Biochemical Genetics of Opossum Aldehyde Dehydrogenase 3: Evidence for Three ALDH3A-Like Genes and an ALDH3B-Like Gene

Mammalian ALDH3 isozymes participate in peroxidic and fatty aldehyde metabolism, and in anterior eye tissue UV-filtration. BLAT analyses were undertaken of the opossum genome using rat ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2 amino acid sequences. Two predicted opossum ALDH3A1-like genes and an ALDH3A2-like gene were observed on chromosome 2, as well as an ALDH3B-like gene, which showed similar intron–exon boundaries with other mammalian ALDH3-like genes. Opossum ALDH3 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for rat ALDH3A1. Eleven glycine residues were conserved for all of the opossum ALDH3-like sequences examined, including two glycine residues previously located within the stem of the rat ALDH3A1 active site funnel. Phylogeny studies of human, rat, opossum, and chicken ALDH3-like sequences indicated that the common ancestor for ALDH3A- and ALDH3B-like genes predates the appearance of birds during vertebrate evolution.     

Opossum Aldehyde Dehydrogenases: Evidence for Four ALDH1A1-like Genes on Chromosome 6 and ALDH1A2 and ALDH1A3 Genes on Chromosome 1

Evidence is presented for six opossum ALDH1A genes, including four ALDH1A1-like genes on chromosome 6 and ALDH1A2- and ALDH1A3-like genes on chromosome 1. Predicted structures for the opossum aldehyde dehydrogenase (ALDH) subunits and the intron–exon boundaries for opossum ALDH genes showed a high degree of similarity with other mammalian ALDHs. Phylogenetic analyses supported the proposed designation of these opossum class 1 ALDHs as ALDH1A-like, ALDH1A2-like, and ALDH1A3-like and are therefore likely to play important roles in retinal and peroxidic aldehyde metabolism. Alignments of predicted opossum ALDH1A amino acid sequences with sheep ALDH1A1 and rat ALDH1A2 sequences demonstrated conservation of key residues previously shown to participate in catalysis and coenzyme binding. Amino acid substitution rates observed for family 1A ALDHs during vertebrate evolution indicated that ALDH1A2-like genes are evolving slower than ALDH1A1- and ALDH1A3-like genes. It is proposed that the common ancestor for ALDH1A genes predates the appearance of birds during vertebrate evolution.     

Evolutionary analysis of vertebrate Notch genes

We have conducted an evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes resulted from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences showed there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2-5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the Caenorhabditis elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal of the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function.     

Phylogenetic Analysis of the Cytochrome P450 3 (CYP3) Gene Family

Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya. The CYP3 gene family includes the CYP3A and CYP3B subfamilies. Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates. The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds. To understand vertebrate CYP3 diversity better, we determined the killifish (Fundulus heteroclitus) CYP3A30 and CYP3A56 and the ball python (Python regius) CYP3A42 sequences. We performed phylogenetic analyses of 45 vertebrate CYP3 amino acid sequences using a Bayesian approach. Our analyses indicate that teleost, diapsid, and mammalian CYP3A genes have undergone independent diversification and that the ancestral vertebrate genome contained a single CYP3A gene. Most CYP3A diversity is the product of recent gene duplication events. There is strong support for placement of the guinea pig CYP3A genes within the rodent CYP3A diversification. The rat, mouse, and hamster CYP3A genes are mixed among several rodent CYP3A subclades, indicative of a complex history involving speciation and gene duplication. Phylogenetic analyses suggest two CYP3A gene duplication events early in rodent history, with the rat CYP3A9 and mouse Cyp3a13 clade having a sister relationship to all other rodent CYP3A genes. In primate history, the human CYP3A43 gene appears to have a sister relationship to all other known primate CYP3A genes. Other, more recent gene duplications are hypothesized to have occurred independently within the human, pig, rat, mouse, guinea pig, and fish genomes. Functional analyses suggest that gene duplication is strongly tied to acquisition of new function and that convergent evolution of CYP3A function may be frequent among independent gene copies. Current address (Rachel L. Cox): Laboratory of Aquatic Biomedicine, Marine Biology Laboratory, Woods Hole, MA 02543, USA     

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     

Evolution of the vertebrate glucose-dependent insulinotropic polypeptide (GIP) gene

The glucose-dependent insulinotropic polypeptide (GIP) gene is believed to have originated from a gene duplication event very early in vertebrate evolution that also produced the proglucagon gene, yet so far GIP has only been described within mammals. Here we report the identification of GIP genes in chicken, frogs, and zebrafish. The chicken and frog genes are organized in a similar fashion to mammalian GIP genes and contain 6 exons and 5 introns in homologous locations. These genes can also potentially be proteolytically processed in identical patterns as observed in the mammalian sequences that would yield a GIP hormone that is only one amino shorter than the mammalian sequences due to the removal of an extra basic residue by carboxypeptidase E. The zebrafish GIP gene and precursor protein is shorter than other vertebrate GIP genes and is missing exon 5. The predicted zebrafish GIP hormone is also shorter, being only 31 amino acids in length. The zebrafish GIP hormone is similar in length to the proglucagon-derived peptide hormones, peptides encoded from the gene most closely related to GIP. We suggest that the structure of zebrafish GIP is more similar to the ancestral gene, and that tetrapod GIP has been extended. The mammalian GIP hormone has also undergone a period of rapid sequence evolution early in mammalian evolution. The discovery of a conserved GIP in diverse vertebrate suggests that it has an essential role in physiology in diverse vertebrates, although it may have only recently evolved a role as an incretin hormone.     

Cytochrome P450 1 genes in early deuterostomes (tunicates and sea urchins) and vertebrates (chicken and frog): origin and diversification of the CYP1 gene family

Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.     

Stepwise origin and functional diversification of the AFL subfamily B3 genes during land plant evolution

The AFL genes (ABI3/VP1, FUS3 and LEC2) belong to the plant-specific B3 superfamily, playing important roles in regulating seed development and maturation. It is unclear, however, whether these genes appeared at the same time as the origin of seed plants and if all these genes are necessary and sufficient for seed development for all seed plants. By conducting a genome-wide comparative analysis of the putative AFL genes in various plant species, we found that the ABI3 homologous genes existed in all land plant genomes, but the FUS3 homologous were present only in seed plant genomes and the LEC2-like sequences only in dicot genomes. Phylogenetic analysis indicated that the AFL genes had undergone successive rounds of gene duplication and subsequent diversification during land plant evolution, resulting in the stepwise origin of the ABI3, FUS3 and LEC2 genes. Comparison of gene structure of the AFL genes revealed a trend of decreasing in the number of conserved domains from ABI3 to FUS3 and LEC2.     

Long-term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals--a birth-and-death evolution model

Functional glycosyltransferase 6 (GT6) family members catalyze the transfer of galactose or N-acetylgalactosamine in alpha1,3 linkage to various substrates and synthesize structures related to the A and B histo-blood group antigens, the Forssman antigen, alphaGal epitope, and iGb3 glycolipid. In rat, mouse, dog, and cow genomes, we have identified three new mammalian genes (GT6m5, GT6m6, and GT6m7) encoding putative proteins belonging to the GT6 family. Among these, GT6m6 protein does not display major alterations of the GT6 motifs involved in binding of the divalent cation and the substrate. Based on protein sequence comparison, gene structure, and synteny, GT6 homologous sequences were also identified in bird, fish, and amphibian genomes. Strikingly, the number and type of GT6 genes varied widely from species to species, even within phylogenetically related groups. In human, except ABO functional alleles, all other GT6 genes are either absent or nonfunctional. Human, mouse, and cow have only one ABO gene, whereas rat and dog have several. In the chicken, the Forssman synthase-like is the single GT6 family member. Five Forssman synthase-like genes were found in zebrafish, but are absent from three other fishes (fugu, puffer fish, and medaka). Two iGb3 synthase-like genes were found in medaka, which are absent from zebrafish. Fugu, puffer fish, and medaka have an additional GT6 gene that we termed GT6m8, which is absent from all other species analyzed here. These observations indicate that individual GT6 genes have expanded and contracted by recurrent duplications and deletions during vertebrate evolution, following a birth-and-death evolution type.     

Corneal and stomach expression of aldehyde dehydrogenases: from fish to mammals

We have studied the distribution of the ALDH3A1, ALDH1A1 and ALDH2 proteins in the cornea and stomach of several animal species, including mammals (C57BL/6J and SWR/J mice, rat and pig), birds (chicken and turkey), amphibians (frog) and fish (trout and zebrafish). High ALDH3A1 protein levels and catalytic activities were detected in C57BL/6J mouse, rat and pig. We found complete absence of the ALDH3A1 protein in SWR/J mice, which carry the Aldh3a1(c) allele characterized by four amino acid substitutions (G88R, I154N, H305R and I352V) and lack of enzymatic activity. This indicates that the SWR/J mouse strain is a natural gene knockout model for ALDH3A1. Traces of ALDH3A1 were detected in rabbit, whereas expression was absent from chicken, turkey, frog, trout, and zebrafish. Interestingly, significant levels of the cytosolic ALDH1A1 and mitochondrial ALDH2 proteins were detected by immunoblot analysis in all examined species that are deficient in ALDH3A1 expression. In contrast, no ALDH1A1 or ALDH2 protein was detected in the species expressing ALDH3A1. It can, therefore, be concluded that corneal expression of ALDH3A1 or ALDH1A1/ALDH2 occurs in a taxon-specific manner, supporting the protective role of these ALDHs in cornea against the UV-induced oxidative damage.     

Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.     

Evolution of new hormone function: loss and gain of a receptor

The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.     

Nucleotide sequence determination of mouse, chicken and Xenopus laevis rig cDNAs: the rig-encoded protein is extremely conserved during vertebrate evolution

Mouse, chicken and Xenopus laevis homologues to rig (rat insulinoma gene) cDNA were isolated and their nucleotide sequences were determined. Each homologue encoded a 145-amino acid protein; the amino acid sequence remained invariant in the murine and avian genes, and there were only 6 amino acid substitutions in the salientian gene. The evolutionary rate calculated for rig mRNA was sufficiently low to be viewed as evidence that rig is vital to vertebrate species. Southern blot analysis indicated that haploid sets of the mammalian genomes contain several copies of rig or rig-related sequences, whereas there appeared to be only one copy in the amphibian and bird genomes. The possibility that rig belongs to the class of housekeeping genes is discussed.     

Corneal and stomach expression of aldehyde dehydrogenases: from fish to mammals

We have studied the distribution of the ALDH3A1, ALDH1A1 and ALDH2 proteins in the cornea and stomach of several animal species, including mammals (C57BL/6J and SWR/J mice, rat and pig), birds (chicken and turkey), amphibians (frog) and fish (trout and zebrafish). High ALDH3A1 protein levels and catalytic activities were detected in C57BL/6J mouse, rat and pig. We found complete absence of the ALDH3A1 protein in SWR/J mice, which carry the Aldh3a1^c allele characterized by four amino acid substitutions (G88R, I154N, H305R and I352V) and lack of enzymatic activity. This indicates that the SWR/J mouse strain is a natural gene knockout model for ALDH3A1. Traces of ALDH3A1 were detected in rabbit, whereas expression was absent from chicken, turkey, frog, trout, and zebrafish. Interestingly, significant levels of the cytosolic ALDH1A1 and mitochondrial ALDH2 proteins were detected by immunoblot analysis in all examined species that are deficient in ALDH3A1 expression. In contrast, no ALDH1A1 or ALDH2 protein was detected in the species expressing ALDH3A1. It can, therefore, be concluded that corneal expression of ALDH3A1 or ALDH1A1/ALDH2 occurs in a taxon-specific manner, supporting the protective role of these ALDHs in cornea against the UV-induced oxidative damage.     

Phylogenetic Analysis of Zebrafish Basic Helix-Loop-Helix Transcription Factors

The basic helix-loop-helix (bHLH) proteins play important regulatory roles in eukaryotic developmental processes including neurogenesis, myogenesis, hematopoiesis, sex determination, and gut development. Zebrafish is a good model organism for developmental biology. In this study, we identified 139 bHLH genes encoded in the zebrafish genome. Phylogenetic analyses revealed that zebrafish has 58, 29, 21, 5, 19, and 5 bHLH members in groups A, B, C, D, E, and F, respectively, while 2 members were classified as “orphan.” A comparison between zebrafish and human bHLH repertoires suggested that both organisms have a certain number of specific bHLH members. Eight zebrafish bHLH genes were found to have multiple coding regions in the genome. Two of these, Bmal1 and MITF, are good anchor genes for identification of fish-specific whole-genome duplication events in comparison with mouse and chicken genomes. The present study provides useful information for future studies on gene family evolution and vertebrate development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.