Fast Evolution of Interleukin-2 in Mammals and Positive Selection in Ruminants

Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes, as well as in birds. Although we can readily amplify IL-2 gene fragments in placental mammals, no amplification was obtained in other species. This is best explained by very high substitution rates. This suggest that strategies to isolate IL-2 homologous genes outside mammals should involve functional assays, as for the chicken gene, and not hybridization-based techniques. Nonsynonymous substitution rates are especially high in ruminants, due to positive selection acting on regions important in term of structure-function. We suggest that, although globally similar, the immune response of various mammals is not identical, mainly at the level of cytokine-mediated regulations. Received: 27 July 1999 / Accepted: 15 April 2000     

Characterization of Fumarate Transport in Helicobacter pylori

The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. The transport of fumarate at micromolar concentrations was saturable with a K _M of 220 ± 21 μm and V _max of 54 ± 2 nmole/min/mg protein at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors, suggesting the presence of one or more fumarate carriers. The release of fumarate from cells was also saturable with a K _M of 464 ± 71 μm and V _max of 22 ± 2 nmol/min/mg protein at 20°C. The rates of fumarate influx at millomolar concentrations increased linearly with permeant concentration, and depended on the age of the cells. The transport system was specific for dicarboxylic acids suggesting that fumarate is taken up via dicarboxylate transporters. Succinate and fumarate appeared to form an antiport system. The properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential inhibitors. The results provided evidence that influx and efflux of fumarate at low concentrations from H. pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion. The anaerobic C₄-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate transporter. Received: 11 December 1997/Revised: 7 May 1998     

Positive Darwinian Selection Promotes Heterogeneity Among Members of the Antifreeze Protein Multigene Family

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption–inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function. Received: 27 February 2001 / Accepted: 12 September 2001     

Analysis of FcγRIII and IgG Fc Polymorphism Reveals Functional and Evolutionary Implications of Protein–Protein Interaction

Fcγ receptor III (FcγRIII), a low-affinity receptor for the Fc portion of immunoglobulin G (IgG Fc), targets antigen-antibody complexes in a variety of effector cells of the immune system. We have investigated FcγRIII and IgG Fc polymorphism and made comparative analysis of the functional and evolutionary implications of the interaction between these two molecules. Sequence analysis and comparison of the three-dimensional structure suggest that the C-terminal Ig domain of FcγRIII is associated with the binding of IgG. The polymorphic residues of FcγRIII are mainly located in the region of the C-terminal Ig domain that might be involved in IgG binding. Therefore, polymorphism and functional binding affinity seems to be related to each other as has been increasingly implicated in clinical observations. IgG Fcs, the natural ligand of FcγRs, also exhibit significant polymorphism. Three regions have been identified where polymorphism frequently occurs: the putative FcR binding site, the linker region, and the intermolecular domain-domain interface of the second Ig domain. The putative FcγR binding sites where polymorphic, and isotype-specific residues cluster are consistent with the regions that have been identified by mutagenesis and molecular modeling studies. The polymorphic residues of IgG Fc were mainly located in the molecular surface, which could be used in the recognition of other binding molecules. These observations suggest that polymorphic and isotype-specific residues in IgG Fc are closely related to their function and protein-protein interaction. Therefore, the colocalization of the polymorphic residues of FcγRIII and IgG Fcs at their docking sites implies that the polymorphic residues would affect the IgG-FcγRIII binding interactions to optimize their signaling through evolution. Received: 9 December 1999 / Accepted: 15 February 2001     

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     

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. Received: 22 May 1996 / Accepted: 24 November 1996     

Detecting Changes in the Functional Constraints of Paralogous Genes

We describe a new procedure to determine whether regional alterations in the evolutionary constraints imposed on paralogous proteins have occurred. We used as models the A and B (alternatively called α and β) subunits of V/F/A-ATPases, originated by a gene duplication more than 3 billion years ago. Changes associated to three major splits (eubacteria versus Archaea-eukaryotes; Archaea versus eukaryotes; and among free-living bacteria and symbiotic mitochondria) were studied. Only in the first case, when we compared eubacterial or mitochondrial F-ATPases versus eukaryotic vacuolar V-ATPases or archaeal A-ATPases, constraint changes were observed. Modifications in the degree of regional constraining were not detected for the other two types of comparisons (V-ATPases versus A-ATPases and within F-ATPases, respectively). When the rates of evolution of the two subunits were compared, it was found that F-ATPases regulatory subunits evolved faster than catalytic subunits, but the opposite was true for A- and V-ATPases. Our results suggest that, even for universal and essential proteins, selective constraints may be occasionally altered. On the other hand, in some cases no changes were detected after periods of more than 2.2 billion years. Received: 24 February 2000 / Accepted: 12 August 2000     

Interelement Selection in the Regulatory Region of the copia Retrotransposon

We report the results of an analysis of naturally occurring cis-regulatory variation within and between two families of the copia Drosophila long terminal repeat (LTR) retrotransposon. The copia 5′ LTR and adjacent untranslated leader region (ULR) consists of a number of well-characterized sequence motifs which play a role in regulating expression of the element. In order to understand the evolutionary forces which may be responsible for generating and maintaining copia regulatory sequence variation, we have quantified levels of naturally occurring copia LTR-ULR nucleotide variation and subjected the data to a series of tests of neutrality. Our analysis indicates that the copia LTR-ULR has been subject to negative purifying selection within families and positive adaptive selection between families. We discuss these findings with respect to the regulatory evolution of retrotransposons and the phenomenon of interelement selection. Received: 5 February 1998 / Accepted: 14 May 1998     

Molecular Evolution of Cytochrome b of Subterranean Mole Rats, Spalax ehrenbergi Superspecies, in Israel

We describe the molecular evolution of cytochrome b of blind subterranean mole rats. We examined 12 individuals for nucleotide differences in the region of 402 base pairs of mitochondrial cytochrome b. Each individual represents a different population from the entire ecological and speciational range of the four chromosomal species in Israel (2n= 52, 54, 58, and 60) belonging to the Spalax ehrenbergi superspecies. Our results indicate the following. (i) There are seven first-position transitional differences, compared to 34 variable third positions, with no observed second-position substitutions. (ii) A maximum of four amino acids differences occurs across the range. (iii) Within-species diversity increases southward. Only 1 autoapomorphic substitution characterizes either 2n= 52 or 2n= 54, but 6–11 substitutions characterize 2n= 58, and 9–13 substitutions characterize 2n= 60. (iv) Both parsimony and maximum-likelihood trees suggest two monophyletic groups: (a) 2n= 52 and 54, and (b) 2n= 58 and 60, as identified earlier by other protein and DNA markers. (v) Mitochondrial cytochrome b heterogeneity is significantly correlated with climatic factors (rainfall) and biotic factors (body size and allozymes). We hypothesize that two selective regimes direct cytochrome b evolution in the S. ehrenbergi superspecies: (i) purifying selection in the flooded, mesic, hypoxic northern range of 2n= 52 and 54 and (ii) diversifying selection in the climatically spatiotemporal, xeric, and variable southern range of 2n= 58 and 60. Thus, the molecular evolution of mitochondrial cytochrome b in S. ehrenbergi is explicable by opposite selective stresses across the range of S. ehrenbergi in Israel, associated with the ecological adaptive radiation of the complex. Received: 23 October 1998 / Accepted: 2 May 1999     

Structural Comparisons of Muscle and Nonmuscle Actins Give Insights Into the Evolution of Their Functional Differences

Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein. Received: 15 March 1996 / Accepted: 14 July 1996     

Measuring Shifts in Function and Evolutionary Opportunity Using Variability Profiles: A Case Study of the Globins

Variability profiles measured over a set of aligned sequences can be used to estimate evolutionary freedom to vary. Differences in variability profiles between clades can be used to identify shifts in function at the molecular level. We demonstrate such a shift between the alpha and beta subunits of hemoglobin. We also show that the variability profiles for myoglobin are different between whales and primates and speculate that the differences between the two clades may reflect a shift associated with the novel oxygen storage demands in the lineage leading to whales. We discuss the relationship between sequence variability and ``evolutionary opportunity' and explore the utility of Maynard Smith's multidimensional evolutionary opportunity space metaphor for exploring functional constraints, genetic redundancy, and the context dependency of the genotype-phenotype map. This work has implications for quantitatively defining and comparing protein function. Supplementary data is available from bioinfo.mbb.yale.edu/align. Received: 16 September 1999 / Accepted: 19 May 2000     

Variation in the Pattern of Nucleotide Substitution Across Sites

A model of nucleotide substitution that allows the transition/transversion rate bias to vary across sites was constructed. We examined the fit of this model using likelihood-ratio tests by analyzing 13 protein coding genes and 1 pseudogene. Likelihood-ratio testing indicated that a model that allows variation in the transition/transversion rate bias across sites provided a significant improvement in fit for most protein coding genes but not for the pseudogene. When the analysis was repeated with parameters estimated separately for first, second, and third codon positions, strong heterogeneity was uncovered for the first and second codon positions; the variation in the transition/transversion rate was generally weaker at the third codon position. The transition rate bias and branch lengths are underestimated when variation in the transition/transversion rate was not accommodated, suggesting that it may be important to accommodate variation in the pattern of nucleotide substitution for accurate estimation of evolutionary parameters. Received: 4 November 1997 / Accepted: 19 May 1998     

Erratic Evolution of Glycerol-3-Phosphate Dehydrogenase in Drosophila,Chymomyza, and Ceratitis

We have studied the evolution of Gpdh in 18 fruitfly species by sequencing 1,077 nucleotides per species on average. The region sequenced includes four exons coding for 277 amino acids and three variable-length introns. Phylogenies derived by a variety of methods confirm that the nominal genus Zaprionus belongs within the genus Drosophila, whereas Scaptodrosophila and Chymomyza are outside. The rate of GPDH evolution is erratic. The rate of amino acid replacements in a lineage appears to be 1.0 × 10⁻¹⁰/site/year when Drosophila species are considered (diverged up to 55 million years ago), but becomes 2.3 × 10⁻¹⁰ when they are compared to Chymomyza species (divergence around 60 My ago), and 4.6 × 10⁻¹⁰ when species of those two genera are compared with the medfly Ceratitis capitata (divergence around 100 My ago). In order to account for these observations, the rate of amino acid replacement must have been 15 or more times greater in some lineages and at some times than in others. At the nucleotide level, however, Gpdh evolves in a fairly clockwise fashion. Received: 13 June 1996 / Accepted: 16 August 1996     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

Early Detection of G + C Differences in Bacterial Species Inferred from the Comparative Analysis of the Two Completely Sequenced Helicobacter pylori Strains

Identifying the G + C difference between closely related bacterial species or between different strains of the same species is one of the first steps in understanding the evolutionary mechanisms accounting for the differences observed among bacterial species. The G + C content can be one of the most important factors in the evolution of genomic structures. In this paper, we describe a new method for detecting an initial stage of differentiation of the G + C content at the third codon base position between two strains of the same bacterial species. We apply this method to the two strains of Helicobacter pylori. A group of genes is detected with large variations of G + C in the third positions—apparently genes of early response to pressures of changing G + C. We discuss our findings from the viewpoint of genomic evolution. Received: 26 February 2001 / Accepted: 16 May 2001     

Evolution of trophic types in emperor fishes ( Lethrinus,Lethrinidae, Percoidei) based on cytochrome B gene sequence variation

Three trophic categories exist within emperor fishes, genus Lethrinus, relating to body form and dentition type. One group contains low-bodied, high speed, stalking predators with conical teeth. Another group comprises high-bodied, slow speed carnivores with molariform teeth capable of crushing hard-shelled benthic prey. A third group is also high-bodied but with conical teeth feeding mostly on small or soft-shelled benthic prey. Inferring the evolution of these trophic types within Lethrinus using morphology is problematic since these characters are typically correlated with feeding mode and are potentially homoplasious. We use mitochondrial DNA sequences, to independently determine a phylogenetic hypothesis for Lethrinus, which are not dependent on morphological characters relating to trophic categories. We analyzed complete cytochrome b gene sequences (1140 bp) for 20 species of Lethrinus, representing the three trophic types, and for 13 outgroup species, including four other representatives of the Lethrinidae. A monophyletic Lethrinidae did not resolve, but the monophyly of Lethrinus is well supported. In addition, two major clades within Lethrinus are well supported. One of these clades exclusively contains low-bodied species with conical teeth while the other clade only comprises the high-bodied species with molariform teeth. A high-bodied species with conical teeth, Lethrinus miniatus, appears most ancestral and sister to all other Lethrinus species. We hypothesize that this generalist trophic type was the evolutionary precursor to both of the other primary trophic types.     

Evolution of Orthologous Intronless and Intron-Bearing Globin Genes in Two Insect Species

While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species. Received: 31 December 1996 / Accepted: 16 May 1997     

Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Patterns of Gene Duplication in Lepidopteran Pheromone Binding Proteins

We have isolated and characterized cDNAs representing two distinct pheromone binding proteins (PBPs) from the gypsy moth, Lymantria dispar. We use the L. dispar protein sequences, along with other published lepidopteran PBPs, to investigate the evolutionary relationships among genes within the PBP multigene family. Our analyses suggest that the presence of two distinct PBPs in genera representing separate moth superfamilies is the result of relatively recent, independent, gene duplication events rather than a single, ancient, duplication. We discuss this result with respect to the biochemical diversification of moth PBPs. Received: 19 March 1997 / Accepted: 11 July 1997