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The mitochondrial genome is essential for producing ATP (adenosine 5'-triphosphate) via oxidative phosphorylation. The gradual decline of mitochondrial function with age has long been postulated as a factor in aging. More recently, a variety of diseases have been related to molecular defects in human mitochondrial DNA. In both the cases of aging and disease, symptoms were generally neuromuscular, reflecting the tissues most dependent upon mitochondrial function. Also, in both cases novel features of mitochondrial genetics led to complex relations between genotype and phenotype. Little information is yet available about the role of environmental agents in these interactions.

Human brain development follows a unique pattern characterized by a prolonged period of postnatal growth and reorganization, and a postnatal peak in glucose utilization. The molecular processes underlying these developmental changes are poorly characterized. The objectives of this study were to determine developmental trajectories of gene expression and to examine the evolutionary history of genes differentially expressed as a function of age.We used microarrays to determine age-related patterns of mRNA expression in human cerebral cortical samples ranging from infancy to adulthood. In contrast to previous developmental gene expression studies of human neocortex that relied on postmortem tissue, we measured mRNA expression from the nondiseased margins of surgically resected tissue. We used regression models designed to identify transcripts that followed significant linear or curvilinear functions of age and used population genetics techniques to examine the evolution of these genes.We identified 40 transcripts with significant age-related trajectories in expression. Ten genes have documented roles in nervous system development and energy metabolism, others are novel candidates in brain development. Sixteen transcripts showed similar patterns of expression, characterized by decreasing expression during childhood. Comparative genomic analyses revealed that the regulatory regions of three genes have evidence of adaptive evolution in recent human evolution.These findings provide evidence that a subset of genes expressed in the human cerebral cortex broadly mirror developmental patterns of cortical glucose consumption. Whether there is a causal relationship between gene expression and glucose utilization remains to be determined.

Oxidative metabolism is needed for sustained skeletal muscle function. A key component of such metabolism is cytochrome c oxidase, the 13-subunit terminal complex of the mitochondrial electron transport chain. We used mice null for one of the two isoforms of Cox subunit 7a, heart/skeletal muscle-specific Cox7a1, to examine the cellular and functional responses of muscle adaptation in response to mitochondrial dysfunction. Specifically we determined if deletion of Cox7a1 would (1) limit exercise capacity, and (2) alter genes responsible for skeletal muscle capillarity and mitochondrial biogenesis. Sixteen male mice (Cox7a1 null mice, n = 8, and littermate controls, n = 8) performed incremental and run-to-exhaustion treadmill tests. The hindlimb muscles for both groups were analysed. The results indicated that capillary indices were reduced (by 30.7–44.9%) in the Cox7a1 null mice relative to controls. In addition, resting ATP levels and Cox specific activity were significantly reduced (>60%) in both glycolytic and oxidative muscle fibre types despite an increase in a major regulator of mitochondrial biogenesis, PGC-1β. These changes in the skeletal muscle resulted in exercise intolerance for the Cox7a1 null mice. Thus, our data indicate that deletion of the Cox7a1 isoform results in reduced muscle bioenergetics and hindlimb capillarity, helping to explain the observed impairment of muscle structure and function.

Cetaceans (dolphins and whales) have undergone a radical transformation from the original mammalian bodyplan. In addition, some cetaceans have evolved large brains and complex cognitive capacities. We compared approximately 10 000 protein-coding genes culled from the bottlenose dolphin genome with nine other genomes to reveal molecular correlates of the remarkable phenotypic features of these aquatic mammals. Evolutionary analyses demonstrated that the overall synonymous substitution rate in dolphins has slowed compared with other studied mammals, and is within the range of primates and elephants. We also discovered 228 genes potentially under positive selection (d N/ d S > 1) in the dolphin lineage. Twenty-seven of these genes are associated with the nervous system, including those related to human intellectual disabilities, synaptic plasticity and sleep. In addition, genes expressed in the mitochondrion have a significantly higher mean d N/ d S ratio in the dolphin lineage than others examined, indicating evolution in energy metabolism. We encountered selection in other genes potentially related to cetacean adaptations such as glucose and lipid metabolism, dermal and lung development, and the cardiovascular system. This study underlines the parallel molecular trajectory of cetaceans with other mammalian groups possessing large brains.

The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.

Brain ischemia-reperfusion injury (I/R) caused by stroke or cardiac arrest-resuscitation is associated with a high incidence of mortality and, in survivors, often results in profound neurological deficits. Specific regions of the brain (in particular, neurons in the CA1 hippocampus) are especially vulnerable to I/R injury, possibly due to exacerbated production of reactive oxygen species (ROS) and the resultant oxidative damage. However, the molecular mechanisms underlying excessive ROS generation in vulnerable neurons is unknown. Hypothesis: We propose that the hyper-susceptibility of the CA1 hippocampus to I/R injury is due in part to the localized expression of mitochondrial cytochrome c oxidase subunit 4-isoform 2 ( COX4-2 ) - i.e., a specific hyperactive isoform of COX. COX is the rate-limiting enzyme of oxidative phosphorylation and its activity indirectly controls mitochondrial ROS production by regulating mitochondrial membrane potential. Therefore expression of a hyperactive COX isoform (COX4-2) would result in excessive ROS production and promote cell death. Methods and Results: We performed COX activity measurements of isolated COX complexes containing COX4-2 compared to COX without COX4-2 and found COX4-2 expression causes a two-fold increase in activity. Next, we generated COX4-2 knock-out mice to determine the role of COX4-2 in selective vulnerability of neurons. Wild-type mice (WT: with documented COX4-2 expression in the CA1) and COX4-2 knock-out mice (KO) underwent 12 min of global brain ischemia (bilateral carotid occlusion with hypotension) or no intervention (time-matched shams). At 3 days post-reperfusion, the number of viable neurons in the CA1 hippocampus was quantified by cresyl violet staining. As expected: (i) shams displayed 100% neuronal viability; while (ii), in wild-type mice, only 14±2.3% of neurons in the CA1 remained viable following I/R. In contrast, COX4-2 KO mice were characterized by a significant, ∼5-fold increase in the proportion of surviving neurons (68±1.1% viability; pvs WT I/R, n = 3). Conclusion: These data provide novel evidence that the absence of COX4-2 confers protection to the CA1 and suggest that expression of the hyperactive COX4-2 isoform in the CA1 hippocampus contributes to the vulnerability of these neurons to lethal I/R injury.

Mitochondrial energy metabolism has been affected by a broad set of ancient and recent evolutionary events. The oldest example is the endosymbiosis theory that led to mitochondria and a recently proposed example is adaptation to cold climate by anatomically modern human lineages. Mitochondrial energy metabolism has also been associated with an important area in anthropology and evolutionary biology, brain enlargement in human evolution. Indeed, several studies have pointed to the need for a major metabolic rearrangement to supply a sufficient amount of energy for brain development in primates.The genes encoding for the coupled cytochrome c (Cyt c) and cytochrome c oxidase (COX, complex IV, EC 1.9.3.1) seem to have an exceptional pattern of evolution in the anthropoid lineage. It has been proposed that this evolution was linked to the rearrangement of energy metabolism needed for brain enlargement. This hypothesis is reinforced by the fact that the COX enzyme was proposed to have a large role in control of the respiratory chain and thereby global energy production.After summarizing major events that occurred during the evolution of COX and cytochrome c on the primate lineage, we review the different evolutionary forces that could have influenced primate COX evolution and discuss the probable causes and consequences of this evolution. Finally, we discuss and review the co-occurring primate phenotypic evolution.

OXPHOS polymorphisms are known to be population specific and to influence disease. Previous studies have focused on mtDNA polymorphisms. Based on a world sampling of 629 unrelated individuals, we have now studied the polymorphisms of the 80 genes encoding OXPHOS nuclear subunits. We have shown that (i) amino-acid replacement frequencies are significantly correlated with their pathogenicity probability, and (ii) populations can be distinguished based only on amino-acid replacements in nuclear encoded OXPHOS subunits. These results are congruent with the major mtDNA haplogroups, which suggests that OXPHOS complexes are different across the populations in both nuclear and in mitochondrial encoded subunits.

Subunit 7a of mouse cytochrome c oxidase (Cox) displays a contractile muscle-specific isoform, Cox7a1, that is the major cardiac form. To gain insight into the role of this isoform, we have produced a new knockout mouse line that lacks Cox7a1. We show that homozygous and heterozygous Cox7a1 knockout mice, although viable, have reduced Cox activity and develop a dilated cardiomyopathy at 6 weeks of age. Surprisingly, the cardiomyopathy improves and stabilizes by 6 months of age. Cox7a1 knockout mice incorporate more of the “liver-type” isoform Cox7a2 into the cardiac Cox holoenzyme and, also surprisingly, have higher tissue ATP levels.

Cytochrome c (cyt c) participates in two crucial cellular processes, energy production and apoptosis, and unsurprisingly is a highly conserved protein. However, previous studies have reported for the primate lineage (i) loss of the paralogous testis isoform, (ii) an acceleration and then a deceleration of the amino acid replacement rate of the cyt c somatic isoform, and (iii) atypical biochemical behavior of human cyt c. To gain insight into the cause of these major evolutionary events, we have retraced the history of cyt c loci among primates. For testis cyt c, all primate sequences examined carry the same nonsense mutation, which suggests that silencing occurred before the primates diversified. For somatic cyt c, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses yielded the same tree topology. The evolutionary analyses show that a fast accumulation of non-synonymous mutations (suggesting positive selection) occurred specifically on the anthropoid lineage root and then continued in parallel on the early catarrhini and platyrrhini stems. Analysis of evolutionary changes using the 3D structure suggests they are focused on the respiratory chain rather than on apoptosis or other cyt c functions. In agreement with previous biochemical studies, our results suggest that silencing of the cyt c testis isoform could be linked with the decrease of primate reproduction rate. Finally, the evolution of cyt c in the two sister anthropoid groups leads us to propose that somatic cyt c evolution may be related both to COX evolution and to the convergent brain and body mass enlargement in these two anthropoid clades.

It had not previously been known whether synthesis of nuclear-encoded mitochondrial subunits occurs in pre-implantation embryos. We have used cytoplasmic injections of antisense RNA transcribed in vitro to study this question. Capped, in vitro transcribed RNA antisense to either cytochrome c oxidase subunit IV or VIIc injected into each cell at the two-cell stage markedly inhibited synthesis of adenine nucleotide by the 8- to 16-cell stage, whereas injection of the cognate sense RNAs gave levels similar to those previously published for normal embryos. These results strongly suggest that translation of nuclear-encoded mRNAs for mitochondrial subunits is required during pre-implantation development. It was of additional interest that, not only was ATP decreased, but ADP and AMP as well, with the effect that the charge ratio remained constant. The results also suggest, therefore, that the mechanism by which cells normally regulate their charge ratio, though to be with adenylate deaminase, is already in place.

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 COXVHa-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.

Postischemic reperfusion is known to cause iron-mediated peroxidation of polyunsaturated fatty acids in membranes, including mitochondrial membranes, in the brain cortex. Consequently, we tested the hypothesis that this radical-mediated damage would extend to DNA. Mitochondrial DNA (mtDNA) was chosen because of its presence at a known site of free radical formation, its sensitivity and ease of assay, and its known lack of any repair systems. In model experiments we utilized endonuclease III or piperidine to amplify topological form conversions in mtDNA damaged by in vitro reactions with hydroxyl radical. We then applied the amplified detection assays to dog brain mtDNA isolated after 2 or 8 h of reperfusion following a 20-min cardiac arrest. We found that ischemia and reperfusion caused no topological form conversions in mtDNA. Similarly, nucleotide incorporation by a gap-filling reaction showed no sensitivity to digestion of the mtDNA by exonuclease III, an enzyme known to remove blocked 3’ termini at the site of radical-generated nicks. Furthermore, the recovery of mtDNA was similar in all experimental groups, suggesting that putatively damaged forms had not been removed by rapid degradation. Thus, despite mitochondrial membrane damage, brain mtDNA does not accumulate oxygen radical damage during postischemic brain reperfusion.

Mammalian cytochrome c oxidase (COX) is a 13-subunit polypeptide complex that contains 10 subunits coded by the nucleus and 3 by the mitochondria. The nuclear-encoded subunits, though of unknown function, are presumed to play a regulatory role. Three of these (subunits VIa, VIIa, and VIII) generally exist in one of two isoforms--a constitutive (L) isoform or a skeletal muscle/heart-specific (H) isoform. To study the regulation, and possibly function, of these isoforms, we have begun characterizing the genes. In this paper we describe the isolation and characterization of the gene for the bovine COX VIIa-H isoform. The gene consists of four exons spanning 1.58 kb and is associated with a CpG island. There are no canonical TATA or CCAAT boxes immediately upstream of the transcription start site. Putative DNA sequence elements associated with respiratory function, muscle gene activation, and housekeeping function are present both in the upstream regions and within introns.

Specific sets of brain-expressed genes, such as aerobic energy metabolism genes, evolved adaptively in the ancestry of humans and may have evolved adaptively in the ancestry of other large-brained mammals. The recent addition of genomes from two afrotherians (elephant and tenrec) to the expanding set of publically available sequenced mammalian genomes provided an opportunity to test this hypothesis. Elephants resemble humans by having large brains and long life spans; tenrecs, in contrast, have small brains and short life spans. Thus, we investigated whether the phylogenomic patterns of adaptive evolution are more similar between elephant and human than between either elephant and tenrec lineages or human and mouse lineages, and whether aerobic energy metabolism genes are especially well represented in the elephant and human patterns. Our analyses encompassed ≈6,000 genes in each of these lineages with each gene yielding extensive coding sequence matches in interordinal comparisons. Each gene's nonsynonymous and synonymous nucleotide substitution rates and dN/dS ratios were determined. Then, from gene ontology information on genes with the higher dN/dS ratios, we identified the more prevalent sets of genes that belong to specific functional categories and that evolved adaptively. Elephant and human lineages showed much slower nucleotide substitution rates than tenrec and mouse lineages but more adaptively evolved genes. In correlation with absolute brain size and brain oxygen consumption being largest in elephants and next largest in humans, adaptively evolved aerobic energy metabolism genes were most evident in the elephant lineage and next most evident in the human lineage.

Treatment of poly C with methoxyamine leads to formation of 5,6-dihydro-6-methoxyaminocytosine residues in the polymer. These direct incorporation of adenylate residues into the poly G synthesized when the modified poly C is used as a template for RNA polymerase. The process is analogous to that of hydroxylamine-induced errors in replication. Comparison of the uptake of [14C]methoxyamine by the poly C with the percentage of adenylate incorporated in the poly G shows that the induction of errors is a highly efficient process: every modified cytosine residue directs the incorporation of one adenylate residue. A probable explanation for this is that the incorporation is mediated by hydrogen-bonding between the adenine and a predominant imino tautomer of the cytosine adduct.

Subunit VIIa of bovine cytochrome c oxidase occurs in two forms, the so-called heart and liver isoforms, which have been shown by protein analysis to differ in 35% of their amino acids. We have isolated and characterized cDNAs for each isoform. The derived heart-type processed protein is 59 amino acids long, with a 21-residue presequence; the immediate C terminus differs from the established protein sequence. The liver-type processed protein is 60 residues long, with a 23-amino acid presequence. Both presequences are traditional in that they are positively charged and appear amphiphilic when helically arrayed. The presequences are only 22% identical, but they both contain conserved residues indicative of two-step processing of the precursor proteins. Southern blot analysis reveals that the bovine genome contains five to six copies of the liver-type gene as opposed to the presence of a single copy heart-type gene. Transcriptional analysis shows that heart-type message is detectable only in heart and skeletal muscle; the liver type is also seen in heart and muscle and, additionally, in the other tissues examined (liver, brain, and lung). The amino acid sequence EKQKLFQED is conserved in rat and in both isoforms in cow and human and may represent a domain of core subunit function.

Iron-mediated peroxidation of brain lipids is known to occur during reperfusion following cardiac arrest. Since in vitro damage to DNA is caused by similar iron-dependent peroxidation, we tested whether free radical damage to genomic DNA also develops during reperfusion following cardiac arrest and resuscitation. Genomic DNA was isolated from the cerebral cortex in (i) normal dogs, (ii)_dogs subjected to a 20-min cardiac arrest, and (iii)_dogs resuscitated from a 20-min cardiac arrest and then allowed to reperfuse for 2 or 8 h. DNA strand nicks were evaluated by in vitro labeling of newly created 3′ and 5′ termini. DNA base damage was evaluated utilizing with piperidine prior to labeling of 5′ termini. The 3t DNA termini were labeled before and after digestion with exonuclease III, and the 5′ DNA termini were labeled before and after treatment with piperidine. In vitro experiments with genomic DNA damaged by oxygen radicals verified that these labeling methods identified radical damage. In the experimental animal groups, terminal incorporation and electrophoretic mobility of brain nuclear DNA are not significantly changed either by 20 min of complete brain ischermia or during the first 8 h of reperfusion. We conclude that genomic DNA is not extensively damaged during cardiac arrest and early reperfusion, and therefore such DNA damage does not appear to be an important early aspect of the neurologic injury that accompanies cardiac arrest and resuscitation.

The human genome evolution project seeks to reveal the genetic underpinnings of key phenotypic features that are distinctive of humans, such as a greatly enlarged cerebral cortex, slow development, and long life spans. This project has focused predominantly on genotypic changes during the 6-million-year descent from the last common ancestor (LCA) of humans and chimpanzees. Here, we argue that adaptive genotypic changes during earlier periods of evolutionary history also helped shape the distinctive human phenotype. Using comparative genome sequence data from 10 vertebrate species, we find a signature of human ancestry-specific adaptive evolution in 1,240 genes during their descent from the LCA with rodents. We also find that the signature of adaptive evolution is significantly different for highly expressed genes in human fetal and adult-stage tissues. Functional annotation clustering shows that on the ape stem lineage, an especially evident adaptively evolved biological pathway contains genes that function in mitochondria, are crucially involved in aerobic energy production, and are highly expressed in two energy-demanding tissues, heart and brain. Also, on this ape stem lineage, there was adaptive evolution among genes associated with human autoimmune and aging-related diseases. During more recent human descent, the adaptively evolving, highly expressed genes in fetal brain are involved in mediating neuronal connectivity. Comparing adaptively evolving genes from pre- and postnatal-stage tissues suggests that different selective pressures act on the development vs. the maintenance of the human phenotype.

We have isolated a chimpanzee processed pseudogene for subunit IV of cytochrome c oxidase (COX; EC 1.9.3.1) by screening a chimpanzee genomic library in λCharon 32 with a bovine liver cDNA encoding COX subunit IV (COX IV), and localized it to a 1.9-kb Hin dIII fragment. Southern-blot analysis of genomic DNA from five primates showed that DNAs from human, gorilla, and chimpanzee each contained the 1.9-kb pseudogene fragment, whereas orangutan and pigtail macaque monkey DNA did not. This result clearly indicates that the pseudogene arose before the divergence of the chimpanzee and gorilla from the primate lineage. By screening Chinese hamster × human hybrid panels with the human COX4 cDNA, we have mapped COX4 genes to two human chromosomes, 14 and 16. The 1.9-kb Hin dIII fragment containing the pseudogene, COX4P1 , can be assigned to chromosome 14, and by means of rearranged chromosomes in somatic cell hybrids, to 14q21-qter. Similarly, the functional gene, COX4 , has been mapped to 16q22-qter.

Subunit 4 of cytochrome c oxidase (CcO) is a nuclear-encoded regulatory subunit of the terminal complex of the mitochondrial electron transport chain. We have recently discovered an isoform of CcO 4 (CcO4-2) which is specific to lung and trachea, and is induced after birth. The role of CcO as the major cellular oxygen consumer, and the lung-specific expression of CcO4-2, led us to investigate CcO4-2 gene regulation. We cloned the CcO4-2 promoter regions of cow, rat and mouse and compared them with the human promoter. Promoter activity is localized within a 118-bp proximal region of the human promoter and is stimulated by hypoxia, reaching a maximum (threefold) under 4% oxygen compared with normoxia. CcO4-2 oxygen responsiveness was assigned by mutagenesis to a novel promoter element (5'-GGACGTTCCCACG-3') that lies within a 24-bp region that is 79% conserved in all four species. This element is able to bind protein, and competition experiments revealed that, within the element, the four core bases 5'-TCNCA-3' are obligatory for transcription factor binding. CcO isolated from lung showed a 2.5-fold increased maximal turnover compared with liver CcO. We propose that CcO4-2 expression in highly oxygenated lung and trachea protects these tissues from oxidative damage by accelerating the last step in the electron transport chain, leading to a decrease in available electrons for free radical formation.

International audience; Previous molecular analyses of mammalian evolutionary relationships involving a wide range of placental mammalian taxa have been restricted in size from one to two dozen gene loci and have not decisively resolved the basal branching order within Placentalia. Here, on extracting from thousands of gene loci both their coding nucleotide sequences and translated amino acid sequences, we attempt to resolve key uncertainties about the ancient branching pattern of crown placental mammals. Focusing on approximately 1,700 conserved gene loci, those that have the more slowly evolving coding sequences, and using maximum-likelihood, Bayesian inference, maximum parsimony, and neighbor-joining (NJ) phylogenetic tree reconstruction methods, we find from almost all results that a clade (the southern Atlantogenata) composed of Afrotheria and Xenarthra is the sister group of all other (the northern Boreoeutheria) crown placental mammals, among boreoeutherians Rodentia groups with Lagomorpha, and the resultant Glires is close to Primates. Only the NJ tree for nucleotide sequences separates Rodentia (murids) first and then Lagomorpha (rabbit) from the other placental mammals. However, this nucleotide NJ tree still depicts Atlantogenata and Boreoeutheria but minus Rodentia and Lagomorpha. Moreover, the NJ tree for amino acid sequences does depict the basal separation to be between Atlantogenata and a Boreoeutheria that includes Rodentia and Lagomorpha. Crown placental mammalian diversification appears to be largely the result of ancient plate tectonic events that allowed time for convergent phenotypes to evolve in the descendant clades.

Background Many electron transport chain (ETC) genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains. Results In a dataset comprising 29 vertebrate taxa, including representatives from all major groups of primates, there is nearly 100% conservation of the COX5A amino acid sequence among extant, non-anthropoid placental mammals. The most recent common ancestor of these species lived about 100 million years (MY) ago. In contrast, anthropoid primates show markedly elevated rates of nonsynonymous evolution. In particular, branch site tests identify five positively selected codons in anthropoids, and ancestral reconstructions infer that substitutions in these codons occurred predominantly on stem lineages (anthropoid, ape and New World monkey) and on the human terminal branch. Examination of catarrhine brain samples by immunohistochemistry characterizes for the first time COX5A protein distribution in the primate neocortex, and suggests that the protein is most abundant in the mitochondria of large-size projection neurons. Real time quantitative PCR supports previous microarray results showing COX5A is expressed in cerebral cortical tissue at a higher level in human than in chimpanzee or gorilla. Conclusion Taken together, these results suggest that both protein structural and gene regulatory changes contributed to COX5A evolution during humankind's ancestry. Furthermore, these findings are consistent with the hypothesis that adaptations in ETC genes contributed to the emergence of the energetically expensive anthropoid neocortex.

Mitochondria are both the power plant of the cell and a central integrator of signals that govern the lifespan, replication and death of the cell. Perhaps as a consequence, genes that encode components of the mitochondrial electron transport chain (ETC) are generally conserved. Therefore, it is surprising that many of these genes in anthropoid primates (New World monkeys, Old World monkeys and apes, including humans) have been major targets of darwinian positive selection. Sequence comparisons have provided evidence that marked increases of non-synonymous substitution rates occurred in anthropoid ETC genes that encode subunits of Complex III and IV, and the electron carrier molecule cytochrome c (CYC). Two important questions are: (i) how has evolution altered ETC function? and; (ii) how might functional changes in the ETC be linked to evolution of an expanded neocortical brain?

Cytochrome c (CYC) and 9 of the 13 subunits of cytochrome c oxidase (complex IV; COX) were previously shown to have accelerated rates of nonsynonymous substitution in anthropoid primates. Cytochrome b, the mtDNA encoded subunit of ubiquinol-cytochrome c reductase (complex III), also showed an accelerated nonsynonymous substitution rate in anthropoid primates but rate information about the nuclear encoded subunits of complex III has been lacking. We now report that phylogenetic and relative rates analysis of a nuclear encoded catalytically active subunit of complex III, the iron-sulfur protein (ISP), shows an accelerated rate of amino acid replacement similar to cytochrome b. Because both ISP and subunit 9, whose function is not directly related to electron transport, are produced by cleavage into two subunits of the initial translation product of a single gene, it is probable that these two subunits of complex III have essentially identical underlying rates of mutation. Nevertheless, we find that the catalytically active ISP has an accelerated rate of amino acid replacement in anthropoid primates whereas the catalytically inactive subunit 9 does not.

Gene expression profiles from the anterior cingulate cortex (ACC) of human, chimpanzee, gorilla, and macaque samples provide clues about genetic regulatory changes in human and other catarrhine primate brains. The ACC, a cerebral neocortical region, has human-specific histological features. Physiologically, an individual's ACC displays increased activity during that individual's performance of cognitive tasks. Of approximately 45,000 probe sets on microarray chips representing transcripts of all or most human genes, approximately 16,000 were commonly detected in human ACC samples and comparable numbers, 14,000-15,000, in gorilla and chimpanzee ACC samples. Phylogenetic results obtained from gene expression profiles contradict the traditional expectation that the non-human African apes (i.e., chimpanzee and gorilla) should be more like each other than either should be like humans. Instead, the chimpanzee ACC profiles are more like the human than like the gorilla; these profiles demonstrate that chimpanzees are the sister group of humans. Moreover, for those unambiguous expression changes mapping to important biological processes and molecular functions that statistically are significantly represented in the data, the chimpanzee clade shows at least as much apparent regulatory evolution as does the human clade. Among important changes in the ancestry of both humans and chimpanzees, but to a greater extent in humans, are the up-regulated expression profiles of aerobic energy metabolism genes and neuronal function-related genes, suggesting that increased neuronal activity required increased supplies of energy.

The terminal enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX), contains three mitochondrial and ten nuclear encoded subunits in mammals. Three of the nuclear subunits (VIa, VIIa, and VIII) have muscle and non-muscle-specific isoforms, subunit IV contains a lung-specific isoform, and subunit VIb contains a testes-specific isoform. For subunit VIII, the smallest nuclear encoded COX polypeptide, we have now found a third gene (COX 8-3), which has been identified in human, lemur, rat, and mouse, suggesting that it is present in a broad range of Eutherian mammals. Sequence similarity and gene structure support the homology of COX8-3 to the other subunit VIII isoforms, indicating that all three are the product of gene duplications. COX VIII-3 protein is mitochondrially-targeted, as shown by a fluorescent COX VIII3/DsRed fusion protein. Both the mitochondrial targeting and its sequence conservation suggest that COXVIII-3 functions as part of the COX holoenzyme and could have a tissue-specific role, as is the case for the other two isoforms. Questions remain about where COX8-3 is predominantly expressed. However, detection of full-length cDNAs, lower levels of sequence divergence at the first and second codon positions compared to the third, and a conserved gene structure indicate that COX VIII-3 is an expressed gene whose origin dates to at least 91 million years ago.

Sperm motility is highly dependent on aerobic energy metabolism, of which the apparent rate-limiting step of the mitochondrial respiratory chain is catalyzed by cytochrome c oxidase (COX). COX is the only electron transport chain complex to display isoforms, consistent with its suggested rate-limiting role. Isoforms were previously described for four of the 13 subunits. We now report the discovery that COX subunit VIb displays a testes-specific isoform in human, bull, rat, and mouse (COX VIb-2). Analysis of a variety of rat and mouse tissues, including ovaries, demonstrates exclusive expression of VIb-2 in testes, whereas VIb-1 transcripts are absent in rodent testes, even at early developmental stages. In contrast, both isoforms are transcribed in human testes. In situ hybridizations with human, rat, and mouse testes sections reveal VIb-2 transcripts in all testicular cell types. Within the seminiferous tubules, VIb-1 shows stronger signals in the periphery than in the lumen. Previously, cytochrome c was the only component of the mitochondrial respiratory chain known to express a testes-specific isoform in mammals. COX subunit VIb connects the two COX monomers into the physiological dimeric form, and is the only COX subunit that, like cytochrome c, is solely located in the inter-membrane space. Significant differences between the isoform sequences, in particular changes in charged amino acids, suggest interactions with cytochrome c and sperm-specific energy requirements.

What do functionally important DNA sites, those scrutinized and shaped by natural selection, tell us about the place of humans in evolution? Here we compare ≈90 kb of coding DNA nucleotide sequence from 97 human genes to their sequenced chimpanzee counterparts and to available sequenced gorilla, orangutan, and Old World monkey counterparts, and, on a more limited basis, to mouse. The nonsynonymous changes (functionally important), like synonymous changes (functionally much less important), show chimpanzees and humans to be most closely related, sharing 99.4% identity at nonsynonymous sites and 98.4% at synonymous sites. On a time scale, the coding DNA divergencies separate the human–chimpanzee clade from the gorilla clade at between 6 and 7 million years ago and place the most recent common ancestor of humans and chimpanzees at between 5 and 6 million years ago. The evolutionary rate of coding DNA in the catarrhine clade (Old World monkey and ape, including human) is much slower than in the lineage to mouse. Among the genes examined, 30 show evidence of positive selection during descent of catarrhines. Nonsynonymous substitutions by themselves, in this subset of positively selected genes, group humans and chimpanzees closest to each other and have chimpanzees diverge about as much from the common human–chimpanzee ancestor as humans do. This functional DNA evidence supports two previously offered taxonomic proposals: family Hominidae should include all extant apes; and genusHomoshould include three extant species and two subgenera,Homo(Homo)sapiens(humankind),Homo(Pan)troglodytes(common chimpanzee), andHomo(Pan)paniscus(bonobo chimpanzee).

Cytochrome c oxidase (COX) is a 13-subunit protein complex that catalyzes the last step in mitochondrial electron transfer in mammals. Of the 10 subunits encoded by nuclear DNA (three are mtDNA products), some are expressed as tissue- and/or development-specific isoforms. For COX subunit VIII, previous work showed that expression of the contractile muscle-specific isoform gene, COX8H , is absent in humans and Old World monkeys, and the other isoform gene, COX8L , is expressed ubiquitously. Here, we show that COX8H is transcribed in most primate clades, but its expression is absent in catarrhines, that is, in Old World monkeys and hominids (apes, including humans), having become a pseudogene in the stem of the catarrhines. The ubiquitously expressed isoform, COX8L , underwent nonsynonymous rate acceleration and elevation in the ratio of nonsynonymous/synonymous changes in the stem of anthropoid primates (New World monkeys and catarrhines), possibly setting the stage for loss of the heart-type (H) isoform. The most rapidly evolving region of VIII-L is one that interacts with COX I, suggesting that the changes are functionally coadaptive. Because accelerated rates of nonsynonymous substitutions in anthropoids such as observed for COX8L are also shown by genes for at least 13 other electron transport chain components, these encoded amino acid replacements may be viewed as part of a series of coadaptive changes that optimized the anthropoid biochemical machinery for aerobic energy metabolism. We argue that these changes were linked to the evolution of an expanded neocortex in anthropoid primates.

We have isolated and characterized the human gene ( COX7AH ) for the contractile muscle isoform of cytochrome c oxidase (COX) subunit VIIa. This subunit is one of the 10 nuclear encoded subunits of the 13-subunit holoenzyme that carries out the terminal step in the electron transport chain. Using transient transfection assays, we have located a 5′-flanking region sufficient to direct high level, skeletal myotube-specific reporter gene expression. This 792 bp basal promoter, which contains the single transcription start but no canonical TATA or CCAAT boxes, contains one MEF2 site, three E boxes, and an Sp1 site that show binding to their cognate factors, and are all required for full expression. Mutation and transactivation analysis suggest that there is functional interaction between these binding sites.

Mitochondrial DNA (mtDNA)-encoded proteins function in eukaryotes as subunits of respiratory complexes that also contain nuclear DNA (nDNA)-encoded subunits. The importance of functional interactions between mtDNA- and nDNA-encoded proteins was previously demonstrated by testing the survivability of cybrid cells or individuals containing nDNA and mtDNA from different populations or species. This report focuses on the multisubunit respiratory complex cytochrome c oxidase (COX), made up of both mtDNA-encoded and nDNA-encoded subunits. A combination of evolutionary and crystallographic data is employed to determine whether rates of nonsynonymous substitutions have been higher, the same, or lower for residues in close proximity that are encoded by a different genome (nDNA or mtDNA). This determination is performed by simply taking the ratio, called the interaction ratio i, of the nonsynonymous substitution rate of the close-contact residues to the nonsynonymous substitution rate of the noncontact residues. We assume that the close-contact residues (which are more likely to interact) are functionally important and that, therefore, amino acid replacements among these residues cannot escape the scrutiny of natural selection. i = 1 indicates that the close-contact residues have been under neither greater purifying selection nor greater positive selection than the noncontact residues as a specific consequence of their being encoded by separate genomes. i1 indicates that the close-contact residues have been under greater purifying selection but less positive selection than have the noncontact residues. Conversely, i1 indicates that the close-contact residues have been under less purifying but greater positive selection than have the noncontact residues. i1 may be referred to as a constraining interaction; i.e., the close-contact residues compared with the noncontact residues appear to be under greater structural-functional constraints. On the other hand, i1 may be referred to as an optimizing interaction; i.e., apparently many different amino acid replacements are required to optimize this subunit's interaction with the other subunit. A major finding is that the nDNA-encoded residues in close physical proximity to mtDNA-encoded residues evolve more slowly than the other nuclear-encoded residues (and thus display a constraining interaction), whereas the mtDNA-encoded residues in close physical proximity to nDNA-encoded residues evolve more rapidly than the other mitochondrial-encoded residues (and thus display an optimizing interaction). A possible reason for this striking difference between the nuclear- and mitochondrial-encoded COX subunits in how their functional interaction evolves is discussed.

The initial goals of the Human Genome Project (HGP), to sequence the human genome and identify its constituent genes, are on the verge of being achieved. However, the scientific community recognizes that sequencing must be followed by understanding the manifold functions of human genes throughout

We identified a novel human gene, NOC4 (Neighbor Of COX4), located 58 to COX4, the gene for cytochrome c oxidase subunit IV, on Chr16q32-ter. Transcripts from this gene were iden- tified among human expressed sequence tags. A full-length, 1.06- kb human retinal NOC4 cDNA encoded a 24-kDa, 210-amino acid hypothetical protein of unknown function. Northern hybridization analysis of human RNAs from various tissues detected NOC4 transcripts of 2.2 and 1.4 kb in all tissues examined, suggesting that NOC4 expression is ubiquitous. Transcription of both the COX4 and NOC4 genes initiates within a 250-bp intergenic pro- moter and occurs in opposite directions. The bidirectional pro- moter is G + C-rich, lacks TATA and CCAAT elements, and con- tains multiple potential binding sites for Sp1 and NRF-2/GABP. Two of the NRF-2/GABP sites are located within 14-bp direct repeats, a conserved feature of mammalian COX4 promoters. The NOC4 and COX4 genes are also linked in the rat, mouse, and bovine genomes. A NOC4-GFP fusion protein is located in both the nucleus and the cytoplasm, including the mitochondria.

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.

Mitochondria contain a molecular genetic system to express the 13 protein components of the electron transport system encoded in the mitochondrial genome (mtDNA). Defects in the function of this system result in some diaseases, many of which are multisystem disorders, prominently involving highly aerobic, postmitotic tissues. These defects can be caused by large-scale rearrangements of mtDNA, by point mutations, or by nuclear gene mutations resulting in abnormalities in mtDNA. Although any of these mutations would be expected to produce a similar clinical phenotype by compromising oxidative phosphorylation, the surprising and puzzling result is that different clinical phenotypes are generally associated with specific mtDNA mutations. Moreover, the same mutation can produce a distinct clinical phenotype in different individuals or pedigrees. MtDNA rearrangements are also found in aged individuals, but at a subclinical level, suggesting that normal and pathological processes can differ by the effect of genetic or environmental factors on the error rate of mtDNA replication.

In comparison with other primate species, humans have an extended juvenile period during which the brain is more plastic. In the current study we sought to examine gene expression in the cerebral cortex during development in the context of this adaptive plasticity. We introduce an approach designed to discriminate genes with variable as opposed to uniform patterns of gene expression and found that greater inter-individual variance is observed among children than among adults. For the 337 transcripts that show this pattern, we found a significant overrepresentation of genes annotated to the immune system process (pFDR ~/= 0). Moreover, genes known to be important in neuronal function, such as brain-derived neurotrophic factor (BDNF), are included among the genes more variably expressed in childhood. We propose that the developmental period of heightened childhood neuronal plasticity is characterized by more dynamic patterns of gene expression in the cerebral cortex compared to adulthood when the brain is less plastic. That an overabundance of these genes are annotated to the immune system suggests that the functions of these genes can be thought of not only in the context of antigen processing and presentation, but also in the context of nervous system development.

Subunit VIIa of mammalian cytochrome c oxidase is one of three nuclear-encoded subunits that exhibit isoforms, existing predominantly as an H-form in cardiac and skeletal muscle tissues and as an L-form in others. We have isolated and characterized the L-isoform gene (COX7aL). It is 5.4 kb long, consists of four exons, and is located at a CpG island. Sp1 sites and an NRF1 site are located in an approximately 100-bp region immediately upstream of the gene. Comparison of the sequence and organization with the previously described H-isoform gene shows identical intron-exon organizations, with the first intron of both isoform genes splitting the presequence coding region almost identically. These results suggest that the isoform genes arose by duplication from a common ancestor prior to the mammalian radiation and that the ancestor already contained the presequences. In addition, four processed pseudogenes of the L-type have been isolated and characterized, one of which (COX7aLP1) contains no deletions, insertions, or frame-shifts and can encode a precursor protein of 83 amino acids. Construction of a phylogenetic tree employing extant COX7aL cDNA and bovine pseudogene sequences suggests that the expressed bovine gene and COX7aLP1 arose from a gene duplication event 4.6-6.8 Mya.

Rat brain nuclear proteins were examined for tyrosine phosphorylation after resuscitation from a 10-min cardiac arrest. Insulin (1 unit/kg intravenously), given immediately after resuscitation, caused a marked increase in tyrosine phosphorylation of a 90-kDa brain protein. This effect occurred without hypoglycemia and was not observed after insulin administration in previously insulinopenic, diabetic, nonischemic animals. Insulin-responsive tyrosine phosphorylation of a specific 90-kDa protein during reperfusion may represent insulin stimulation of a neuroprotective brain response to an ischemic insult, consistent with recent observations that insulin administration during reperfusion protects selectively vulnerable neurons from postischemic death.

Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase (ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein (hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.

We have compared the DNA sequences of nine mammalian genes for cytochrome c oxidase subunit IV (COX4 genes)--four expressed genes (human, bovine, rat, and mouse) and five pseudogenes (human, chimpanzee, orangutan, squirrel monkey, and bovine)--and constructed the sequence of the ancestral mammalian COX4 gene. By analyzing these sequences to determine the pattern and rate of nucleotide substitution in each branch of the evolutionary tree, we deduced that the human gene has evolved rapidly since the origin of the primate pseudogene approximately 41 million years ago, and we discuss the suggestion that this results from coevolution of nuclear and mitochondrial genes for cytochrome c oxidase.

We have isolated a cDNA that encodes subunit VIIc of bovine cytochrome c oxidase (COX VIIc). The 325-bp cDNA contains sequences encoding the mature 47-amino acid (aa) polypeptide and a 16-aa presequence. The deduced aa sequence of the processed polypeptide is identical to that of the heart protein determined by aa sequencing. Northern-blot analysis reveals a single 525-nucleotide (nt) transcript in all tissues examined, whose levels vary with the corresponding respiratory activities in different tissues; thus, no evidence for isoforms of COX VIIc is seen in adult tissues. Southern-blot analysis of bovine genomic DNA digested with three different restriction enzymes reveals several bands that hybridize with the cDNA. We present here the sequence of one genomic region that contains a processed gene encoding COX VIIc. The genomic and cDNA nt sequences are 99% identical throughout the 189-bp open reading frame; the deduced aa sequences are identical. The sequence of the genomic clone suggests that the cDNA terminates prematurely at an EcoRI site in the 3'-untranslated region. We have compared COXVIIc cDNAs from cow, human and mouse, and find the presequence similarity among them to be 100% at the aa level.