.     

Nucleotide sequence of cDNA encoding subunit VIII of cytochrome c oxidase from rat heart.

In this study, the complete cDNA of subunit VIII-h of rat cytochrome c oxidase is presented. A rat skeletal muscle cDNA library was screened with a 132 bp fragment of the cDNA of rat COX subunit VIII-h. Four positive clones were sequenced in both directions.     

COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini

From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus.     

Assignment of the human cytochrome c1 gene to chromosome 8

We previously isolated a cDNA clone for human cytochrome c1. The insert DNA of approximately 950 bp from this clone was used as a probe to identify the cytochrome c1 gene. High molecular weight DNAs extracted from a panel of 14 independent human-mouse somatic cell hybrids were digested with BamHI and analyzed by Southern blot hybridization. The results indicated that the gene for human cytochrome c1 is located on chromosome 8.