Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Comparisons of the Nucleotide Substitution Process Among Repetitive Segments of the α- and β-Spectrin Genes

The actin–cross-linking protein spectrin is a prominent component of the membrane cytoskeleton. Spectrin is a tetramer of two antiparallel αβ-dimers which share a unique and ancient gene structure. The α-spectrin and β-spectrin genes are composed primarily of tandemly repeated 106-amino-acid segments, each of which forms a triple α-helical coiled coil. Both the genes and the repeats themselves are homologous. The two genes are thought to be the result of a gene duplication event, and each gene is the product of duplications of the 106-amino-acid repeats. In this work we compare the process of molecular evolution across the repeated segments of the α- and β-spectrin genes. We find that the α-spectrin segments have, for the most part, evolved in a homogeneous fashion, while considerable heterogeneity is found among β-spectrin segments. Several segments with unique known functions are found to have evolved differently than the others. On the basis of heterogeneity of the evolutionary process, we suggest that at least one repeat has a unique function that has yet to be documented. We also present new statistical methods for comparing the evolutionary process between different regions of DNA sequences. Received: 27 March 1996 / Accepted: 21 October 1996     

Sequences and Evolution of Human and Squirrel Monkey Blue Opsin Genes

The sequences of the entire blue opsin gene in the squirrel monkey (Saimiri boliviensis) and the five introns of the human blue opsin gene were obtained. Intron 3 of these genes contains an Alu sequence and intron 4 contains a partial mer13 sequence. A comparison of the squirrel monkey opsin sequence with published mammalian opsin sequences shows that features believed to be functionally critical are all conserved. However, the blue opsin has evolved twice as fast as rhodopsin and is only as conservative as the β globin, which has evolved at the average rate of mammalian proteins. Interestingly, the interhelical loops are, on average, actually more conservative than the transmembrane α helical regions. The introns of the blue opsin gene have evolved at the average rate of introns in primate genes. Received: 5 August 1996 / Accepted: 2 October 1996     

Duplication and divergence of the genes of the α-esterase cluster ofDrosophila melanogaster

The α-esterase cluster of D. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37–66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the α-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle. Received: 18 January 1996 / Accepted: 12 March 1996     

Phylogenomic Analysis of the α Proteasome Gene Family from Early-Diverging Eukaryotes

We employed a phylogenomic approach to study the evolution of α subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven α proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple α subunit sequences generated single α proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct α proteasome genes each. The presence of seven distinct α proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the α proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the α proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented. Received: 14 February 2000 / Accepted: 14 August 2000     

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: Mutants with altered channel gating and pressure sensitivity

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic α-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning α-helix. Received: 25 September 1996/Revised: 21 November 1996     

Reconstructing the Diversification of α-Esterases: Comparing the Gene Clusters of Drosophila buzzatii and D. melanogaster

A cluster composed of 10 active α-esterase genes and a pseudogene is distributed over 60 kb in the Drosophila melanogaster genome. This paper describes the corresponding cluster in Drosophila buzzatii, whose lineage diverged from that of D. melanogaster when the subgenera Drosophila and Sophophora diverged about 50 Mya. With three exceptions we find that the composition of the cluster is conserved in the two lineages. The location of αE1 in D. melanogaster differs from that of its nearest relative in D. buzzatii, and αE4 has duplicated independently in the two lineages. The nature of these differences indicates that a mechanism exists whereby copies of genes can be placed in opposite orientation and nonadjacent positions within a gene cluster, although this does not seem to be a feature of earlier events in the cluster's evolution. The rates of amino acid change are not significantly different between orthologs, but the rates differ sevenfold among paralogs, indicating that very different selective forces are acting on the genes of the cluster. Mapping of sequence differences onto a model of the tertiary structure of the enzymes indicates that motifs contributing to substrate binding and catalysis have changed radically in the αE4s and suggest that this subgroup of α-esterases may be evolving into a substantially different functional niche. Received: 4 January 2000 / Accepted: 18 April 2000     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

Molecular Evolution of Nitrogen Fixation: The Evolutionary History of the nifD, nifK, nifE, and nifN Genes

The pairs of nitrogen fixation genes nifDK and nifEN encode for the α and β subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed. Received: 21 June 1999 / Accepted: 1 March 2000     

Phylogenetic Relationships Within the Genus Equus and the Evolution of α and θ Globin Genes

Sequences of the α1, α2 and θ globin genes from six equid species have been determined to investigate relationships within the genus Equus. Analyses using standard phylogenetic methods, or an approach designed to account for the effects of gene conversion between the α genes, gave broadly similar results and show that the horses diverged from the zebra/ass ancestor ∼2.4 million years ago and that the zebra and ass species arose in a rapid radiation ∼0.9 million years ago. These results from the α genes are corroborated by θ gene data and are in contrast to mitochondrial DNA studies of the phylogeny of this genus, which suggest a more gradual set of speciation events. Received: 22 April 1997 / Accepted: 20 July 1998     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Phylogenetic Analysis of Three Lipocalin-Like Proteins Present in the Milk of Trichosurus vulpecula (Phalangeridae, Marsupialia)

Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents. Received: 28 October 1996 / Accepted: 19 May 1997     

Thyroid Hormone Receptor Genes of Neotenic Amphibians

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to α and β genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3′,5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRα in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRβ expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRα and TRβ may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis. Received: 23 April 1996 / Accepted: 20 January 1997     

Primary Structure and Phylogenetic Relationships of a Malate Dehydrogenase Gene from Giardia lamblia

The lactate and malate dehydrogenases comprise a complex protein superfamily with multiple enzyme homologues found in eubacteria, archaebacteria, and eukaryotes. In this study we describe the sequence and phylogenetic relationships of a malate dehydrogenase (MDH) gene from the amitochondriate diplomonad protist, Giardia lamblia. Parsimony, distance, and maximum-likelihood analyses of the MDH protein family solidly position G. lamblia MDH within a eukaryote cytosolic MDH clade, to the exclusion of chloroplast, mitochondrial, and peroxisomal homologues. Furthermore, G. lamblia MDH is specifically related to a homologue from Trichomonas vaginalis. This MDH topology, together with published phylogenetic analyses of β-tubulin, chaperonin 60, valyl-tRNA synthetase, and EF-1α, suggests a sister-group relationship between diplomonads and parabasalids. Since these amitochondriate lineages contain genes encoding proteins which are characteristic of mitochondria and α-proteobacteria, their shared ancestry suggests that mitochondrial properties were lost in the common ancestor of both groups. Received: 14 September 1998 / Accepted: 29 December 1998     

Extensive Gene Duplication in the Early Evolution of Animals Before the Parazoan–Eumetazoan Split Demonstrated by G Proteins and Protein Tyrosine Kinases from Sponge and Hydra

To know whether genes involved in cell–cell communication typical of multicellular animals dramatically increased in concert with the Cambrian explosion, the rapid evolutionary burst in the major groups of animals, and whether these genes exist in the sponge lacking cell cohesiveness and coordination typical of eumetazoans, we have carried out cloning of the G-protein α subunit (Gα) and the protein tyrosine kinase (PTK) cDNAs from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We obtained 13 Gα and 20 PTK cDNAs. Generally animal gene families diverged first by gene duplication (subtype duplication) that gave rise to diverse subtypes with different primary functions, followed by further gene duplication in the same subtype (isoform duplication) that gave rise to isoform genes with virtually identical function. Phylogenetic trees of Gα and PTK families including cDNAs from sponge and hydra revealed that most of the present-day subtypes had been established in the very early evolution of animals before the parazoan–eumetazoan split, the earliest branching among the extant animal phyla, by extensive subtype duplication: for PTK and Gα families, 23 and 9 subtype duplications were observed in the early stage before the parazoan–eumetazoan split, respectively, and after that split, only 2 and 1 subtype duplications were found, respectively. After the separation from arthropods, vertebrates underwent frequent isoform duplications before the fish–tetrapod split. Furthermore, rapid amino acid changes appear to have occurred in concert with the extensive subtype duplication and isoform duplication. Thus the pattern of gene diversification during animal evolution might be characterized by bursts of gene duplication interrupted by considerably long periods of silence, instead of proceeding gradually, and there might be no direct link between the Cambrian explosion and the extensive gene duplication that generated diverse functions (subtypes) of these families. Received: 4 November 1998 / Accepted: 17 November 1998     

Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Phylogenetic Affinity of Mitochondria of Euglena gracilis and Kinetoplastids Using Cytochrome Oxidase I and hsp60

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5′ and 3′ untranslated transcribed sequences and lack a 3′ poly[A] tail. The COI gene does not require uridine insertion/deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [α-³²P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3′ end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences. Received: 5 March 1996 / Accepted: 31 July 1996     

Ancient Origin of Human Complement Factor H

We studied the evolutionary history of two homologous proteins of the human complement system, factor H (FH) and the α chain of the C4b binding protein (C4bpα), and included in this study the related proteins from the barred sand bass (P. nebulifer) and the nematode C. elegans. Phylogenetic trees inferred from individual short consensus repeats (SCRs) and divergence among repeats from different genes suggest that human FH has a much closer evolutionary relationship to putative complement components from P. nebulifer and C. elegans than does the C4bpα. This indicates that a member of the alternative pathway of the complement system (FH) has an ancient origin, while a homologous member of the classical pathway (C4bpα) appeared later in evolutionary history as a result of gene duplication. The ancient evolutionary position of FH is in agreement with the suggestion that the alternative pathway of the complement system is older than the classical pathway. Phylogenetic analysis also shows that the sand bass cofactor protein SBP1 and cofactor related protein SBCRP-1 have diverged very recently. Received: 1 December 1997 / Accepted: 3 June 1998     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 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