Evolution of the Aldose Reductase-Related Gecko Eye Lens Protein ρB-Crystallin: A Sheep in Wolf's Clothing

ρB-crystallin (AJ245805) is a major protein component (20%) in the eye lens of the gecko Lepidodactylus lugubris. Limited peptide sequence analysis earlier revealed that it belongs to the aldo-keto reductase superfamily, as does the frog lens ρ-crystallin. We have now determined the complete cDNA sequence of ρB-crystallin and established that it is more closely related to the aldose reductase branch of the superfamily than to frog ρ-crystallin. These gecko and frog lens proteins have thus independently been recruited from the same enzyme superfamily. Aldose reductase is implicated in the development of diabetic cataract in mammals, and, if active, ρB-crystallin might be a potential risk for the gecko lens. Apart from a replacement 298 Cys → Tyr, ρB-crystallin possesses all amino acid residues thought to be required for catalytic activity of the aldose reductases. However, modeling studies of the ρB-crystallin structure indicate that substrate specificity and nicotinamide cofactor affinity might be affected. Indeed, neither recombinant ρB-crystallin nor the reverse mutant 298 Tyr → Cys showed noticeable activity toward aliphatic and aromatic substrates, although cofactor binding was retained. Various other oxidoreductases are known to be recruited as abundant lens proteins in many vertebrate species; ρB-crystallin demonstrates that an aldose reductase-related enzyme also can be modified to this end. Received: 18 July 2000 / Accepted: 3 November 2000     

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     

Identification of Major Phylogenetic Branches of Inhibitory Ligand-Gated Channel Receptors

The gene superfamily of ligand-gated ion channel (LGIC) receptors is composed of members of excitatory LGIC receptors (ELGIC) and inhibitory LGIC receptors (ILGIC), all using amino acids as ligands. The ILGICs, including GABA_A, Gly, and GluCl receptors, conduct Cl⁻ when the ligand is bound. To evaluate the phylogenetic relationships among ILGIC members, 90 protein sequences were analyzed by both maximum-parsimony and distance matrix-based methods. The strength of the resulting phylogenetic trees was evaluated by means of bootstrap. Four major phylogenetic branches are recognized. Branch I, called BZ, for the majority of the members are known to be related to benzodiazepine binding, is subdivided into IA, composed of all GABA_A receptor α subunits, and IB, composed of the γ and ε subunits, which are shown to be tightly linked. Branch II, named NB for non–benzodiazepine binding, and consisting of GABA_A receptor β, δ, π, and ρ subunits, is further subdivided into IIA, containing β subunits; IIB, containing δ, and π subunits; and IIC, containing ρ subunits. Branch IIIA, composed of vertebrate Gly receptors, is loosely clustered with Branch IIIB, composed of invertebrate GluCl receptors, to form Branch III, which is designated NA for being non–GABA responsive. Branch IV is called UD for being undefined in specificity. The existence of primitive forms of GABA_A receptor non-β subunits in invertebrates is first suggested by the present analysis, and the identities of sequences p25123 from Drosophila melanogaster, s34469 from Lymnaea stagnalis, and u14635 and p41849 from C. aenorhabditis elegans are determined to be different from their previously given annotations. The proposed branching classification of ILGICs provides a phylogenetic map, based on protein sequences, for tracing the evolutionary pathways of ILGIC receptor subunits and determining the identities of newly discovered subunits on the basis of their protein sequences. Received: 15 April 1997 / Accepted: 11 March 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     

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     

Monophyly of Lampreys and Hagfishes Supported by Nuclear DNA–Coded Genes

The phylogenetic position of hagfishes in vertebrate evolution is currently controversial. The 18S and 28S rRNA trees support the monophyly of hagfishes and lampreys. In contrast, the mitochondrial DNAs suggest the close association of lampreys and gnathostomes. To clarify this controversial issue, we have conducted cloning and sequencing of the four nuclear DNA–coded single-copy genes encoding the triose phosphate isomerase, calreticulin, and the largest subunit of RNA polymerase II and III. Based on these proteins, together with the Mn superoxide dismutase for which hagfish and lamprey sequences are available in database, phylogenetic trees have been inferred by the maximum likelihood (ML) method of protein phylogeny. It was shown that all the five proteins prefer the monophyletic tree of cyclostomes, and the total log-likelihood of the five proteins significantly supports the cyclostome monophyly at the level of ±1 SE. The ML trees of aldolase family comprising three nonallelic isoforms and the complement component group comprising C3, C4, and C5, both of which diverged during vertebrate evolution by gene duplications, also suggest the cyclostome monophyly. Received: 28 April 1999 / Accepted: 30 June 1999     

Phylogeny of Japanese Papilionid Butterflies Inferred from Nucleotide Sequences of the Mitochondrial ND5 Gene

Phylogenetic relationships among the Japanese papilionid butterflies were analyzed by comparing 783 nucleotide sequences of the mitochondrial gene encoding NADH dehydrogenase subunit 5 (ND5). Phylogenetic trees of the representative species from each family in the superfamily Papilionoidea revealed that the species of the family Papilionidae and those of all other families formed distinct clusters, with a few species of the family Hesperiidae (Hesperioidea) as an outgroup. In the phylogenetic trees of most Japanese species of the family Papilionidae with Nymphalis xanthomelas (Nymphalidae) as an outgroup, the tribe Parnassiini (Parnassiinae) formed a cluster, and the rest formed the other cluster in which the tribe Zerynthiini (Parnassiinae) and the subfamily Papilioninae formed different subclusters. In the Papilioninae cluster, the tribes Troidini and Graphiini formed a subcluster, and the tribe Papilionini formed the other subcluster. These results generally agree with the traditional classification of the papilionid butterflies based on their morphological characteristics and support the proposed evolutionary genealogy of the butterflies based on their morphology, behavior, and larval host plants, except that the tribes Parnasiini and Zerynthiini (both Parnassiinae) are not in the same cluster. Received: 16 March 1998 / Accepted: 28 April 1998     

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     

Sucrose Phosphate Synthase Genes in Plants Belong to Three Different Families

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date. Received: 17 April 2001 / Accepted: 27 August 2001     

Close Evolutionary Relatedness of α-Amylases from Archaea and Plants

The amino acid sequences of 22 α-amylases from family 13 of glycosyl hydrolases were analyzed with the aim of revealing the evolutionary relationships between the archaeal α-amylases and their eubacterial and eukaryotic counterparts. Two evolutionary distance trees were constructed: (i) the first one based on the alignment of extracted best-conserved sequence regions (58 residues) comprising β2, β3, β4, β5, β7, and β8 strand segments of the catalytic (α/β)₈-barrel and a short conserved stretch in domain B protruding out of the barrel in the β3 →α3 loop, and (ii) the second one based on the alignment of the substantial continuous part of the (α/β)₈-barrel involving the entire domain B (consensus length: 386 residues). With regard to archaeal α-amylases, both trees compared brought, in fact, the same results; i.e., all family 13 α-amylases from domain Archaea were clustered with barley pI isozymes, which represent all plant α-amylases. The enzymes from Bacillus licheniformis and Escherichia coli, representing liquefying and cytoplasmic α-amylases, respectively, seem to be the further closest relatives to archaeal α-amylases. This evolutionary relatedness clearly reflects the discussed similarities in the amino acid sequences of these α-amylases, especially in the best-conserved sequence regions. Since the results for α-amylases belonging to all three domains (Eucarya, Eubacteria, Archaea) offered by both evolutionary trees are very similar, it is proposed that the investigated conserved sequence regions may indeed constitute the ``sequence fingerprints' of a given α-amylase. Received: 3 June 1998 / Accepted: 20 August 1998     

Did Archaeal and Bacterial Cells Arise Independently from Noncellular Precursors? A Hypothesis Stating That the Advent of Membrane Phospholipid with Enantiomeric Glycerophosphate Backbones Caused the Separation of the Two Lines of Descent

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively. Received: 24 March 1997 / Accepted: 21 May 1997     

Evolution of Chordate Actin Genes: Evidence from Genomic Organization and Amino Acid Sequences

The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates. Received: 20 June 1996 / Accepted: 16 October 1996     

Phylogenetic Position of the Subgenus Lordiphosa of the Genus Drosophila (Diptera: Drosophilidae) Inferred from Alcohol Dehydrogenase (Adh) Gene Sequences

We analyzed the phylogenetic relationship between the species of Lordiphosa and other Drosophilidae using alcohol dehydrogenase (Adh) gene sequences. The phylogenetic trees consistently show that the four species Drosophila kurokawai, D. collinella, D. stackelbergi, and D. clarofinis, which include three species groups of Lordiphosa, form a monophyletic clade. This clade is placed as a sister group to the willistoni and saltans groups of Sophophora. On the other hand, three species of Lordiphosa, D. tenuicauda, D. pseudotenuicauda, and D. acutissima, all of which belong to the tenuicauda group, are not shown to be related to the major Lordiphosa lineage. In the phylogenetic trees, these species are included into the clade comprised of Drosophila and Hirtodrosophila, although it remains uncertain whether the tenuicauda group is a monophyletic group or not. These results indicate that Lordiphosa is polyphyletic and that most of the members of the subgenus have a close relationship to the neotropical groups of Sophophora. The above conclusion is compatible with the hypothesis of Okada (Mushi [1963] 37:79–100) and Lastovka and Máca (Acta Ent Bohemoslov [1978] 75:404–420) that Lordiphosa is most closely related to Sophophora; in contrast, our results contradict the hypothesis of Grimaldi (Bull Am Mus Nat Hist [1990] 197:1–139) that Lordiphosa is a sister group to the genus Scaptomyza. Received: 12 May 1999 / Accepted: 14 April 2000     

The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): Genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae

The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids. Received: 22 August 1995 / Accepted: 6 September 1995     

The cytochrome b gene as a phylogenetic marker: the limits of resolution for analyzing relationships among cichlid fishes

The mitochondrial cytochrome b (cyt-b) gene is widely used in systematic studies to resolve divergences at many taxonomic levels. The present study focuses mainly on the utility of cyt-b as a molecular marker for inferring phylogenetic relationship at various levels within the fish family Cichlidae. A total of 78 taxa were used in the present analysis, representing all the major groups in the family Cichlidae (72 taxa) and other families from the suborders Labroidei and Percoidei. Gene trees obtained from cyt-b are compared to a published total evidence tree derived from previous studies. Minimum evolution trees based on cyt-b data resulted in topologies congruent with all previous analyses. Parsimony analyses downweighting transitions relative to transversions (ts1:tv4) or excluding transitions at third codon positions resulted in more robust bootstrap support for recognized clades than unweighted parsimony. Relative rate tests detected significantly long branches for some taxa (LB taxa) which were composed mainly by dwarf Neotropical cichlids. An improvement of the phylogenetic signal, as shown by the four-cluster likelihood mapping analysis, and higher bootstrap values were obtained by excluding LB taxa. Despite some limitations of cyt-b as a phylogenetic marker, this gene either alone or in combination with other data sets yields a tree that is in agreement with the well-established phylogeny of cichlid fish. Received: 11 October 2000 / Accepted: 26 February 2001     

Molecular Phylogenetic Inference of the Woolly Mammoth Mammuthus primigenius, Based on Complete Sequences of Mitochondrial Cytochrome b and 12S Ribosomal RNA Genes

Complete sequences of cytochrome b (1,137 bases) and 12S ribosomal RNA (961 bases) genes in mitochondrial DNA were successfully determined from the woolly mammoth (Mammuthus primigenius), African elephant (Loxodonta africana), and Asian elephant (Elephas maximus). From these sequence data, phylogenetic relationships among three genera were examined. Molecular phylogenetic trees reconstructed by the neighbor-joining and the maximum parsimony methods provided an identical topology both for cytochrome b and 12S rRNA genes. These results support the ``Mammuthus-Loxodonta' clade, which is contrary to some previous morphological reports that Mammuthus is more closely related to Elephas than to Loxodonta. Received: 8 April 1997 / Accepted: 23 July 1997     

Protein Tyrosine Kinase cDNAs from Amphioxus, Hagfish, and Lamprey: Isoform Duplications Around the Divergence of Cyclostomes and Gnathostomes

Animals evolved a variety of gene families involved in cell–cell communication and developmental control by gene duplication and domain shuffling. Each family is made up of several subtypes or subfamilies with distinct structures and functions, which diverged by gene duplications and domain shufflings before the divergence of parazoans and eumetazoans. Since the separation from protostomes, vertebrates expanded the multiplicity of members (isoforms) in the same subfamily by further gene duplications in their early evolution before the fish–tetrapod split. To know the dates of isoform duplications more closely, we have conducted isolation and sequencing cDNAs encoding the fibroblast growth factor receptor, Eph, src, and platelet-derived growth factor receptor subtypes belonging to the protein tyrosine kinase family from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Lampetra reissneri, a lamprey. From a phylogenetic tree of each subfamily inferred from a maximum likelihood (ML) method, together with a bootstrap analysis based on the ML method, we have shown that the isoform duplications frequently occurred in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes by gene duplications and possibly chromosomal duplications. Received: 28 April 1998 / Accepted: 30 June 1999     

The Molecular Evolution of the Vertebrate Trypsinogens

We expand the already large number of known trypsinogen nucleotide and amino acid sequences by presenting additional trypsinogen sequences from the tunicate (Boltenia villosa), the lamprey (Petromyzon marinus), the pufferfish (Fugu rubripes), and the frog (Xenopus laevis). The current array of known trypsinogen sequences now spans the entire vertebrate phylogeny. Phylogenetic analysis is made difficult by the presence of multiple isozymes within species and rates of evolution that vary highly between both species and isozymes. We nevertheless present a Fitch-Margoliash phylogeny constructed from pairwise distances. We employ this phylogeny as a vehicle for speculation on the evolution of the trypsinogen gene family as well as the general modes of evolution of multigene families. Unique attributes of the lamprey and tunicate trypsinogens are noted. Received: 12 July 1997     

A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms

Fungi have evolved a unique α-aminoadipate pathway for lysine biosynthesis. The fungal-specific enzyme homoaconitate hydratase from this pathway is moderately similar to the aconitase-family proteins from a diverse array of taxonomic groups, which have varying modes of obtaining lysine. We have used the similarity of homoaconitate hydratase to isopropylmalate isomerase (serving in leucine biosynthesis), aconitase (from the tricarboxylic acid cycle), and iron-responsive element binding proteins (cytosolic aconitase) from fungi and other eukaryotes, eubacteria, and archaea to evaluate possible evolutionary scenarios for the origin of this pathway. Refined sequence alignments show that aconitase active site residues are highly conserved in each of the enzymes, and intervening sequence sites are quite dissimilar. This pattern suggests strong purifying selection has acted to preserve the aconitase active site residues for a common catalytic mechanism; numerous other substitutions occur due to adaptive evolution or simply lack of functional constraint. We hypothesize that the similarities are the remnants of an ancestral gene duplication, which may not have occurred within the fungal lineage. Maximum likelihood, neighbor joining, and maximum parsimony phylogenetic comparisons show that the α-aminoadipate pathway enzyme is an outgroup to all aconitase family proteins for which sequence is currently available. Received: 7 October 1997