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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26919/1/0000485.pd

Molecular Evolution of Cytochrome c Oxidase Subunit IV: Evidence for Positive Selection in Simian Primates

Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of ``neutral DNA'' nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42366/1/239-44-5-477_44n5p477.pd

OCPAT: an online codon-preserved alignment tool for evolutionary genomic analysis of protein coding sequences

<p>Abstract</p> <p>Background</p> <p>Rapidly accumulating genome sequence data from multiple species offer powerful opportunities for the detection of DNA sequence evolution. Phylogenetic tree construction and codon-based tests for natural selection are the prevailing tools used to detect functionally important evolutionary change in protein coding sequences. These analyses often require multiple DNA sequence alignments that maintain the correct reading frame for each collection of putative orthologous sequences. Since this feature is not available in most alignment tools, codon reading frames often must be checked manually before evolutionary analyses can commence.</p> <p>Results</p> <p>Here we report an online codon-preserved alignment tool (OCPAT) that generates multiple sequence alignments automatically from the coding sequences of any list of human gene IDs and their putative orthologs from genomes of other vertebrate tetrapods. OCPAT is programmed to extract putative orthologous genes from genomes and to align the orthologs with the reading frame maintained in all species. OCPAT also optimizes the alignment by trimming the most variable alignment regions at the 5' and 3' ends of each gene. The resulting output of alignments is returned in several formats, which facilitates further molecular evolutionary analyses by appropriate available software. Alignments are generally robust and reliable, retaining the correct reading frame. The tool can serve as the first step for comparative genomic analyses of protein-coding gene sequences including phylogenetic tree reconstruction and detection of natural selection. We aligned 20,658 human RefSeq mRNAs using OCPAT. Most alignments are missing sequence(s) from at least one species; however, functional annotation clustering of the ~1700 transcripts that were alignable to all species shows that genes involved in multi-subunit protein complexes are highly conserved.</p> <p>Conclusion</p> <p>The OCPAT program facilitates large-scale evolutionary and phylogenetic analyses of entire biological processes, pathways, and diseases.</p

Molecular evolution of the cytochrome c oxidase subunit 5A gene in primates

Abstract 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\u27s ancestry. Furthermore, these findings are consistent with the hypothesis that adaptations in ETC genes contributed to the emergence of the energetically expensive anthropoid neocortex

Rapid purification of yeast mitochondrial DNA in high yield

A procedure is presented for the rapid isolation of mitochondrial DNA (mtDNA) in high yield from Saccharomyces cerevisiae. Yeast cells, which may be grown to late stationary phase, are broken by a combination of enzymatic and mechanical means; mtDNA is then isolated from a crude mitochondrial lysate by a single cycle of bisbenzimide-CsCl buoyant density centrifugation. mtDNA so isolated is at least 99.5% pure, and has a mean duplex molecular weight of 24.5 [middle dot] 106. In addition to mtDNA and bulk nuclear DNA, several other yeast nucleic acid species, identified as ribosomal DNA and a mixture of duplex RNAs, form discrete bands in these gradients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23088/1/0000005.pd

Non-coding regions of nuclear-DNA-encoded mitochondrial genes and intergenic sequences are targeted by autoantibodies in breast cancer

Autoantibodies against mitochondrial-derived antigens play a key role in chronic tissue inflammation in autoimmune disorders and cancers. Here, we identify autoreactive nuclear genomic DNA (nDNA)-encoded mitochondrial gene products (GAPDH, PKM2, GSTP1, SPATA5, MFF, TSPOAP1, PHB2, COA4, and HAGH) recognized by breast cancer (BC) patients\u27 sera as nonself, supporting a direct relationship of mitochondrial autoimmunity to breast carcinogenesis. Autoreactivity of multiple nDNA-encoded mitochondrial gene products was mapped to protein-coding regions, 3\u27 untranslated regions (UTRs), as well as introns. In addition, autoantibodies in BC sera targeted intergenic sequences that may be parts of long non-coding RNA (lncRNA) genes, including LINC02381 and other putative lncRNA neighbors of the protein-coding genes ERCC4, CXCL13, SOX3, PCDH1, EDDM3B, and GRB2. Increasing evidence indicates that lncRNAs play a key role in carcinogenesis. Consistent with this, our findings suggest that lncRNAs, as well as mRNAs of nDNA-encoded mitochondrial genes, mechanistically contribute to BC progression. This work supports a new paradigm of breast carcinogenesis based on a globally dysfunctional genome with altered function of multiple mitochondrial and non-mitochondrial oncogenic pathways caused by the effects of autoreactivity-induced dysregulation of multiple genes and their products. This autoimmunity-based model of carcinogenesis will open novel avenues for BC treatment

Location and structure of the var1 gene on yeast mitochondrial DNA: Nucleotide sequence of the 40.0 allele

Alleles of the var1 locus on yeast mitochondrial DNA specify the size of var1 ribosomal protein. We report the nucleotide sequence of a var1 allele that determines the smallest var1 protein. It contains an open reading frame of 396 codons, which we identify as the structural gene for var1 protein. The var1 protein specified by this allele has an amino acid composition in close agreement with that predicted by the DNA sequence. The var1 coding region is highly unusual: it is 89.6% AT and contains a 46 bp GC-rich palindromic cluster that accounts for 38% of the total GC residues. Our results strongly suggest that like mammalian mitochondria but unlike those from Neurospora, yeast mitochondria use AUA as a methionine codon. Comparison with the sequence of a var1 allele specifying a larger protein suggests that some size polymorphism of var1 protein results from in-frame insertions of a variable number of AAT (Asn) codons.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23885/1/0000124.pd

Mitochondrial DNAs of Suillus: three fold size change in molecules that share a common gene order

We constructed restriction-site and gene maps for mitochondrial DNAs from seven isolates of five species of Suillus (Boletaceae, Basidiomycotina). Each mitochondrial genome exists as a single circular chromosome, ranging in size from 36 to 121 kb. Comparisons within species and between two closely related species revealed that insertions and deletions are the major form of genome change, whereas most restriction sites are conserved. Among more distantly related species, size and restriction-site differences were too great to allow precise alignments of maps, but small clusters of putatively homologous restriction sites were found. Two mitochondrial gene orders exist in the five species. These orders differ only by the relative positions of the genes for ATPase subunit 9 and the small ribosomal RNA and are interconvertible by a single transposition. One of the two gene arrangements is shared by four species whose mitochondrial DNAs span the entire size range of 36 to 121 kb. The conservation of gene order in molecules that vary over three-fold in size and share few restriction sites demonstrates a low frequency of rearrangements relative to insertions, deletions, and base substitutions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46963/1/294_2004_Article_BF00365756.pd

Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis

Cytochrome c (Cytc) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cytc has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cytc is tightly regulated by allosteric mechanisms, tissue‐specific isoforms, and post‐translational modifications (PTMs). Distinct residues of Cytc are modified by PTMs, primarily phosphorylations, in a highly tissue‐specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨm), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia–reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨm hyperpolarization, excessive ROS generation, and the release of Cytc. It is also the dephosphorylated form of the protein that leads to maximum caspase activation. We discuss the complex regulation of Cytc and propose that it is a central regulatory step of the mammalian ETC that can be rate limiting in normal conditions. This regulation is important because it maintains optimal intermediate ΔΨm, limiting ROS generation. We examine the role of Cytc PTMs, including phosphorylation, acetylation, methylation, nitration, nitrosylation, and sulfoxidation and consider their potential biological significance by evaluating their stoichiometry.—Kalpage, H. A., Bazylianska, V., Recanati, M. A., Fite, A., Liu, J., Wan, J., Mantena, N., Malek, M. H., Podgorski, I., Heath, E. I., Vaishnav, A., Edwards, B. F., Grossman, L. I., Sanderson, T. H., Lee, I., Hüttemann, M. Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis. FASEB J. 33, 1540–1553 (2019). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154496/1/fsb2fj201801417r.pd