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BMC Genomics
2014 Mar 20;15:216. doi: 10.1186/1471-2164-15-216.
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The Xenopus alcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes.
Borràs E
,
Albalat R
,
Duester G
,
Parés X
,
Farrés J
.
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BACKGROUND: The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family.
RESULTS: Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not present in amphibians and reptiles.
CONCLUSIONS: The study of the ancient forms of ADH2 and ADH7 sheds new light on the evolution of the vertebrate ADH system, whereas the special features showed by the novel forms point to the acquisition of new functions following the ADH gene family expansion which occurred in amphibians.
Figure 1. Detection of ADH classes in X. laevis. (A) Northern blot analysis of ADH1B, ADH3 and ADH9 from intestine (I), kidney (K), liver (L) and stomach (S), performed on 15-μg samples of total RNA. (B) Ethidium bromide-stained gel, from the same electrophoresis as in panel (A), showing 18S and 28S rRNAs next to the RNA molecular weight marker (0.24-9.5 kb, Invitrogen). The estimated molecular size of the RNA hybrids detected was ~1.6 kb. (C) RT-PCR of ADH8B from liver (L), esophagus (E), stomach (S) and intestine (I) next to DNA molecular weight marker VIII (Roche). Esophagus, stomach and intestine show an amplification product of 603 bp, indicating the presence of the ADH8B cDNA.
Figure 2. Detection of ADH activity in X. laevis tissues. Starch gel electrophoresis of tissue homogenates (15 μl) from different animals. (A) ADH1 or ADH1-like activity staining using 2-buten-1-ol as a substrate and NAD+ as a coenzyme. (B) Glutathione (GSH)-dependent formaldehyde dehydrogenase (ADH3) activity staining. Lanes: S, stomach; L, liver; O, ovary (pool of oocytes at different maturation stages). All detected ADH forms showed anodic mobility and different band patterns. ADH1 or ADH1-like activity is more abundant in liver extracts than in stomach and is absent in ovary, whereas ADH3 is more abundant in ovary.
Figure 3. Chromosomal location and synteny of ADH loci.X. tropicalis scaffolds GL172747.1 and GL172865.1 are compared to human syntenic chromosomes 4 and 9, A. carolinensis scaffolds GL343323.1 and GL343307.1, and P. sinensis JH210661.1 and JH209104.1. All the identified genes are shown transcriptionally oriented (ADH genes in black and others in grey). The genes marked with an asterisk lack the first exon in the assembly. The opposite orientation of several orthologous genes (underlined) in X. tropicalis and human suggests a past inversion and posterior rearrangements involving the ADH cluster. In contrast, frog genes between NPNT (nephronectin, not shown) and NFKB1, located at 0.58-1.30 Mb of scaffold GL172747.1, have the same orientation as their human orthologues (not shown). Gene symbols in human chromosomes are NFKB1: Nuclear factor of kappa light polypeptide gene enhancer in B cells 1, SLC39A8: Solute carrier family 39 (zinc transporter) member 8, BANK1: B-cell scaffold protein with ankyrin repeats 1, PPP3CA: Serine/threonine phosphatase 2B catalytic subunit (alpha isoform), DDIT4L: DNA-damage inducible transcript 4-like, H2AFZ: Histone H2A family member Z, DNAJB14: DnaJ homolog subfamily B member 14, MTTP: Microsomal triglyceride transfer protein, DAPP1: Dual adaptor for phosphotyrosine and 3′-phosphoinositides, METAP1: Methionine aminopeptidase 1, EIF4E: Eukaryotic translation initiation factor 4E, TSPAN5: Tetraspanin 5, TDRD7: Tudor domain containing protein 7, TMOD1: Tropomodulin-1, NCBP1: 80 kDa nuclear cap binding protein, XPA: DNA-repair protein complementing XP-A cells, ANP32B: Acidic leucine-rich nuclear phosphoprotein 32 family member B, CORO2A: Coronin-2A.
Figure 4. Phylogeny of vertebrate ADHs. Seven amphibian classes of ADH can be differentiated phylogenetically, where branches of each class are shown in a different color. The reliability of the Neighbour-joining (NJ) tree was tested by bootstrap analysis (1000 replicates). Within each class, branches were collapsed when bootstrap values were <80 with the exception of X. tropicalis ADH7. A second tree constructed following the Maximum-likelihood (ML) method (500 replicates) produced a similar topology. Figures at nodes are the scores from bootstrap resampling of the data, NJ values are in bold and ML values in italics. ADH sequences from X. laevis, X. tropicalis, A. carolinensis and P. sinensis are those described in the present manuscript and their accession numbers are provided in Tables 3 and 4. Accession numbers of other ADH sequences are compiled in Table 2. Alignment of all vertebrate ADHs included in the phylogenetic tree is presented in Additional file 15. Scale-bar represents substitutions per nucleotide.
Figure 5. Hypothetical evolutionary pathways leading to tetrapod ADH multiplicity. At the base of vertebrate radiation, an initial tandem duplication of the ancestral ADH3 led to a two-gene cluster. Actinopterygia (ray-finned fish) and sarcopterygia (lobe-finned fish and tetrapods) acquired ADH1 activity by the most 5â² member of the cluster [4]. Before the amniota/amphibian split (360 Mya), ADH2 and ADH7 would have arisen in tetrapods as a consequence of gene duplication events. In reptiles and birds, no additional ADH classes have been found. In contrast, ADH1 tandem duplications led to further class multiplicity in the amphibian lineage; thus, ADH8, ADH9, and more recently ADH10 forms would derive from the ancestral ADH1. Close to the origin of mammals, ADH7 was lost while gene duplications generated ADH5 and ADH6, and tandem duplication of ADH1 gave rise to ADH4. Only in primates, ADH6 was lost simultaneously or close to ADH1 duplications generating ADH1A-C isozymes [13]. Likewise, additional duplications occurred in other vertebrate lineages, and those ADH genes leading to isoenzyme multiplicity in at least one member of that lineage are underlined (in reptiles, multiple ADH1 and ADH7 are found in lizards, but not in turtles). In some organisms, ADH pseudogenes are also observed.
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