Origin of the NEFA and Nuc Signal Sequences

The human protein NEFA binds calcium, contains a leucine zipper repeat that does not form a homodimer, and is proposed (along with the homologous Nuc protein) to have a common evolutionary history with an EF-hand ancestor. We have isolated and characterized the N-terminal domain of NEFA that contains a signal sequence inferred from both endoproteinase Asp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to the conserved multiple domains of the mitochondrial carrier family (MCF) proteins. The leader sequence of Nuc is, however, most similar to the signal sequences of membrane and/or secreted proteins (e.g., mouse insulin-like growth factor receptor). We suggest that the divergent NEFA and Nuc N-terminal sequences may have independent origins and that the common high hydrophobicity governs their targeting to the ER. These results provide insights into signal sequence evolution and the multiple origins of protein targeting. Received: 20 February 1997 / Accepted: 28 July 1997     

On the Origin of Protein Synthesis Factors: A Gene Duplication/Fusion Model

Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor. We evaluate this proposition and whether the relationship can be extended to other factors of protein synthesis. Analysis of amino acid sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G and suggests further that this association is a result of gene duplication/fusion events. In support of this mechanism, the three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity. The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes. The root of this protein superfamily tree would be an ancestor of the modern IF-1 gene sequence. The repeated fundamental motif of this protein superfamily is a small RNA binding domain composed of two α-helices packed along side of an antiparallel β-sheet. Received: 17 October 1996 / Accepted: 10 June 1997     

Estimation of the Transition/Transversion Rate Bias and Species Sampling

The transition/transversion (ti/tv) rate ratios are estimated by pairwise sequence comparison and joint likelihood analysis using mitochondrial cytochrome b genes of 28 primate species, representing both the Strepsirrhini (lemurs and lories) and the Anthropoidea (monkeys, apes, and humans). Pairwise comparison reveals a strong negative correlation between estimates of the ti/tv ratio and the sequence distance, even when both are corrected for multiple substitutions. The maximum-likelihood estimate of the ti/tv ratio changes with the species included in the analysis. The ti/tv bias within the lemuriform taxa is found to be as strong as in the anthropoids, in contradiction to an earlier study which sampled only one lemuriform. Simulations show the surprising result that both the pairwise correction method and the joint likelihood analysis tend to overcorrect for multiple substitutions and overestimate the ti/tv ratio, especially at low sequence divergence. The bias, however, is not large enough to account for the observed patterns. Nucleotide frequency biases, variation of substitution rates among sites, and different evolutionary dynamics at the three codon positions can be ruled out as possible causes. The likelihood-ratio test suggests that the ti/tv rate ratios may be variable among evolutionary lineages. Without any biological evidence for such a variation, however, we are left with no plausible explanations for the observed patterns other than a possible saturation effect due to the unrealistic nature of the model assumed. Received: 1 October 1997 / Accepted: 29 September 1998     

Algae or Protozoa: Phylogenetic Position of Euglenophytes and Dinoflagellates as Inferred from Mitochondrial Sequences

The chloroplasts of euglenophytes and dinoflagellates have been suggested to be the vestiges of endosymbiotic algae acquired during the process of evolution. However, the evolutionary positions of these organisms are still inconclusive, and they have been tentatively classified as both algae and protozoa. A representative gene of the mitochondrial genome, cytochrome oxidase subunit I (coxI), was chosen and sequenced to clarify the phylogenetic positions of four dinoflagellates, two euglenophytes and one apicomplexan protist. This is the first report of mitochondrial DNA sequences for dinoflagellates and euglenophytes. Our COXI tree shows clearly that dinoflagellates are closely linked to apicomplexan parasites but not with algae. Euglenophytes and algae appear to be only remotely related, with euglenophytes sharing a possible evolutionary link with kinetoplastids. The COXI tree is in general agreement with the tree based on the nuclear encoded small subunit of ribosomal RNA (SSU rRNA) genes, but conflicts with that based on plastid genes. These results support the interpretation that chloroplasts present in euglenophytes and dinoflagellates were captured from algae through endosymbioses, while their mitochondria were inherited from the host cell. We suggest that dinoflagellates and euglenophytes were originally heterotrophic protists and that their chloroplasts are remnants of endosymbiotic algae. Received: 24 March 1997 / Accepted: 21 April 1997     

The Complete Mitochondrial DNA Sequence of the Pig (Sus scrofa)

The complete mitochondrial genome sequence of the pig, Sus scrofa, was determined. The length of the sequence presented is 16,679 nucleotides. This figure is not absolute, however, due to pronounced heteroplasmy caused by variable numbers of the motif GTACACGTGC in the control region of different molecules. A phylogenetic study was performed on the concatenated amino acid and nucleotide sequences of 12 protein-coding genes of the mitochondrial genome. The analysis identified the pig (Suiformes) as a sister group of a cow/whale clade, making Artiodactyla paraphyletic. The split between pig and cow/whale was molecularly dated at 65 million years before present. Received: 2 December 1997 / Accepted: 20 February 1998     

The Peperomia Mitochondrial coxI Group I Intron: Timing of Horizontal Transfer and Subsequent Evolution of the Intron

The Peperomia polybotrya coxI gene intron is the only currently reported group I intron in a vascular plant mitochondrial genome and it likely originated by horizontal transfer from a fungal donor. We provide a clearer picture of the horizontal transfer and a portrayal of the evolution of the group I intron since it was gained by the Peperomia mitochondrial genome. The intron was transferred recently in terms of plant evolution, being restricted to the single genus Peperomia among the order Piperales. Additional support is presented for the suggestion that a recombination/repair mechanism was used by the intron for integration into the Peperomia mitochondrial genome, as a perfect 1:1 correspondence exists between the intron's presence in a species and the presence of divergent nucleotide markers flanking the intron insertion site. Sequencing of coxI introns from additional Peperomia species revealed that several mutations have occurred in the intron since the horizontal transfer, but sequence alterations have not caused frameshifts or created stop codons in the intronic open reading frame. In addition, two coxI pseudogenes in Peperomia cubensis were discovered that lack a large region of coxI exon 2 and contain a truncated version of the group I intron that likely cannot be spliced out. Received: 29 May 1997 / Accepted: 1 November 1997     

A Reexamination of the Phylogenetic Position of Callimico (Primates) Incorporating New Mitochondrial DNA Sequence Data

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNA^His, tRNA^Ser, and tRNA^Leu genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets (Callithrix and Cebuella) and the tamarins (Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico. Received: 20 March 1997 / Accepted: 17 December 1997     

Synonymous and nonsynonymous rate variation in nuclear genes of mammals

A maximum likelihood approach was used to estimate the synonymous and nonsynonymous substitution rates in 48 nuclear genes from primates, artiodactyls, and rodents. A codon-substitution model was assumed, which accounts for the genetic code structure, transition/transversion bias, and base frequency biases at codon positions. Likelihood ratio tests were applied to test the constancy of nonsynonymous to synonymous rate ratios among branches (evolutionary lineages). It is found that at 22 of the 48 nuclear loci examined, the nonsynonymous/synonymous rate ratio varies significantly across branches of the tree. The result provides strong evidence against a strictly neutral model of molecular evolution. Our likelihood estimates of synonymous and nonsynonymous rates differ considerably from previous results obtained from approximate pairwise sequence comparisons. The differences between the methods are explored by detailed analyses of data from several genes. Transition/transversion rate bias and codon frequency biases are found to have significant effects on the estimation of synonymous and nonsynonymous rates, and approximate methods do not adequately account for those factors. The likelihood approach is preferable, even for pairwise sequence comparison, because more-realistic models about the mutation and substitution processes can be incorporated in the analysis. Received: 17 May 1997 / Accepted: 28 September 1997     

The rate of mitochondrial 12S rRNA gene evolution is similar in freshwater turtles and marsupials

Assertions that the ``conventional' rate of mitochondrial DNA (mtDNA) evolution is reduced in poikilotherms in general and turtles in particular were tested for side-necked turtles (Pleurodira: Chelidae). Homologous data sets of mitochondrial 12S rRNA gene sequences were used to compare the average divergence between the Australian and South American species for two Gondwanan groups: the chelid turtles and the marsupials. The mean nucleotide divergences between continental groups for both the turtles and the marsupials are remarkably similar. These data suggest that the rate of evolution of mitochondrial 12S rRNA gene is not substantially slower in turtles than in the homeothermic marsupials. Received: 24 February 1997 / Accepted: 30 June 1997     

The major histocompatability complex (MHC) contains conserved polymorphic genomic sequences that are shuffled by recombination to form ethnic-specific haplotypes

The major histocompatibility complex (MHC) consists of polymorphic frozen blocks (PFBs) that are linked to form megabase haplotypes. These blocks consist of polymorphic sequences and define regions where recombination appears to be inhibited. We have been able to show, using a highly polymorphic sequence centromeric of HLA-B (within the beta block), that PFBs are conserved and contain specific insertions/deletions and substitutions that are the same for individuals with the same MHC haplotype but that differ between at least most different haplotypes. A sequence comparison between ethnic-specific haplotypes shows that these sequences have remained stable and predate the formation of these haplotypes. To determine whether the same conserved block has been involved in the generation of multiple haplotypes, we compared the block typing profiles of different ethnic specific haplotypes. Block typing profiles have previously been shown to be identical in individuals with the same MHC haplotype but, generally, to differ between different haplotypes. It was found that some PFBs are common to more than one haplotype, implying a common ancestry. Subsequently, haplotypes have been generated by the shuffling and exchange of these PFBs. The regions between these PFBs appear to permit the recombination sites and therefore could be expected to exhibit either low polymorphism or a localized ``hotspot.' Received: 20 January 1997 / Accepted: 11 March 1997     

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase

It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved β-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the β-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase. Received: 25 April 1997 / Accepted: 29 July 1997     

Tail-to-Head Arrangement of a Partial Chicken Glyceraldehyde-3-Phosphate Dehydrogenase Processed Pseudogene

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5′ region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization. Received: 15 November 1996 / Accepted: 1 April 1997     

Estimation of the Transition/Transversion Ratio

A simple method for estimating the transition/transversion ratio was developed. This method can be applied to not only two sequences but also more than two sequences. The statistical properties of the method and some other methods were examined by numerical computation and computer simulation. The results obtained showed that, in terms of bias and variance, the new method gives a better estimate of the transition/transversion ratio than do the other examined methods. The new method was applied to human and chimpanzee mitochondrial control region sequences. Received: 22 September 1997 / Accepted: 1 November 1997     

Deuterostomic Actin Genes and the Definition of the Chordates: cDNA Cloning and Gene Organization for Cephalochordates and Hemichordates

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed. Received: 4 July 1997 / Accepted: 29 August 1997     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998     

Interactions of Primary Amphipathic Vector Peptides with Membranes. Conformational Consequences and Influence on Cellular Localization

The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, α-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of β-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides. Received: 10 October 1997/Revised: 15 January 1998     

Rapid Rate of Control-Region Evolution in Pacific Butterflyfishes (Chaetodontidae)

Sequence differences in the tRNA-proline (tRNA^pro) end of the mitochondrial control-region of three species of Pacific butterflyfishes accumulated 33–43 times more rapidly than did changes within the mitochondrial cytochrome b gene (cytb). Rapid evolution in this region was accompanied by strong transition/transversion bias and large variation in the probability of a DNA substitution among sites. These substitution constraints placed an absolute ceiling on the magnitude of sequence divergence that could be detected between individuals. This divergence ``ceiling' was reached rapidly and led to a decay in the relative rate of control-region/cytb b evolution. A high rate of evolution in this section of the control-region of butterflyfishes stands in marked contrast to the patterns reported in some other fish lineages. Although the mechanism underlying rate variation remains unclear, all taxa with rapid evolution in the 5′-end of the control-region showed extreme transition biases. By contrast, in taxa with slower control-region evolution, transitions accumulated at nearly the same rate as transversions. More information is needed to understand the relationship between nucleotide bias and the rate of evolution in the 5′-end of the control-region. Despite strong constraints on sequence change, phylogenetic information was preserved in the group of recently differentiated species and supported the clustering of sequences into three major mtDNA groupings. Within these groups, very similar control-region sequences were widely distributed across the Pacific Ocean and were shared between recognized species, indicating a lack of mitochondrial sequence monophyly among species. Received: 30 June 1996 / Accepted: 15 May 1997     

The Complete Mitochondrial Genome of the Hagfish Myxine glutinosa: Unique Features of the Control Region

The complete sequence of the mitochondrial DNA of the hagfish Myxine glutinosa has been determined. The hagfish mtDNA (18,909 bp) is the longest vertebrate mtDNA determined so far. The gene arrangement conforms to the consensus vertebrate type and differs from that of lampreys. The exceptionally long (3628-bp) control region of the hagfish contains the typical conserved elements found in other vertebrate mtDNAs but is characterized by a large number of putative hairpins, which can potentially fold into a highly compact secondary structure that appears to be unique to hagfish. The comparison of the mtDNAs of two M. glutinosa specimens, excluding the control region, shows a 0.6% divergence at the nucleotide level as a sample of intraspecies polymorphism. Received: 21 August 2000 / Accepted: 2 March 2001