Exploring the Evolutionary History of the Alcohol Dehydrogenase Gene (<Emphasis Type="Italic">Adh</Emphasis>) Duplication in Species of the Family Tephritidae

In the olive fruit fly Bactrocera oleae and the med fly Ceratitis capitata previous studies have shown the existence of two Adh genes in each species. This observation, in combination with the former finding that various Drosophila species of virilis and repleta group encode two isozymes of ADH which are the result of a gene duplication, challenged us to address a scenario dealing with the evolutionary history of the Adh gene duplication in Tephritidae. In our lab we proceeded to the cloning and sequence analysis of Adh genes from more tephritid species, a prerequisite for further study of this issue. Here we show that phylogenetic trees produced from either the nucleotide or the amino acid sequences of 14 tephritid Adh genes consisted of two main clusters, with Adh sequences of the same type grouping together (i.e., Adh1 sequences form a cluster and Adh2 sequences form a second one), as expected if there was one duplication event before speciation within the family Tephritidae. We used the amount of divergence between the two isozymic forms of Adh of the species carrying both Adh1 and Adh2 genes to obtain an estimate of the age of the duplication event. Interestingly, our data again support the hypothesis that the duplication of an ancestral Adh single gene in the family Tephritidae occurred before the emergence of the genera Bactrocera and Ceratitis, thus suggesting that Adh duplication was based on a prespeciation rather than a postspeciation event that might have involved two independent duplication events, one in each of the two genera.     

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     

Functional Constraints of Alcohol Dehydrogenase (ADH) of Tephritidae and Relationships with Other Dipteran Species

Alcohol dehydrogenase is considered a very important enzyme in insect metabolism because it is involved (in its homodimeric form) in the catalysis of the reversible conversion of various alcohols in larval feeding sites to their corresponding aldehydes and ketones, thus contributing to detoxification and metabolic purposes. Using 14 amino acid ADH sequences recently determined in our laboratory, we constructed a three-dimensional (3D) model of olive fruit fly Bactrocera oleae ADH1 and ADH2, based on the known homologous Drosophila lebanonensis ADH structure, and the amino acid residues that have been proposed as being responsible for catalysis were located on it. Moreover, in a comparative study of the ADH sequences, the residues occupying characteristic positions in the ADH of species of the Bactrocera and Ceratitis genera (called genus-specific) as well as residues appearing only in ADH1 or ADH2 (called isozymic-specific) were defined and localized on the 3D model. All regions important for catalytic activity, such as those forming the substrate- and coenzyme-binding sites, are highly conserved in all tephritid species examined. Genus-specific amino acids are located on the outside of the protein, on loops and regions predicted to be antigenic. The higher percentage of genus-specific amino acid variation seems to be centered in the NAD adenine-binding site, located near the surface of the protein molecule. Nine of 12 isozymic-specific positions are lined along an arc on the surface of the protein, thus linking the two monomer bases of the dimer via the C-terminal interacting loops. Furthermore, the distribution of isozymic- and genus-specific amino acids on the monomer–monomer interface may have some evolutionary significance. Most amino acids predicted to be antigenic are positioned in peripheral regions of nonfunctional importance, but surprisingly, an additional antigenic region is contained within the (highly conserved in tephritids) C-terminal tail.     

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     

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     

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     

Phylogenetic Analysis of Vertebrate Lactate Dehydrogenase (LDH) Multigene Families

In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human–killifish pair than a human–lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60–70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. Received: 5 October 2001 / Accepted: 24 October 2001     

Enzymic and Structural Studies on Drosophila Alcohol Dehydrogenase and Other Short-Chain Dehydrogenases/Reductases

Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters k_cat and K_m. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(−) alcohols. At a high concentration of R(−) alcohols substrate activation occurs. The k_cat and K_m values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD⁺. Probably these lyases descend from an SDR, which has lost the capability to bind NAD⁺, but the enzyme reaction mechanisms may still be similar. Received: 23 May 2000 / Accepted: 4 January 2001     

Identification of Glyoxalase I Sequences in Brassica oleracea and Sporobolus stapfianus: Evidence for Gene Duplication Events

Glyoxalase I (GlxI) is the first of two enzymes involved in the cellular detoxification of methylglyoxal. A recent search of the National Center for Biotechnology Information (NCBI) databases with the protein sequence of Salmonella typhimurium GlxI identified two new hypothetical proteins with unassigned function. These two sequences, from Brassica oleracea and Sporobolus stapfianus, have significant sequence similarity to known GlxI sequences, suggesting that these two open reading frames encode for GlxI in these plants. Interestingly, analysis of these two new sequences indicates that they code for a protein composed of two fused monomers, a situation previously found solely in the yeast GlxI enzymes. Received: 10 May 1997 / Accepted: 15 October 1997     

Duplication and Quadruplication of Arabidopsis thaliana Cysteinyl- and Asparaginyl-tRNA Synthetase Genes of Organellar Origin

Two cysteinyl-tRNA synthetases (CysRS) and four asparaginyl-tRNA synthetases (AsnRS) from Arabidopsis thaliana were characterized from genome sequence data, EST sequences, and RACE sequences. For one CysRS and one AsnRS, sequence alignments and prediction programs suggested the presence of an N-terminal organellar targeting peptide. Transient expression of these putative targeting sequences joined to jellyfish green fluorescent protein (GFP) demonstrated that both presequences can efficiently dual-target GFP to mitochondria and plastids. The other CysRS and AsnRSs lack targeting sequences and presumably aminoacylate cytosolic tRNAs. Phylogenetic analysis suggests that the four AsnRSs evolved by repeated duplication of a gene transferred from an ancestral plastid and that the CysRSs also arose by duplication of a transferred organelle gene (possibly mitochondrial). These case histories are the best examples to date of capture of organellar aminoacyl-tRNA synthetases by the cytosolic protein synthesis machinery. Received: 8 October 1999 / Accepted: 23 January 2000     

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     

Biochemical Differences Between Products of the ADH Locus in Olive Fruit Fly (Bactrocera oleae)

Purified alcohol dehydrogenases from olive fruitflies of genotypes SS, II, and SI were biochemicallycompared. The enzymes were found to differ in thespecific activity, in the influence of pH andtemperature on activity, and in the affinity with differentsubstrate-alcohols. The probable relationships of thesefindings with the dramatic changes in allele frequenciesobserved when natural populations are introduced in the laboratory are discussed.     

Multiple Lineages of the Mitochondrial Gene NADH Dehydrogenase Subunit 1 (ND1) in Parasitic Helminths: Implications for Molecular Evolutionary Studies of Facultatively Anaerobic Eukaryotes

The task of using partial ND1 sequences to infer a phylogeny for species of the genus Paragonimus (Trematoda: Digenea) was complicated by the discovery of at least two ND1 lineages within individual worms. The divergence of the ND1 lineages is shown by phylogenetic analysis not only to predate the divergence of the three Paragonimus species or species groups investigated but also the divergence of some trematode families. Some sequences are clearly pseudogenes as they contain single base deletions and/or premature termination codons. The presence of both pseudogenes and/or mitochondrial heteroplasmy are invoked to explain the presence of multiple and divergent ND1 lineages in these trematodes, which have two distinct cytochemical types of mitochondria. The implications for phylogenetic studies generally and of parasitic helminths specifically, using ND1 sequence data, are discussed. The ability of these organisms to adapt their metabolic processes to the variable availability of oxygen as an electron acceptor are proposed to explain some of the molecular diversity observed in parasitic helminths and possibly also in other anaerobically adapted eukaryotes. Received: 18 October 1999 / Accepted: 23 June 2000     

The Phylogeny of Glyceraldehyde-3-Phosphate Dehydrogenase Indicates Lateral Gene Transfer from Cryptomonads to Dinoflagellates

Sequence analysis of two nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes isolated from the dinoflagellate Gonyaulax polyedra distinguishes them as cytosolic and chloroplastic forms of the enzyme. Distance analysis of the cytosolic sequence shows the Gonyaulax gene branching early within the cytosolic clade, consistent with other analyses. However, the plastid sequence forms a monophyletic group with the plastid isoforms of cryptomonads, within an otherwise cytosolic clade, distinct from all other plastid GAPDHs. This is attributed to lateral gene transfer from an ancestral cryptomonad to a dinoflagellate, providing the first example of genetic exchange accompanying symbiotic associations between the two, which are common in present day cells. Received: 29 April 1998 / Accepted: 7 July 1998     

Characterization of Novel Minisatellite Repeat Loci in Atlantic Salmon (Salmo salar) and Their Phylogenetic Distribution

We present here the sequence and characterization of various minisatellite-like tandem repeat loci isolated from the genome of Atlantic salmon (Salmo salar). Their diversity of sequence and lack of core motifs common to minisatellites of other species suggest the presence of numerous and previously unidentified simple sequence repeat families in this salmonid. Evidence for their ubiquity was provided by screening of a salmon genomic library. Southern blot analysis of the phylogenetic distribution of a subset of the minisatellites found one sequence to be pervasive among vertebrates, others present only in Salmoninae or Salmonidae species, and one amplified only in Atlantic salmon. There is evidence for the positioning of microsatellite and minisatellite arrays in close proximity at many loci. Furthermore, one tandem repeat appears to have been inserted into the transposase coding region of a copy of the Tc1 transposon-like element recently identified in salmonids. Received: 9 October 1996 / Accepted: 20 May 1997     

Evidence for Gene Conversion Between Tandemly Duplicated Cytoplasmic Actin Genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly eclosed adults. A region 5′ of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs. Received: 10 January 1996 / Accepted: 12 March 1996     

Phylogenetic Relationships of the Glycolytic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, from Parabasalid Flagellates

Over 90% of the open reading frame of gap genes for glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was obtained with PCR from five species of Parabasala. With gap1 from Trichomonas vaginalis obtained earlier, the data include two sequences each for three species. All sequences were colinear with T. vaginalis gap1 and shared with it as a synapomorphy a 10- to 11-residue insertion not found in any other gap and an S-loop with characteristic features of eubacterial GAPDH. All residues known to be highly conserved in this enzyme were present. The parabasalid sequences formed a robust monophyletic group in phylogenetic reconstructions with distance-based, maximum-parsimony, and maximum-likelihood methods. The two genes of the amphibian commensal, Trichomitus batrachorum, shared a common ancestor with the rest, which separate into two well-supported lineages. T. vaginalis and Tetratrichomonas gallinarum (both representatives of Trichomonadinae) formed one, while Monocercomonas sp. and Tritrichomonas foetus formed the other. These data agreed with and/or were close to published reconstructions based on other macromolecules. They did not support the ancestral position of Monocercomonas sp. proposed on the basis of morphological characteristics but confirmed an early emergence of Trichomitus batrachorum. The sequence pairs obtained from three species indicated either gene duplications subsequent to the divergence of the corresponding lineages or a strong gene conversion later in these lineages. The parabasalid clade was a robust part of the eubacterial radiation of GAPDH and showed no relationships to the clade that contained all other eukaryotic gap genes. The data clearly reveal that the members of this lineage use in their glycolytic pathway a GAPDH species with properties and an evolutionary history that are unique among all eukaryotes studied so far. Received: 28 April 1997 / Accepted: 14 October 1997     

Molecular Characterization and Evolution of the Amylase Multigene Family of Drosophila ananassae

Drosophila ananassae is known to produce numerous alpha-amylase variants. We have cloned seven different Amy genes in an African strain homozygous for the AMY1,2,3,4 electrophoretic pattern. These genes are organized as two main clusters: the first one contains three intronless copies on the 2L chromosome arm, two of which are tandemly arranged. The other cluster, on the 3L arm, contains two intron-bearing copies. The amylase variants AMY1 and AMY2 have been assigned to the intronless cluster, and AMY3 and AMY4 to the second one. The divergence of coding sequences between clusters is moderate (6.1% in amino acids), but the flanking regions are very different, which could explain their differential regulation. Within each cluster, coding and noncoding regions are conserved. Two very divergent genes were also cloned, both on chromosome 3L, but very distant from each other and from the other genes. One is the Amyrel homologous (41% divergent), the second one, Amyc1 (21.6% divergent) is unknown outside the D. ananassae subgroup. These two genes have unknown functions. Received: 30 May 2000 / Accepted: 17 July 2000     

Gene Duplication and Gene Conversion in the Caenorhabditis elegans Genome

A comprehensive analysis of duplication and gene conversion for 7394 Caenorhabditis elegans genes (about half the expected total for the genome) is presented. Of the genes examined, 40% are involved in duplicated gene pairs. Intrachromosomal or cis gene duplications occur approximately two times more often than expected. In general the closer the members of duplicated gene pairs are, the more likely it is that gene orientation is conserved. Gene conversion events are detectable between only 2% of the duplicated pairs. Even given the excesses of cis duplications, there is an excess of gene conversion events between cis duplicated pairs on every chromosome except the X chromosome. The relative rates of cis and trans gene conversion and the negative correlation between conversion frequency and DNA sequence divergence for unconverted regions of converted pairs are consistent with previous experimental studies in yeast. Three recent, regional duplications, each spanning three genes are described. All three have already undergone substantial deletions spanning hundreds of base pairs. The relative rates of duplication and deletion may contribute to the compactness of the C. elegans genome. Received: 30 July 1998 / Accepted: 12 October 1998