Universal Protein Families and the Functional Content of the Last Universal Common Ancestor

The phylogenetic distribution of Methanococcus jannaschii proteins can provide, for the first time, an estimate of the genome content of the last common ancestor of the three domains of life. Relying on annotation and comparison with reference to the species distribution of sequence similarities results in 324 proteins forming the universal family set. This set is very well characterized and relatively small and nonredundant, containing 301 biochemical functions, of which 246 are unique. This universal function set contains mostly genes coding for energy metabolism or information processing. It appears that the Last Universal Common Ancestor was an organism with metabolic networks and genetic machinery similar to those of extant unicellular organisms.     

Respiratory Chains in the Last Common Ancestor of Living Organisms

Sequences in current databases show that a number of proteins involved in respiratory processes are homologous in archaeal and bacterial species. In particular, terminal oxidases belonging to oxygen, nitrate, sulfate, and sulfur respiratory pathways have been sequenced in members of both domains. They include cytochrome oxidase, nitrate reductase, adenylylsulfate reductase, sulfite reductase, and polysulfide reductase. These proteins can be assigned to the last common ancestor of living organisms assuming that the deepest split of the three domains of life occurred between Archaea and Bacteria and that they were not acquired through lateral gene transfer by one of these domains. These molecular data indicate that several of the most important respiratory pathways arose early in evolution and that the last common ancestor of living organisms was not a simple organism in its energetic metabolism. Rather, it may have been able to gain energy by means of at least four electron transport chains, and therefore it may have been prepared to face a wide range of environmental conditions.     

The Evolution of the Calpain Family as Reflected in Paralogous Chromosome Regions

Calpains, the Ca²⁺-dependent intracellular proteinases, are involved in the regulation of distinct cellular pathways including signal transduction and processing, cytoskeleton dynamics, and muscle homeostasis. To investigate the evolutionary origin of diverse calpain subfamilies, a phylogenetic study was carried out. The topology of the calpain phylogenetic tree has shown that some of the gene duplications occurred before the divergence of the protostome and deuterostome lineages. Other gene doublings, leading to vertebrate-specific calpain forms, took place during early chordate evolution and coincided with genome duplications as disclosed by the localization of calpain genes to paralogous chromosome regions in the human genome. On the basis of the phylogenetic tree, the time of gene duplications, and the localization of calpain genes, we propose a model of tandem and chromosome duplications for the evolution of vertebrate-specific calpain forms. The data presented here are consistent with scenarios proposed for the evolution of other multigene families. Received: 17 November 1998 / Accepted: 30 April 1999     

The Root of the Universal Tree of Life Inferred from Anciently Duplicated Genes Encoding Components of the Protein-Targeting Machinery

The key protein of the signal recognition particle (termed SRP54 for Eucarya and Ffh for Bacteria) and the protein (termed SRα for Eucarya and Ftsy for bacteria) involved in the recognition and binding of the ribosome SRP nascent polypeptide complex are the products of an ancient gene duplication that appears to predate the divergence of all extant taxa. The paralogy of the genes encoding the two proteins (both of which are GTP triphosphatases) is argued by obvious sequence similarities between the N-terminal half of SRP54(Ffh) and the C-terminal half of SRα(Ftsy). This enables a universal phylogeny based on either protein to be rooted using the second protein as an outgroup. Phylogenetic trees inferred by various methods from an alignment (220 amino acid positions) of the shared SRP54(Ffh) and SRα(Ftsy) regions generate two reciprocally rooted universal trees corresponding to the two genes. The root of both trees is firmly positioned between Bacteria and Archaea/Eucarya, thus providing strong support for the notion (Iwabe et al. 1989; Gogarten et al. 1989) that the first bifurcation in the tree of life separated the lineage leading to Bacteria from a common ancestor to Archaea and Eucarya. None of the gene trees inferred from the two paralogues support a paraphyletic Archaea with the crenarchaeota as a sister group to Eucarya. Received: 19 March 1998 / Accepted: 5 June 1998     

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     

Recent Evolutionary History of Human Immunodeficiency Virus Type 1 Subtype B: Reconstruction of Epidemic Onset Based on Sequence Distances to the Common Ancestor

We obtained and studied HIV-1 sequences with a known sampling year from three outbreaks of the HIV-1 epidemic: 141 env V3 (270 nt) sampled between 1984 and 1992 and 117 pol prot/RT (804 nt) sequences sampled between 1986 and 1999 from Dutch homosexual men and injecting drug users (IDUs), as well as 77 env V3 sequences sampled between 1983 and 1994 in the United States. Since retrospective serological and/or epidemiological data on these populations are available, providing estimates of the dates of the onset of the HIV-1 epidemics, we had the opportunity to test different phylogenetic models for their accuracy in deriving the recent evolutionary history of HIV-1 subtype B and the onset date of the HIV-1 epidemic. We observed that, in any given year, individual sequences vary widely in their distances to the common ancestor, and sequences close to the ancestors were found decades after the onset of the epidemic. Nevertheless, the mean evolutionary distances of virus strains to ancestors were increasing significantly during the course of the studied epidemics, which indicates that the molecular clock is operational in the recent evolution of HIV-1. When the relationship between the sampling years of sequences and their nucleotide distances to the common ancestor was extrapolated to the past, analysis of pol sequences provided accurate estimates of the onset years of the epidemics, whereas analysis of V3 sequences by the maximum-likelihood or neighbor-joining methods led to an overestimation of the age of the epidemics. Separate analysis of nonsynonymous and synonymous distances revealed that this overestimation results from nonsynonymous substitutions, whose numbers were not increasing significantly in all three virus populations over the observation period. In contrast, analysis of synonymous env V3 distances provided accurate estimates of the onset years for the outbreaks we studied. Received: 26 October 2001 / Accepted: 8 November 2001     

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     

The Presence of GSI-Like Genes in Higher Plants: Support for the Paralogous Evolution of GSI and GSII Genes

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. Received: 4 May 1999 / Accepted: 17 September 1999     

Reconstructing the Complex Evolutionary History of Hepatitis B Virus

A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus. Received: 5 December 1998 / Accepted: 23 February 1999     

Phylogenies of Developmentally Important Proteins Do Not Support the Hypothesis of Two Rounds of Genome Duplication Early in Vertebrate History

It has been proposed that two rounds of duplication of the entire genome (polyploidization) occurred early in vertebrate history (the 2R hypothesis); and the observation that certain gene families important in regulating development have four members in vertebrates, as opposed to one in Drosophila, has been adduced as evidence in support of this hypothesis. However, such a pattern of relationship can be taken as support of the 2R hypothesis only if (1) the four vertebrate genes can be shown to have diverged after the origin of vertebrates, and (2) the phylogeny of the four vertebrate genes (A–D) exhibits a topology of the form (AB) (CD), rather than (A) (BCD). In order to test the 2R hypothesis, I constructed phylogenies for nine protein families important in development. Only one showed a topology of the form (AB) (CD), and that received weak statistical support. In contrast, four phylogenies showed topologies of the form (A) (BCD) with statistically significant support. Furthermore, in two cases there was significant support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes: in one case there was significant support for duplication of the vertebrate genes at least prior to the divergence of vertebrates and urochordates, and in one case there was weak support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes. Taken together with other recently published phylogenies of developmentally important genes, these results provide strong evidence against the 2R hypothesis. Received: 22 December 1997 / Accepted: 5 October 1998     

On the Origin of Operons and Their Possible Role in Evolution Toward Thermophily

We present a hypothesis suggesting that close linkage of functionally related anabolic genes and their ultimate integration into operons developed under selective pressure as a molecular strategy which contributed to the viability of ancestral thermophilic cells. Cotranslation of functionally related proteins is viewed as having facilitated the formation of multienzyme complexes channeling thermolabile substrates and the mutual stabilization of inherently thermolabile proteins. In this perspective, the evolutionary scheme considered the most probable is the evolution of both Bacteria and Archaea by thermoreduction (Forterre 1995) from a mesophilic, protoeukaryotic last common ancestor (LCA) endowed with appreciable genetic redundancy.     

Evolutionary History of MHC Class I Genes in the Mammalian Order Perissodactyla

We carried out an analysis of partial sequences from expressed major histocompatibility complex (MHC) class I genes isolated from a range of equid species and more distantly related members of the mammalian order Perissodactyla. Phylogenetic analysis revealed a minimum of six groups, five of which contained genes and alleles that are found in equid species and one group specific to the rhinoceros. Four of the groups contained only one, or very few sequences, indicating the presence of relatively nonpolymorphic loci, while another group contained the majority of the equid sequences identified. These data suggest that a diversification of MHC genes took place after the split between the Equidae and the Rhinocerotidae yet before the speciation events within the genus Equus. Received: 17 November 1998 / Accepted: 7 April 1999     

The Evolutionary History of Prosaposin: Two Successive Tandem-Duplication Events Gave Rise to the Four Saposin Domains in Vertebrates

Prosaposin is a multifunctional protein encoded by a single-copy gene. It contains four saposin domains (A, B, C, and D) occurring as tandem repeats connected by linker sequences. Because the saposin domains are similar to one another, it is deduced that they were created by sequential duplications of an ancestral domain. There are two types of evolutionary scenarios that may explain the creation of the four-domain gene: (1) two rounds of tandem internal gene duplication and (2) three rounds of duplications. An evolutionary and phylogenetic analysis of saposin DNA and amino acid sequences from human, mouse, rat, chicken, and zebrafish indicates that the first evolutionary scenario is the most likely. Accordingly, an ancestral saposin-unit duplication produced a two-domain gene, which, subsequently, underwent a second complete tandem duplication to give rise to the present four-domain structure of the prosaposin gene. Received: 8 February 2001 / Accepted: 29 June 2001     

Molecular Evolution of Calmodulin and Calmodulin-Like Genes in the Cephalochordate Branchiostoma

Calmodulin is a calcium-binding EF-hand protein that is an activator of many enzymes as well as ion pumps and channels. Due to its multiple targets and its central role in the cell, understanding the evolutionary history of calmodulin genes should provide insights into the origin of genetic complexity in eukaryotes. We have previously isolated and characterized a calmodulin gene from the early-diverging chordate Branchiostoma lanceolatum (CaM1). In this paper, we report the existence of a second calmodulin gene (CaM2) as well as two CaM-like genomic fragments (CaML-2, CaML-3) in B. lanceolatum and a CaM2 and three CaM-like genes (CaML-1, CaML-2, CaML-3) in B. floridae. The CaM-like genes were isolated using low-stringency PCR. Surprisingly, the nucleotide sequences of the B. lanceolatum CaM1 and CaM2 cDNAs differ by 19.3%. Moreover, the CaM2 protein differs at two positions from the amino acid sequence of CaM1; the latter is identical to calmodulins in Drosophila melanogaster, the mollusc Aplysia californica, and the tunicate Halocynthia roretzi. The two B. lanceolatum CaM-like genes are more closely related to the CaM2 than to the CaM1 gene. This relationship is supported by the phylogenetic analyses and the identical exon/intron organization of these three genes, a relationship unique among animal CaM sequences. These data demonstrate the existence of a CaM multigene family in the cephalochordate Branchiostoma, which may have evolved independently from the multigene family in vertebrates. Received: 2 November 1999 / Accepted: 25 April 2000     

Evolutionary Patterns of Gene Families Generated in the Early Stage of Vertebrates

In this paper we have analyzed 49 vertebrate gene families that were generated in the early stage of vertebrates and/or shortly before the origin of vertebrates, each of which consists of three or four member genes. We have dated the first (T₁) and second (T₂) gene duplications of 26 gene families with 3 member genes. The means of T₁ (594 mya) and T₂ (488 mya) are largely consistent to a well-cited version of two-round (2R) genome duplication theory. Moreover, in most cases, the time interval between two successive gene duplications is large enough that the fate of duplicate genes generated by the first gene duplication was likely to be determined before the second one took place. However, the phylogenetic pattern of 23 gene families with 4 members is complicated; only 5 of them are predicted by 2R model, but 11 families require an additional gene (or genome) duplication. For the rest (7 families), at least one gene duplication event had occurred before the divergence between vertebrate and Drosophila, indicating a possible misleading of the 4:1 rule (member gene ratio between vertebrates and invertebrates). Our results show that Ohno's 2R conjecture is valid as a working hypothesis for providing a most parsimonious explanation. Although for some gene families, additional gene duplication is needed, the credibility of the third genome duplication (3R) remains to be investigated. Received: 13 December 1999 / Accepted: 7 April 2000     

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     

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     

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     

Evolution of 28S Ribosomal DNA in Chaetognaths: Duplicate Genes and Molecular Phylogeny

The chaetognaths are an extraordinarily homogeneous phylum of animals at the morphological level, with a bauplan that can be traced back to the Cambrian. Despite the attention of zoologists for over two centuries, there is little agreement on classification within the phylum. We have used a molecular biological approach to investigate the phylogeny of extant chaetognaths. A rapidly evolving expansion segment toward the 5′ end of 28S ribosomal DNA (rDNA) was amplified using the polymerase chain reaction (PCR), cloned, and sequenced from 26 chaetognath samples representing 18 species. An unusual finding was the presence of two distinct classes of 28S rDNA gene in chaetognaths; our analyses suggest these arose by a gene (or gene cluster) duplication in a common ancestor of extant chaetognaths. The two classes of chaetognath 28S rDNA have been subject to different rates of molecular evolution; we present evidence that both are expressed and functional. In phylogenetic reconstructions, the two classes of 28S rDNA yield trees that root each other; these clearly demonstrate that the Aphragmophora and Phragmophora are natural groups. Within the Aphragmophora, we find good support for the groupings denoted Solidosagitta, Parasagitta, and Pseudosagitta. The relationships between several well-supported groups within the Aphragmophora are uncertain; we suggest this reflects rapid, recent radiation during chaetognath evolution. Received: 19 March 1996 / Accepted: 5 August 1996     

Ascidian and Amphioxus Adh Genes Correlate Functional and Molecular Features of the ADH Family Expansion During Vertebrate Evolution

The alcohol dehydrogenase (ADH) family has evolved into at least eight ADH classes during vertebrate evolution. We have characterized three prevertebrate forms of the parent enzyme of this family, including one from an urochordate (Ciona intestinalis) and two from cephalochordates (Branchiostoma floridae and Branchiostoma lanceolatum). An evolutionary analysis of the family was performed gathering data from protein and gene structures, exon–intron distribution, and functional features through chordate lines. Our data strongly support that the ADH family expansion occurred 500 million years ago, after the cephalochordate/vertebrate split, probably in the gnathostome subphylum line of the vertebrates. Evolutionary rates differ between the ancestral, ADH3 (glutathione-dependent formaldehyde dehydrogenase), and the emerging forms, including the classical alcohol dehydrogenase, ADH1, which has an evolutionary rate 3.6-fold that of the ADH3 form. Phylogenetic analysis and chromosomal mapping of the vertebrate Adh gene cluster suggest that family expansion took place by tandem duplications, probably concurrent with the extensive isoform burst observed before the fish/tetrapode split, rather than through the large-scale genome duplications also postulated in early vertebrate evolution. The absence of multifunctionality in lower chordate ADHs and the structures compared argue in favor of the acquisition of new functions in vertebrate ADH classes. Finally, comparison between B. floridae and B. lanceolatum Adhs provides the first estimate for a cephalochordate speciation, 190 million years ago, probably concomitant with the beginning of the drifting of major land masses from the Pangea. Received: 10 April 2001 / Accepted: 23 May 2001