BACKGROUND
vertebrate alcohol dehydrogenases  are dimeric zinc-containing enzymes with a 40-kda subunit and 373– <dig> amino acid residues.
structurally, they belong to the medium-chain dehydrogenase/reductase  superfamily  <cit> .
adhs catalyze the reversible oxidation of a wide range of alcohol substrates to the corresponding aldehydes or ketones, and can be grouped in eight enzyme classes   <cit> , according to their primary structure and function.
the human adh gene nomenclature used throughout the text is the enzyme class-based nomenclature currently used for vertebrate adh  <cit>  and differs from that approved by the human genome organization  gene nomenclature committee  <cit> , as the former facilitates comparisons with adhs from other mammals and lower vertebrate species.
tandem gene duplications gave rise to the multiplicity of forms in the adh family, including isoenzymes and allelic forms in particular lineages.
adh <dig> is the most ancient form and the only class present before chordates.
it is a glutathione-dependent formaldehyde dehydrogenase , a highly conserved and ubiquitous detoxifying enzyme.
duplication of the ancestral adh <dig> gene near the agnathan/gnathostome split originated adh <dig>  which evolved independently in the fish and tetrapod lines becoming the classical hepatic ethanol dehydrogenase  <cit> .
in tetrapods, a second duplication of the gene coding for adh <dig> generated adh <dig>  also hepatic but active at higher ethanol concentrations  <cit> .
close to the origin of mammals, adh <dig> duplicated giving rise to adh <dig>  a highly retinoid-active enzyme  <cit>  present in eye, skin and gastric tissues  <cit> .
the most evolutionarily recent classes in mammals are adh <dig> and adh <dig>  <cit> , the latter being absent in primates  <cit> .
these two classes, identified at dna level, are the most divergent within mammalian adhs.
on the other hand, adh <dig>  previously named adh-f due to its fetal expression, is a steroid/retinoid dehydrogenase that was first described in chicken  <cit> .
finally, adh <dig> is a unique nadp+-dependent adh isolated from the stomach of the frog rana perezi and its proposed function is the reduction of retinaldehyde to retinol  <cit> .
studies on amphibian adh genetics have been scarce.
isozyme patterns of x. laevis liver ethanol dehydrogenase suggested the existence of two polymorphic genes encoding adh subunits that did not form heterodimers and were located in different linkage groups  <cit> .
the enzymes adh <dig>  adh <dig> and adh <dig> from the frog rana perezi were purified and characterized by our group, and the adh <dig> and adh <dig> proteins were also sequenced  <cit> .
the cloning of the cdna of r. perezi adh <dig>  <cit>  allowed to perform mutagenesis studies on coenzyme specificity  <cit>  and to obtain the crystal structure of the enzyme  <cit> .
partial cdnas of x. laevis adh <dig> and an adh4-like form were cloned and used for expression analysis in embryonic and adult tissues  <cit> .
later, two reviews on mdr-adh evolution  <cit> , which included genomic data, provided some partial information on the amphibian adh system.
here the adh system of the development model frog x. laevis has been further investigated, especially the retinaldehyde-active adh <dig>  tetraploidy of x. laevis  was a handicap for genetic studies, thus the present work was restricted to expression patterns and extended with additional information from expressed sequence tag  collections.
on the other hand, its diploid relative x. tropicalis , the subject of the only amphibian genome project, was used for a genomic approach to the amphibian adh family.
since the reptile genome of the anole lizard  and the turtle  had been sequenced at the time of the study, the adh gene sequences from these organisms could be identified and used in phylogenetic analyses and genomic comparisons.
the joint analysis of xenopus genome-wide data and the results of the expression analysis described herein provide an integrated view of the amphibian adh system.
moreover, since this organism occupies a key phylogenetic position, this work provides insight into the molecular evolution of the adh gene family in vertebrates.
methods
animal tissues
tissues were obtained from adult x. laevis females  provided by horst kähler .
the animals were kept in an ice bath for 15 min to diminish their metabolism prior to euthanasia.
after decapitation, the head was immersed in liquid nitrogen to assure total unconsciousness, as recommended  <cit> .
the organs were then removed, cleaned, rinsed in distilled water and stored at –80°c.
prior to analysis, frozen tissues were pulverized in liquid nitrogen and homogenized.
this study was approved by the ethical committee of the universitat autònoma de barcelona.
isolation and cloning of x. laevis cdnas
stomach poly+ rna  was isolated with the “quickprep micro mrna purification kit”  and a cdna pool was synthesized using the “first strand cdna synthesis kit for rt-pcr ”  with the oligo 17riro primer adaptor  <cit> .
nested-pcrs combined degenerate primers based on r. perezi adh <dig>  and amplification products were cloned into pbluescript ii sk  and sequenced.
the partial cloned sequences were later identified as adh1b and adh <dig>  whereas the adh8b and adh <dig> partial cdnas were obtained as described  <cit> .
the 3′-ends were amplified by rapid amplification of cdna ends  <cit>  combining the adaptor-specific primers ro and ri with specific forward primers , and then cloned and sequenced as described above.
x. laevis
adhs
1
2
1degenerate nucleotides are r , w , h  and i .
2numbering refers to amino acid residues unless otherwise indicated.
northern blot analysis of x. laevis adh1b, adh <dig> and adh9
total rna from stomach, liver, kidney and intestine was isolated by the acid guanidinium thiocyanate method  <cit> .
samples  were electrophoresed on a 1% agarose gel containing  <dig>  m formaldehyde and transferred onto a nylon filter.
18s rrna  and 28s rrna  were used to check the integrity and amount of loaded rna and to estimate the size of the rna hybrids.
probes included x. laevis adh1b, adh <dig> and adh <dig> cdnas , labeled with dctp  by a random hexamer-primed method using the “prime-a-gene labeling system”  except for the klenow enzyme .
after a 45-min prehybridization at 60°c in  <dig>  m sodium phosphate, ph  <dig> , 1 mm edta, 1% bsa and 1% sds, filters were hybridized for 18–24 h at 60°c in the presence of 106 cpm/ml of radiolabeled probe.
final 30-min washes at 60°c were performed twice in 40 mm sodium phosphate, ph  <dig> , 1 mm edta and 1% sds.
autoradiography was carried out at –80°c for 2–5 days with hyperfilm-mp  using an intensifying screen.
hybridization signals were then scanned in a bio-rad gs- <dig> imaging densitometer.
rt-pcr of x. laevis adh8b
cdna pools from esophagus, stomach, intestine and liver were prepared from 3–8 μg of total rna using the adh8b-specific reverse outer primer described in table  <dig>  first pcr amplification combined this primer with the adh8b forward outer primer, and a second round used the inner primer pair , generating a 603-bp adh8b cdna fragment.
starch gel electrophoresis and activity staining of x. laevis adh <dig> and adh3
tissues were homogenized  in 30 mm tris–hcl, ph  <dig> ,  <dig>  mm dithiothreitol, centrifuged at  <dig> ×g for 30 min and supernatants were used for analysis by starch gel electrophoresis  <cit> .
total protein was determined by the bio-rad protein assay method, using bovine serum albumin as standard.
in order to discriminate between the nad+-dependent classes adh <dig> and adh <dig>  gel slices were stained for adh activity with  <dig>  m 2-buten-1-ol and  <dig>  mm nad+ , to mainly detect adh <dig>  and for glutathione-dependent fdh activity with  <dig>  mm formaldehyde and 1 mm glutathione, to specifically stain adh <dig>
identification of xenopus adh sequences in protein and expression databases
several adh protein sequences from both xenopus species were gathered at uniprot  <cit> , first by name search and then by exploring clusters with 90% or 50% identity.
ests of x. laevis and x. tropicalis were obtained by blast   <cit>  search, using x. laevis and other vertebrate adhs as protein queries against translated nucleotide sequences , at the sanger institute x. tropicalis est project  <cit>  and tgi gene indices  <cit>  websites.
additional information on the expression sites of xenopus adh clustered transcripts was obtained by name search at the ncbi unigene data bank  <cit> .
identification and analysis of adh genes in xenopus tropicalis, anolis carolinensis and pelodiscus sinensis genomes
x. tropicalis genome assembly  <dig> , a. carolinensis anocar <dig>  and p. sinensis pelsin_ <dig>  were interrogated with blast-based search tools to identify possible adh gene locations.
for x. tropicalis, tblastn searches were undertaken at the joint genome institute x. tropicalis genome assembly  <dig>  website  <cit>  using x. laevis adhs as protein queries.
for a. carolinensis and p. sinensis, tblastn searches were conducted using the ensembl genome browser  <cit>  to compare x. tropicalis adhs against genomic databases, allowing some local mismatch.
protein blat  analyses  <cit>  using the ucsc web browser  <cit>  were also performed and the same scaffolds producing significant alignments were obtained.
these adh-containing scaffolds were then exported with ensembl, 3-frame translated in both orientations, and manually screened for the presence of adh genes either using exon sequences derived from x. tropicalis ests or consensus adh sequences.
the ensembl genome browser  <cit>  was also used to align syntenic regions of the genomes studied.
exon-intron boundaries were determined according to the general gt/ag consensus, and intron lengths and discontinuities in the dna sequence were annotated.
the first 600 bp of the 5′-non-coding regions of x. tropicalis adh genes were checked for potential transcription factor binding sites using match  <cit> , based on the transfac database of position weight matrices, using 100% coincidence for the core and 95% for the whole matrix.
in addition, the first 650 bp of the 3′-non-coding regions were screened for polyadenylation signals.
sequence alignment and phylogenetic analyses
sequence analysis and manipulation were carried out using bioedit sequence alignment editor, version  <dig> .
<dig>   <cit> .
multiple sequence alignments were performed with clustal omega  <cit> .
gaps and missing positions were not removed from the alignment and trees were constructed considering partial deletions for the pairwise comparisons.
phylogenetic analyses were conducted using mega version  <dig>  <cit> .
unrooted phylogenetic trees were constructed by neighbour-joining   <cit>  using the jtt  matrix  <cit>  for amino acid distance calculations.
evolutionary rates among sites were considered γ-distributed and the α parameter was calculated with tree-puzzle  <dig>   <cit> .
bootstrap analysis  <cit>  with  <dig> replicates was performed to assess the relative confidence on the topology obtained.
a second tree was constructed following the maximum-likelihood  method  <cit> , by using the phyml  <dig> .
<dig> program  <cit>  included in the macgde software package.
the tree parameters were the same as those used for the nj tree; in this case, the reliability of the inferred phylogeny was assessed by  <dig> bootstrap repetitions.
accession numbers of adh sequences
the accession numbers of the vertebrate adh sequences used in alignments and phylogenetic analyses are listed in table  <dig>  except for those of x. laevis, x. tropicalis, a. carolinensis and p. sinensis adhs, which are provided in tables  <dig> and  <dig>
1
the table lists the accession numbers for the protein sequences, other than those of x. laevis, x. tropicalis, a. carolinensis and p. sinensis, used in alignments and phylogenetic analyses.
1accession numbers refer to uniprot database, unless otherwise indicated.
xenopus laevis
and
xenopus tropicalis
x. laevis
x. tropicalis
all xenopus adhs supported by evidence at the protein, transcript or gene level, are included, together with their accession numbers and expression sites.
a single adh1a gene exists in x. tropicalis while two genes are found in x. laevis.
in contrast, no x. laevis adh <dig> sequence has been found to the date.
accession numbers are taken from uniprot or embl .
expression sites were mostly obtained from unigene , but also from genbank .
for x. laevis, results of expression experiments from our group  and other sources, are also included.
1partial sequence, probably another allele of this gene; 2hoffmann et al.
<cit> ; 3partial sequence, containing frameshift mutations; n.a.
: not available.
a. carolinensis
and
p. sinensis
a. carolinensis
p. sinensis
all the adh classes and genes identified in a. carolinensis  and p. sinensis  genomes, together with their embl accession numbers , are included.
multiple adh <dig> and adh <dig> loci exist in a. carolinensis, while no adh <dig> gene has been found.
in contrast, p. sinensis has a single gene for each class.
RESULTS
isolation, cloning and identification of x. laevis cdnas
with the initial aim of studying the amphibian nadp+-dependent adh <dig> in the development model x. laevis, degenerated primers were designed, based on the r. perezi adh <dig> sequence.
by rt-pcr amplification from a x. laevis stomach cdna pool, four cdnas were cloned and sequenced, and, on the basis of amino acid sequence identity, they were assigned to adh <dig>  adh <dig>  adh <dig> and a novel adh <dig> class.
adh <dig>  adh <dig> and adh <dig> were similar to their r. perezi orthologues and showed the typical residues of each class, while the low sequence identity values  between adh <dig> and other classes pointed it to be a new class.
the adh <dig> cdna was likely to be an allele of the same gene which partial sequence had been reported by hoffmann et al.
<cit> , while adh <dig> was identical to the alleged adh4-like form reported in the same study.
our studies here indicate that xenopus does not possess an adh <dig> ortholog.
since the x. tropicalis genome project provided reliable genomic data from this organism, a parallel study was conducted to identify x. tropicalis adh genes, and the same nomenclature was used for the two xenopus species.
some adh genes appear to be closely related, probably encoding isozymes of the same enzymatic class.
therefore, our gene notation includes an arabic number indicating the adh class, followed by a capital letter corresponding to the encoded isozyme, assigned in ascending order by the gene location in the scaffold.
for those genes that had been previously identified in x. tropicalis genomic studies  <cit> , their names were conserved whenever it was possible.
moreover, putative duplicated genes in x. laevis relative to x. tropicalis were denoted with a “1” or “2” tag after the name field.
according to this nomenclature, subsequent screening of data banks retrieved the following x. laevis adh sequences, as detailed in table 3: four adh <dig> forms ; one form each of adh <dig>  adh <dig> and adh9; two novel sequences that were named adh <dig> ; and a partial sequence similar to that of chicken adh <dig>  the here cloned x. laevis adh <dig> and adh <dig> cdnas were identical to the sequences retrieved while adh <dig> cdna corresponded to adh1b.
the only adh <dig> transcript found in databases showed a nonsense mutation after codon  <dig>  and notably differed from the cloned sequence from gastric tissue.
thus, this new sequence was considered to be a different gene, designed as adh8a, while that previously cloned was named adh8b.
class assignment was mainly based on amino acid identities in pairwise comparisons with other vertebrate adh enzymes , considering an intraclass identity >65% for amphibian adh sequences .
the presence of class-specific residues and phylogenetic relationships were also taken into account for xenopus adh <dig> and adh <dig>  which were considered as separate classes despite their high amino acid identities with adh <dig> enzymes.
expression of x. laevis adhs
northern blot analysis performed on intestine, kidney, liver and stomach from x. laevis, with cdna probes for cloned adh1b, adh <dig> and adh <dig>  showed a  <dig> -kb mrna in positive samples .
adh <dig> transcripts were present in all the tissues analyzed at relatively low levels.
starch gel electrophoresis confirmed its generalized expression and revealed high glutathione-dependent formaldehyde dehydrogenase activity in the ovary as compared with esophagus, stomach and liver .
adh1b expression followed the typical pattern found for adh <dig> genes in vertebrates, as it was differentially expressed in the tissues analyzed , while adh <dig> was only detected in stomach.
moreover, adh8b transcripts were detected by rt-pcr in x. laevis esophagus, stomach and intestine but not in liver , confirming the gastrointestinal location of this enzyme reported in r. perezi  <cit> .
the diffuse band of low molecular weight observed in the liver corresponds to an unspecific amplification product, as confirmed by the lack of nadp+-dependent activity observed in electrophoresed liver extracts .
additional data on the expression profile of the identified adhs was obtained from xenopus est libraries.
although est evidence is not quantitative, it can reflect very low transcript amounts, undetectable by less sensitive methods, but which may be physiologically relevant.
table  <dig> combines the results of localization studies with data obtained from expression libraries of adult tissues and embryonic stages.
chromosomal location and structure of x. tropicalis, a. carolinensis and p. sinensis adh genes
a total of eleven loci encoding adh enzymes  were identified in scaffolds gl <dig>  and gl <dig>  of the x. tropicalis genome assembly  <dig> .
scaffold gl <dig>  comprises the overlapping scaffolds  <dig> and  <dig> from previous version  <dig> , referred in prior publications  <cit> .
scaffold gl <dig>  contains nine genes that constitute the main adh cluster  and one isolated gene , while scaffold gl <dig>  contains another single adh gene , probably located in a different chromosome.
likewise, twelve adh loci  were identified in scaffolds gl <dig>  and gl <dig>  of the a. carolinensis assembly anocar <dig> .
scaffold gl <dig>  contains eleven genes  ascribed to adh <dig> and adh <dig> classes; and a single gene is located in scaffold gl <dig>  .
in the p. sinensis assembly pelsin_ <dig> , four adh loci  were identified in scaffolds jh <dig>  , and jh <dig>  .
therefore, although no adh <dig> locus has been found in a. carolinensis , the analysis of the p. sinensis genome confirms the presence of class ii in reptiles.
sequence data of x. laevis, x. tropicalis, a. carolinensis and p. sinensis adhs were deposited in the european molecular biology laboratory  nucleotide sequence database  <cit> , and accession numbers are provided in tables  <dig> and  <dig>
synteny of amphibian, reptile and human regions containing adh genes is shown in figure  <dig>  scaffolds gl <dig>  of x. tropicalis, gl <dig>  of a. carolinensis, and jh <dig>  and jh <dig>  of p. sinensis are syntenic to human chromosome  <dig>  where all seven human adh genes are closely linked.
on the other hand, x. tropicalis scaffold gl <dig>  may be syntenic to human chromosome  <dig>  arrangement of several orthologous genes flanking the adh loci in x. tropicalis scaffold gl <dig>   indicates a past inversion that isolated adh10a from the main adh cluster, presumably followed by other rearrangements in the same region.
in contrast, reptiles show a single cluster although adh genes are found in both orientations in the a. carolinensis genome.
all x. tropicalis genes contain nine exons and eight introns and the insertion points are those conserved in animal adhs  <cit> .
comparison of the gene structures shows a wide range of intron sizes; therefore this parameter has not been conserved, even among close genes.
the fact that no stop codons or reading frame alterations were detected in the coding sequences, together with evidence from x. tropicalis est collections, support the idea that all these genes are transcriptionally active.
moreover, all the x. tropicalis ests found could be assigned to an adh genomic sequence.
the eleven genes were phylogenetically classified in seven classes: adh <dig> ,  <dig>   <dig>   <dig>   <dig> ,  <dig>  and  <dig> .
furthermore, sequences from uniprot  <cit>  provided supporting evidence and sometimes complementary information, like the first exon of adh <dig>  missing in the assembly but present in sequence q5m7k <dig> .
promoter analysis of x. tropicalis adh genes
regions including 600 bp upstream of the translation start codon were screened for proximal regulatory elements .
putative tata boxes were established for all genes except for adh <dig>  upstream tata box has been described for most adh genes, such as those encoding human and mouse class i  <cit>  and human class ii  <cit> ; whereas the promoters of human and mouse class iii contain gc boxes clustered near the start site  <cit> .
putative transcription factor binding sites were also predicted, although they should be functionally validated in vivo since competition, chromatin structure and other influences are as important as binding affinity.
promoters of adh1a and adh1b contain putative sites for hnf3beta, gata- <dig>  overlapping half-sites for estrogen and retinoid receptors , three ap <dig> sites in adh1a and one site for sp <dig> in adh1b.
positive regulation of the human adh1a gene was reported to be influenced by gata- <dig>  while differences in hnf3beta binding could be related with tissue specificity of adh <dig>  <cit> .
in addition, ap1-responsive genes are susceptible to be negatively regulated by retinoic acid  <cit> .
adh1c promoter has a single site for oct <dig> and reverse sites for hnf3beta and sp <dig>  the adh <dig> promoter contains one nf1-binding site in forward orientation and two reverse sites for gata- <dig>  nf <dig> and ap <dig>  and one for gfi <dig>  the adh <dig> tata-less promoter shows single sites for oct <dig>  c-myb and hnf3beta, a reverse ccaat box and reverse sites for usf, gata- <dig> and ap <dig>  both adh <dig> promoters exhibit a ccaat box, and gata- <dig> , xfd and hnf3beta sites.
their differential traits are cre-bp <dig> and ap <dig> sites in adh8a, and overlapping half-sites for estrogen and retinoid receptors in adh8b.
the adh <dig> promoter has also a ccaat box, and gata- <dig> and xfd sites, in addition to a site for oct <dig>  finally, adh10a promoter has one usf, one chop:c/ebpalpha and three hnf3beta sites, while adh10b shows a putative site for estrogen receptor, and gata- <dig> and ap1-binding sites.
furthermore, single polyadenylation signals were found within the first 650 bp of the 3′-non-coding region of the three adh <dig> genes, adh <dig>  adh <dig> and adh8b; two signals were observed in the case of adh <dig>  adh8a, adh <dig> and adh10b, and up to five signals were located at the 3′-end of adh10a.
sequence analysis and evolutionary relationships
available amino acid sequences of r. perezi, x. laevis and x. tropicalis were aligned  and key residues for substrate and coenzyme binding are summarized in table  <dig>
all available sequences, full-length and partial, are considered.
sequence annotation consists of source organism abbreviation  followed by the assigned class number.
position numbering is based on horse adh1e.
residues at positions 223– <dig>  are involved in the interaction with the extra phosphate group of nadp+ in adh <dig> and determine preference for this coenzyme  <cit> .
xenopus orthologs are closely related phylogenetically, with an intraclass identity of 85-97% .
evidence of x. laevis genome duplication was exclusively found for x. tropicalis adh1a, which corresponds to x. laevis adh1a <dig> and adh1a <dig> genes .
although only partial sequences were found for x. laevis adh1a <dig>  identical key residues in x. tropicalis adh1a , which are not found in x. laevis adh1a <dig>  suggest that the two former sequences would be functionally closer.
the percentage of identity between x. laevis adh1a <dig> and adh1a <dig> is  <dig> % in  <dig> residues.
when rana and xenopus class i sequences were compared, r. perezi adh <dig> showed the highest identity with xenopus adh1b forms  and identical residues at most important positions .
for adh <dig>  the percent identity between the r. perezi and xenopus forms is 71-73% and all possess ser <dig>  ser <dig>  phe <dig>  gly <dig> and leu <dig>
amphibian sequences were also compared to other vertebrate adhs, including those identified in a. carolinensis  and p. sinensis , in unrooted phylogenetic trees .
molecular phylogenetic analysis on the deduced protein sequences using nj and ml estimations produced similar topologies.
for each tree construction, among-site rate heterogeneity was taken into account and confidence in each node was assessed by  <dig> and  <dig> bootstrap replicates, respectively.
in the tree of figure  <dig>  xenopus adh <dig>  adh <dig>  adh <dig>  adh <dig> and adh <dig> sequences cluster with other extant members of their classes, whereas adh <dig> branches separately.
the tree topology pictures the constant nature of class iii, in contrast to the other adh classes.
related to class i, amphibian adh <dig>  adh <dig>  adh <dig> and adh <dig> form a protein cluster presumably derived from a common ancestor.
an important radiation would have occurred in amphibians from a primitive adh <dig> gene, originating the four classes mentioned above, although the order and genes involved in each duplicatory event cannot be ascertained.
likewise, the presence of eight adh <dig> and three adh <dig> forms in a. carolinensis suggests that specific duplications could have occurred in lizards but not in turtles, as these organisms belong to different reptilian lineages.
percent identity within anole class i sequences ranges from  <dig> % to  <dig> %, and adh1a and adh1b show the highest identity with uromastyx  adh1a  and adh1b , respectively.
within anole adh <dig> sequences, the percent identity is around 71-78%.
interestingly, multiple alignments reveal that all class i enzymes from reptiles to humans, as well as other classes derived from amniote adh <dig>  such as adh <dig> and adh <dig>  show a deletion at position  <dig> with respect to amphibian adh <dig> proteins and the remaining adh classes .
phylogenetic data supporting the results of this study  are available from the treebase repository .
discussion
a total of eleven adh genes with a conserved structure have been identified in the x. tropicalis genome, and grouped in seven enzyme classes: adh  <dig> ,  <dig>   <dig>   <dig>   <dig> ,  <dig>  and  <dig> .
these loci are distributed in two scaffolds, one containing the main discontinuous adh cluster, syntenic to human 4q21- <dig>  but broken by several rearrangements, and the other showing the single adh1a locus.
the amphibian adh system represents a unique organization among tetrapods since sequencing data and comparative analysis of genomes describe single clusters in human, rat, mouse and chicken  <cit> .
genes similar to those of x. tropicalis have been identified in x. laevis, indicating that the multiplicity of adh forms was present prior to the divergence of the two species.
duplication of the x. laevis genome   <cit>  affected a great number of gene families, such as those of globin or α-actin  <cit> , but the only adh duplicates found to date correspond to the x. tropicalis adh1a gene, which were named adh1a <dig> and adh1a <dig> in x. laevis.
this suggests that many adh duplicated loci could have been lost.
nevertheless, further identification of other gene duplicates in x. laevis should not be discarded.
in this regard, activity staining of hepatic adhs revealed the existence of two polymorphic genes coding for ethanol dehydrogenase subunits that did not heterodimerize and were placed in separate genetic linkage groups  <cit> .
in the following description of the amphibian adh properties, we include functional features of forms not yet characterized, predicted from the wide information of the structure/function relationships available for the adh family.
however, the proposed functions have to be confirmed by the expression and kinetic characterization of the novel enzymes, especially adh <dig> and adh <dig>
ancient forms of vertebrate adh classes in amphibians adh1
amphibian class adh <dig> clusters with the novel amphibian classes adh <dig>  adh <dig> and adh <dig>  since all of them derive from a primitive adh <dig> gene ancestor, also common to the amniote class i line.
later, adh <dig> duplications generated adh1a, 1b and 1c ; adh8a and 8b; and adh10a and 10b from their corresponding ancestors.
xenopus adh1a is the most similar to other vertebrate adh <dig> enzymes, whereas xenopus adh1b shows the highest identity with r. perezi adh <dig>  xenopus adh1a and adh1b show arg <dig>  his <dig>  phe <dig> and phe <dig>  typical class i residues that are associated with ethanol dehydrogenase activity.
the substrate-binding pocket of r. perezi adh1b is extremely hydrophobic and space-restricted, resulting in low km values for aliphatic alcohols, it has wide substrate specificity and is moderately active with retinoids  <cit> .
in adh1a, smaller substrate-binding residues val <dig> and val <dig> anticipate higher km values for this isozyme, and substitution by his <dig>  suggests an increased rate of nad+ dissociation and higher kcat values.
x. laevis adh1a <dig> has many atypical residues, such as gly <dig> or thr <dig>  which suggest an alternative proton-relay pathway in comparison with all the other class i enzymes, showing his <dig>  moreover, voluminous residues phe <dig>  met <dig> and met <dig> would increase hydrophobicity and would narrow the substrate cleft even more than in adh1b.
these residue exchanges predict different substrate specificity and suggest that adh1a <dig> may have acquired a new function after gene duplication, while adh1a <dig> would have maintained the original one.
adh1c has unique features among substrate-binding residues.
at position  <dig>  the lack of an aromatic ring expands the substrate cleft and permits the accommodation of large substrates, as in human adh1a and chicken adh <dig>  <cit> .
in contrast, an unusual phe <dig> would narrow the entrance, although still may allow productive binding of retinoids as occurs in x. laevis adh8b  <cit> .
these features predict that adh1c binds large alcohol substrates better than ethanol.
substitutions in the coenzyme-binding site, in relation to amniote class i, are his <dig> instead of arg , asp <dig>  and asn/thr <dig>  which could weaken the coenzyme binding and increase kcat values.
expression pattern of amphibian class i in adult and embryonic tissues resembles that of other vertebrates, and transcripts of adh1b are abundant in the developing tadpole .
regarding reptiles, specific duplications of the adh <dig> gene occurred in lizards.
anole adh1a is the ortholog of uromastyx adh1a and clusters with other known reptilian and avian class i enzymes, while anole adh1b is the ortholog of uromastyx adh1b and clusters with the rest of anole class i forms.
thus, a. carolinensis adh1c-1h genes may have arisen from further tandem duplications of adh1b in this organism, although the existence of additional adh <dig> genes in uromastyx cannot be discarded.
interestingly, a. carolinensis adh1d, adh1e and adh1g share the residues gln-arg-ser instead of the typical class i triad asp-ile-gln at positions 223– <dig>  which interact with the adenosine moiety of the coenzyme.
similar residues are found in nadp+-dependent mdr enzymes such as sulfolobus solfataricus glucose dehydrogenase , xilella fastidiosa cinnamyl alcohol dehydrogenase  or saccharomyces cerevisiae adh <dig> , among others, thus suggesting a higher preference for this coenzyme.
given the phylogenetic distance between these adh <dig> forms and amphibian class adh <dig>  this suggests that the nadp+ specificity could have arisen at least twice during the vertebrate adh evolution.
adh2
amphibians are the most ancient organisms that possess a class ii enzyme.
therefore, the duplicatory event that generated adh <dig> from adh <dig> can be placed between fish/tetrapod and amphibian/amniote splits,  <dig> to  <dig> mya  <cit> .
moreover, x. tropicalis adh <dig> already has the four-residue insertion, considered as the most distinctive trait of class ii enzymes.
the phylogenetic proximity of amphibian and avian adh <dig> enzymes, in spite of the overall variability of this class, predicts similar structure and kinetic behaviour.
the ostrich enzyme has been described as a mixed-class, structurally similar to mammalian class ii but resembling class-i kinetic properties, since it is notably active with short-chain alcohol substrates such as ethanol.
ostrich adh <dig> shares  <dig> %,  <dig> % and  <dig> % identity with turtle, frog and human adh <dig>  respectively.
these four sequences show ser/thr <dig>  phe <dig>  tyr <dig>  leu <dig>  phe <dig>  val <dig>  ile/leu <dig> and phe <dig> in the substrate-binding site ; and also ser <dig> and ser <dig>  which distinguish human class ii from class i  <cit> .
all the residues involved in the substrate interaction are almost identical in frog, turtle and ostrich adh <dig>  moreover, the three enzymes show arg <dig>  ser/thr <dig>  his <dig> and ile <dig> at the coenzyme-binding site, the same residues which are found in human adh1b <dig>  concluding that the primitive class ii forms may share common kinetic properties with class i enzymes.
human adh1b <dig> is included for comparison with all available adh <dig> enzymes.
the four-residue insertion of class ii enzymes is also shown.
residues of the coenzyme-binding site  are common to human adh1b <dig>  residues at positions 223-224- <dig> are d-i-n in all cases.
*human adh <dig> is polymorphic at position  <dig> and shows ser or thr  <cit> .
class ii can be divided in two structurally and functionally distinct subgroups  <cit> .
the first one exhibits a low activity with ethanol and comprises mouse and rat adh <dig>  both showing pro <dig>  and rabbit adh2b, which lacks a his residue at both positions  <dig> or  <dig> .
in contrast, the second group is constituted by rabbit adh2a and amphibian, reptilian and avian adh <dig>  all of them possessing his51; and human, marmoset and bovine adh <dig>  which show his <dig> .
these forms may share not only the ethanol dehydrogenase activity but also the ability of metabolizing retinoids, as reported for human adh <dig>  <cit> .
adh3
x. laevis and x. tropicalis adh <dig> sequences show the  <dig> functionally important residues strictly conserved in class iii enzymes from reptiles to mammals  <cit> .
adh <dig> is a glutathione-dependent formaldehyde dehydrogenase, as seen spectrophotometrically for the purified enzyme of r. perezi <cit>  and by activity staining of electrophoresed tissue homogenates from r. perezi and x. laevis.
expression of amphibian adh <dig> is detected in every stage and tissue studied, although it is more abundant in some organs such as the ovary, suggesting that oocytes may store large amounts of maternal adh <dig> for its later use during the embryonic development.
the maternal origin of adh <dig> mrna has been previously described in drosophila  <cit>  and zebrafish  <cit>  embryos.
adh7
the novel reptilian genomic sequences supported the class assignment of x. tropicalis adh <dig>  sharing identity percentages of  <dig> % and  <dig> % with turtle adh <dig> and anole adh7b, respectively.
as listed in table  <dig>  all adh <dig> enzymes show thr <dig>  involved in the stereospecificity for secondary alcohols; a small residue such as cys at position  <dig> , indicative of high km values for ethanol and correct positioning of steroid substrates  <cit> ; phe <dig> and leu <dig> ; and similar coenzyme-binding residues.
positions  <dig> to  <dig> are almost identical, and his <dig> and trp <dig> are common for all adh <dig> enzymes.
these two positions were reported to affect the conformation of the loop 112– <dig>  widening the entrance of the substrate-binding site and conferring to adh <dig> the ability to oxidize large hydrophobic alcohols  <cit> .
residues  <dig> and  <dig>  involved in the rearrangement of the loop 112– <dig> at the entrance of the substrate-binding pocket and present in all adh <dig> forms, are also included.
residues at positions 223-224- <dig> are d-i-n in all cases.
among the three forms identified in a. carolinensis, adh7b is the most similar to turtle, chicken and frog adh <dig>  with identity percentages of  <dig> %,  <dig> % and  <dig> %, respectively.
already present in amphibians, adh <dig> appeared between the tetrapod/fish and the amniote/amphibian splits, 450– <dig> mya  <cit> .
on the other hand, the common position of turtle and chicken adh <dig> and vertebrate adh5-adh <dig> loci within the adh cluster  <cit> , together with the fact that they do not coexist in any organism, suggests that the loss of adh <dig> might have occurred close to the origin of adh <dig> and adh <dig>  or even in the same genetic event.
novel adh classes in amphibians adh8
a total of five members are now known from the nadp+-dependent class viii: adh <dig> from r. perezi, and adh8a and adh8b from both x. laevis and x. tropicalis.
they show the conserved triad gly223-ser/thr224-gln/his <dig> that interacts with the 2′-phosphate of the adenosine moiety of nadp+  <cit> .
despite their different substrate-binding sites, especially concerning large hydrophobic residues, both x. laevis adh8b and r. perezi adh <dig> reduce retinaldehyde and medium-chain aldehydes  <cit> .
one major difference is the substitution of phe <dig> by cys that correlates with the poor ethanol oxidizing activity of x. laevis adh8b .
residues gly <dig>  ser <dig> and ser <dig> would determine the proposed catalytic mechanism and proton-relay pathway of r. perezi adh <dig>  <cit> .
while gly <dig> is conserved in all adh <dig> members, ser <dig> and ser <dig>  also found in adh8a, are substituted by thr <dig> and ala <dig> in adh8b enzymes.
remarkably, several deletions exist in adh <dig> with respect to amphibian class i. the first one is found at position  <dig> of r. perezi adh <dig> and both xenopus adh8b enzymes, and it may account to some extent for the wide substrate pocket observed in r. perezi adh <dig>  <cit> .
the second one, at position  <dig>  is common for all adh <dig> sequences.
and the third, located at position  <dig>  is only found in xenopus adh8b enzymes.
regarding coenzyme-interacting positions, the presence of gly <dig> and the lack of a typical hydrophobic residue in position  <dig>  usually val or ile, suggest a lower affinity for the coenzyme and increased kcat values for adh <dig> enzymes.
adult expression pattern of adh <dig> includes the gastrointestinal tract and skin, where it may participate in cell differentiation through regulation of retinoic acid levels acting as a retinaldehyde reductase  <cit> .
in embryos, adh <dig> expression is also observed after neurulation, although est evidences are too scarce to suggest a possible function .
adh8a and adh8b promoters are closely related, both exhibiting a putative ccaat box, and common hnf-3beta, xfd and gata-binding sites.
adh9
x. laevis and x. tropicalis adh <dig> are the only members known from this novel class, and none of them has been characterized at the protein level.
these enzymes share a percentage of amino acid identity lower than 60% with any other adh, and show many special sequence features, such as cys <dig>  met <dig>  phe <dig>  val <dig>  met <dig>  and phe318; together with his residues at both positions  <dig> and  <dig>  his <dig> indicates the same proton-relay pathway as in class i enzymes, while his <dig> suggests rapid coenzyme dissociation.
asp <dig> at the cofactor-binding site indicates nad+-dependence.
cys <dig>  phe <dig>  met <dig>  and phe <dig> predict a substrate pocked enlarged at its inner part, but narrow and hydrophobic at the middle and the entrance, similar to that of adh <dig>  substrate preferences may be large substrates rather than ethanol, but probably not steroids.
northern blot studies detect adh <dig> transcripts in adult stomach, esophagus and skin, while discard the expression of the enzyme at embryonic stages .
colocalization with adh <dig>  in spite of their sequence divergence and different cofactor specificity, may obey to the common regulatory elements found in their gene promoters, and it is consistent with the adjacent chromosomal location of their genes.
adh <dig> was initially described as an adh4-like form in x. laevis, likely because its tissue localization  <cit> .
based on phylogenetic analyses and class-specific sequence signatures, it is now clear that adh <dig> constitutes a separate class and thus adh <dig> is not present in amphibians.
the absence of adh <dig> forms in reptiles and birds, and its presence in marsupials  <cit>  supports its emergence at the origin of mammals .
adh10
two isoenzymatic forms occur in this class, adh10a and adh10b, which are closely related to adh <dig>  common residues of adh <dig> and adh <dig> enzymes are ser <dig>  phe <dig>  met <dig> and leu <dig>  for substrate interaction; and arg <dig>  his <dig> and leu <dig>  for coenzyme binding .
val <dig> and his <dig> are particular of adh <dig>  substitution of typical ile <dig> by smaller val can affect the strength of coenzyme interaction, and substitution of arg <dig> by his was suggested to increase kcat values in human adh <dig>  <cit> .
three residue exchanges characterize adh10b: val <dig>  phe <dig> and arg <dig>  val <dig> results in a wider bottom of the substrate pocket and it has not been found in any other adh.
phe <dig>  present in most class ii enzymes, narrows the middle region and increases its hydrophobicity, but hydrophilic arg <dig> should compensate for this fact.
adh <dig> is the only class with a charged residue at position  <dig>  also interesting is the basic residue found at position  <dig> which, similarly to arg <dig> in class iii enzymes  <cit> , could contribute to a substrate-binding site with a wider entrance and higher volume.
a deletion at position  <dig> of x. tropicalis adh10b could also participate in this rearrangement.
the substrate pocket of adh10b with a widened entrance and inner part  could accommodate large substrates, such as steroids, provided that phe <dig> was not a major steric constraint.
ser <dig> can also be found in horse adh1s and human adh1c, both able to oxidize steroids.
adrenal and gonadal steroids are therefore proposed as adh <dig> substrates, in agreement with its predominant expression in xenopus mesonephric kidney and testis, and the presence of a putative site for estrogen receptor in the adh10b promoter.
CONCLUSIONS
in conclusion, the complex xenopus adh system is composed of the vertebrate classes adh <dig>  adh <dig>  adh <dig> and adh <dig>  together with novel class i-derived enzymes, adh <dig>  adh <dig> and adh <dig>  exclusively found in amphibians.
adh <dig> is not present in amphibians and reptiles.
the study of the ancient forms of adh <dig> and adh <dig>  also found in reptiles, led to significant conclusions about the evolutionary history of the adh family , whereas the special features showed by the novel forms described herein point to the acquisition of new functions following the adh gene family expansion occurred in amphibians.
availability of supporting data
the data sets supporting the results of this article are included within the article and its additional files.
abbreviations
adh: alcohol dehydrogenase; ap1: activator protein 1; camp: cyclic adenosine monphosphate; c/ebpalpha: ccaat-enhancer binding protein alpha; chop: c/ebp homologous protein; c-myb: mieloblastosis viral oncogene homolog; cre-bp1: camp-responsive element binding protein; est: expressed sequence tag; fdh: formaldehyde dehydrogenase; gata-1: gata-binding protein 1; gfi1: growth factor independence 1; hnf3beta: hepatic nuclear factor  <dig> beta; mdr: medium-chain dehydrogenase/reductase; nad: nicotinamide adenine dinucleotide ; nf1: nuclear factor 1; oct1: octamer-binding factor 1; rt-pcr: reverse transcription-polymerase chain reaction; sds: sodium dodecyl sulfate; usf: upstream stimulating factor; ssp1: stimulating protein 1; xfd: xenopus fork head domain.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
eb carried out the cloning and detection of x. laevis adh forms, identified adh sequences in genomic and expression databases, performed comparative analyses, constructed nj trees, and drafted the manuscript.
ra calculated evolutionary parameters of vertebrate adh, constructed ml trees, and participated in drafting of the manuscript.
gd provided partial sequences of x. laevis adh8b and adh <dig> cdnas, and critically reviewed the manuscript.
xp participated in the design and coordination of the study.
jf conceived the study, participated in its design and coordination, and provided critical review of the manuscript.
all authors have read and approved the final manuscript.
supplementary material
additional file 1
percentage of amino acid identity between
xenopus tropicalis
and representative vertebrate adh sequences.
click here for file
additional file 2
percentage of amino acid identity between amphibian adh sequences.
click here for file
additional file 3
xenopus tropicalis adh1a cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 4
xenopus tropicalis adh1b cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 5
xenopus tropicalis adh1c cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 6
xenopus tropicalis adh <dig> cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 7
xenopus tropicalis adh <dig> cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 8
xenopus tropicalis adh <dig> cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
click here for file
additional file 9
xenopus tropicalis adh8a cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 10
xenopus tropicalis adh8b cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 11
xenopus tropicalis adh <dig> cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 12
xenopus tropicalis adh10a cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 13
xenopus tropicalis adh10b cdna sequence.
the sequence includes the translated coding exons, intron flanking regions , and the proximal promoter  and 3′-untranslated region  with predicted regulatory elements.
putative tata boxes and polyadenylation signals are in bold and underlined.
putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics ; and the orientation  is given in parentheses.
click here for file
additional file 14
alignment of amphibian adh amino acid sequences.
click here for file
additional file 15
alignment of vertebrate adhs included in the phylogenetic tree.
click here for file
acknowledgements
this work was supported by grants from the spanish ministerio de economía y competitividad  and generalitat de catalunya .
we thank dr. ines hoffmann for her preliminary work on x. laevis adh1b and adh <dig>  we are also grateful to dr. salvador bartolomé, from the laboratori de luminiscència i espectroscòpia de biomolècules of the universitat autònoma de barcelona, for image capture and processing.

 vertebrate alcohol dehydrogenases are dimeric zinc-containing enzymes with a 40-kda subunit and 373– amino acid residues. structurally, they belong to the medium-chain dehydrogenase/reductase superfamily. adhs catalyze the reversible oxidation of a wide range of alcohol substrates to the corresponding aldehydes or ketones, and can be grouped in eight enzyme classes, according to their primary structure and function. the human adh gene nomenclature used throughout the text is the enzyme class-based nomenclature currently used for vertebrate adh and differs from that approved by the human genome organization gene nomenclature committee, as the former facilitates comparisons with adhs from other mammals and lower vertebrate species. tandem gene duplications gave rise to the multiplicity of forms in the adh family, including isoenzymes and allelic forms in particular lineages. adh is the most ancient form and the only class present before chordates. it is a glutathione-dependent formaldehyde dehydrogenase, a highly conserved and ubiquitous detoxifying enzyme. duplication of the ancestral adh gene near the agnathan/gnathostome split originated adh which evolved independently in the fish and tetrapod lines becoming the classical hepatic ethanol dehydrogenase. in tetrapods, a second duplication of the gene coding for adh generated adh also hepatic but active at higher ethanol concentrations. close to the origin of mammals, adh duplicated giving rise to adh a highly retinoid-active enzyme present in eye, skin and gastric tissues. the most evolutionarily recent classes in mammals are adh and adh, the latter being absent in primates. these two classes, identified at dna level, are the most divergent within mammalian adhs. on the other hand, adh previously named adh-f due to its fetal expression, is a steroid/retinoid dehydrogenase that was first described in chicken. finally, adh is a unique nadp+-dependent adh isolated from the stomach of the frog rana perezi and its proposed function is the reduction of retinaldehyde to retinol. studies on amphibian adh genetics have been scarce. isozyme patterns of x. laevis liver ethanol dehydrogenase suggested the existence of two polymorphic genes encoding adh subunits that did not form heterodimers and were located in different linkage groups. the enzymes adh adh and adh from the frog rana perezi were purified and characterized by our group, and the adh and adh proteins were also sequenced. the cloning of the cdna of r. perezi adh allowed to perform mutagenesis studies on coenzyme specificity and to obtain the crystal structure of the enzyme. partial cdnas of x. laevis adh and an adh4-like form were cloned and used for expression analysis in embryonic and adult tissues. later, two reviews on mdr-adh evolution, which included genomic data, provided some partial information on the amphibian adh system. here the adh system of the development model frog x. laevis has been further investigated, especially the retinaldehyde-active adh tetraploidy of x. laevis was a handicap for genetic studies, thus the present work was restricted to expression patterns and extended with additional information from expressed sequence tag collections. on the other hand, its diploid relative x. tropicalis, the subject of the only amphibian genome project, was used for a genomic approach to the amphibian adh family. since the reptile genome of the anole lizard and the turtle had been sequenced at the time of the study, the adh gene sequences from these organisms could be identified and used in phylogenetic analyses and genomic comparisons. the joint analysis of xenopus genome-wide data and the results of the expression analysis described herein provide an integrated view of the amphibian adh system. moreover, since this organism occupies a key phylogenetic position, this work provides insight into the molecular evolution of the adh gene family in vertebrates. methods animal tissues tissues were obtained from adult x. laevis females provided by horst kähler. the animals were kept in an ice bath for 15 min to diminish their metabolism prior to euthanasia. after decapitation, the head was immersed in liquid nitrogen to assure total unconsciousness, as recommended. the organs were then removed, cleaned, rinsed in distilled water and stored at –80°c. prior to analysis, frozen tissues were pulverized in liquid nitrogen and homogenized. this study was approved by the ethical committee of the universitat autònoma de barcelona. isolation and cloning of x. laevis cdnas stomach poly+ rna was isolated with the “quickprep micro mrna purification kit” and a cdna pool was synthesized using the “first strand cdna synthesis kit for rt-pcr ” with the oligo 17riro primer adaptor. nested-pcrs combined degenerate primers based on r. perezi adh and amplification products were cloned into pbluescript ii sk and sequenced. the partial cloned sequences were later identified as adh1b and adh whereas the adh8b and adh partial cdnas were obtained as described. the 3′-ends were amplified by rapid amplification of cdna ends combining the adaptor-specific primers ro and ri with specific forward primers, and then cloned and sequenced as described above. x. laevis adhs 1 2 1degenerate nucleotides are r, w, h and i. 2numbering refers to amino acid residues unless otherwise indicated. northern blot analysis of x. laevis adh1b, adh and adh9 total rna from stomach, liver, kidney and intestine was isolated by the acid guanidinium thiocyanate method. samples were electrophoresed on a 1% agarose gel containing  m formaldehyde and transferred onto a nylon filter. 18s rrna and 28s rrna were used to check the integrity and amount of loaded rna and to estimate the size of the rna hybrids. probes included x. laevis adh1b, adh and adh cdnas, labeled with dctp by a random hexamer-primed method using the “prime-a-gene labeling system” except for the klenow enzyme. after a 45-min prehybridization at 60°c in  m sodium phosphate, ph , 1 mm edta, 1% bsa and 1% sds, filters were hybridized for 18–24 h at 60°c in the presence of 106 cpm/ml of radiolabeled probe. final 30-min washes at 60°c were performed twice in 40 mm sodium phosphate, ph , 1 mm edta and 1% sds. autoradiography was carried out at –80°c for 2–5 days with hyperfilm-mp using an intensifying screen. hybridization signals were then scanned in a bio-rad gs- imaging densitometer. rt-pcr of x. laevis adh8b cdna pools from esophagus, stomach, intestine and liver were prepared from 3–8 μg of total rna using the adh8b-specific reverse outer primer described in table  first pcr amplification combined this primer with the adh8b forward outer primer, and a second round used the inner primer pair, generating a 603-bp adh8b cdna fragment. starch gel electrophoresis and activity staining of x. laevis adh and adh3 tissues were homogenized in 30 mm tris–hcl, ph ,  mm dithiothreitol, centrifuged at ×g for 30 min and supernatants were used for analysis by starch gel electrophoresis. total protein was determined by the bio-rad protein assay method, using bovine serum albumin as standard. in order to discriminate between the nad+-dependent classes adh and adh gel slices were stained for adh activity with  m 2-buten-1-ol and  mm nad+, to mainly detect adh and for glutathione-dependent fdh activity with  mm formaldehyde and 1 mm glutathione, to specifically stain adh identification of xenopus adh sequences in protein and expression databases several adh protein sequences from both xenopus species were gathered at uniprot, first by name search and then by exploring clusters with 90% or 50% identity. ests of x. laevis and x. tropicalis were obtained by blast search, using x. laevis and other vertebrate adhs as protein queries against translated nucleotide sequences, at the sanger institute x. tropicalis est project and tgi gene indices websites. additional information on the expression sites of xenopus adh clustered transcripts was obtained by name search at the ncbi unigene data bank. identification and analysis of adh genes in xenopus tropicalis, anolis carolinensis and pelodiscus sinensis genomes x. tropicalis genome assembly, a. carolinensis anocar and p. sinensis pelsin_ were interrogated with blast-based search tools to identify possible adh gene locations. for x. tropicalis, tblastn searches were undertaken at the joint genome institute x. tropicalis genome assembly website using x. laevis adhs as protein queries. for a. carolinensis and p. sinensis, tblastn searches were conducted using the ensembl genome browser to compare x. tropicalis adhs against genomic databases, allowing some local mismatch. protein blat analyses using the ucsc web browser were also performed and the same scaffolds producing significant alignments were obtained. these adh-containing scaffolds were then exported with ensembl, 3-frame translated in both orientations, and manually screened for the presence of adh genes either using exon sequences derived from x. tropicalis ests or consensus adh sequences. the ensembl genome browser was also used to align syntenic regions of the genomes studied. exon-intron boundaries were determined according to the general gt/ag consensus, and intron lengths and discontinuities in the dna sequence were annotated. the first 600 bp of the 5′-non-coding regions of x. tropicalis adh genes were checked for potential transcription factor binding sites using match, based on the transfac database of position weight matrices, using 100% coincidence for the core and 95% for the whole matrix. in addition, the first 650 bp of the 3′-non-coding regions were screened for polyadenylation signals. sequence alignment and phylogenetic analyses sequence analysis and manipulation were carried out using bioedit sequence alignment editor, version.. multiple sequence alignments were performed with clustal omega. gaps and missing positions were not removed from the alignment and trees were constructed considering partial deletions for the pairwise comparisons. phylogenetic analyses were conducted using mega version. unrooted phylogenetic trees were constructed by neighbour-joining using the jtt matrix for amino acid distance calculations. evolutionary rates among sites were considered γ-distributed and the α parameter was calculated with tree-puzzle. bootstrap analysis with replicates was performed to assess the relative confidence on the topology obtained. a second tree was constructed following the maximum-likelihood method, by using the phyml. program included in the macgde software package. the tree parameters were the same as those used for the nj tree; in this case, the reliability of the inferred phylogeny was assessed by bootstrap repetitions. accession numbers of adh sequences the accession numbers of the vertebrate adh sequences used in alignments and phylogenetic analyses are listed in table  except for those of x. laevis, x. tropicalis, a. carolinensis and p. sinensis adhs, which are provided in tables  and 1 the table lists the accession numbers for the protein sequences, other than those of x. laevis, x. tropicalis, a. carolinensis and p. sinensis, used in alignments and phylogenetic analyses. 1accession numbers refer to uniprot database, unless otherwise indicated. xenopus laevis and xenopus tropicalis x. laevis x. tropicalis all xenopus adhs supported by evidence at the protein, transcript or gene level, are included, together with their accession numbers and expression sites. a single adh1a gene exists in x. tropicalis while two genes are found in x. laevis. in contrast, no x. laevis adh sequence has been found to the date. accession numbers are taken from uniprot or embl. expression sites were mostly obtained from unigene, but also from genbank. for x. laevis, results of expression experiments from our group and other sources, are also included. 1partial sequence, probably another allele of this gene; 2hoffmann et al.; 3partial sequence, containing frameshift mutations; n.a.: not available. a. carolinensis and p. sinensis a. carolinensis p. sinensis all the adh classes and genes identified in a. carolinensis and p. sinensis genomes, together with their embl accession numbers, are included. multiple adh and adh loci exist in a. carolinensis, while no adh gene has been found. in contrast, p. sinensis has a single gene for each class.  isolation, cloning and identification of x. laevis cdnas with the initial aim of studying the amphibian nadp+-dependent adh in the development model x. laevis, degenerated primers were designed, based on the r. perezi adh sequence. by rt-pcr amplification from a x. laevis stomach cdna pool, four cdnas were cloned and sequenced, and, on the basis of amino acid sequence identity, they were assigned to adh adh adh and a novel adh class. adh adh and adh were similar to their r. perezi orthologues and showed the typical residues of each class, while the low sequence identity values between adh and other classes pointed it to be a new class. the adh cdna was likely to be an allele of the same gene which partial sequence had been reported by hoffmann et al., while adh was identical to the alleged adh4-like form reported in the same study. our studies here indicate that xenopus does not possess an adh ortholog. since the x. tropicalis genome project provided reliable genomic data from this organism, a parallel study was conducted to identify x. tropicalis adh genes, and the same nomenclature was used for the two xenopus species. some adh genes appear to be closely related, probably encoding isozymes of the same enzymatic class. therefore, our gene notation includes an arabic number indicating the adh class, followed by a capital letter corresponding to the encoded isozyme, assigned in ascending order by the gene location in the scaffold. for those genes that had been previously identified in x. tropicalis genomic studies, their names were conserved whenever it was possible. moreover, putative duplicated genes in x. laevis relative to x. tropicalis were denoted with a “1” or “2” tag after the name field. according to this nomenclature, subsequent screening of data banks retrieved the following x. laevis adh sequences, as detailed in table 3: four adh forms; one form each of adh adh and adh9; two novel sequences that were named adh; and a partial sequence similar to that of chicken adh the here cloned x. laevis adh and adh cdnas were identical to the sequences retrieved while adh cdna corresponded to adh1b. the only adh transcript found in databases showed a nonsense mutation after codon and notably differed from the cloned sequence from gastric tissue. thus, this new sequence was considered to be a different gene, designed as adh8a, while that previously cloned was named adh8b. class assignment was mainly based on amino acid identities in pairwise comparisons with other vertebrate adh enzymes, considering an intraclass identity >65% for amphibian adh sequences. the presence of class-specific residues and phylogenetic relationships were also taken into account for xenopus adh and adh which were considered as separate classes despite their high amino acid identities with adh enzymes. expression of x. laevis adhs northern blot analysis performed on intestine, kidney, liver and stomach from x. laevis, with cdna probes for cloned adh1b, adh and adh showed a -kb mrna in positive samples. adh transcripts were present in all the tissues analyzed at relatively low levels. starch gel electrophoresis confirmed its generalized expression and revealed high glutathione-dependent formaldehyde dehydrogenase activity in the ovary as compared with esophagus, stomach and liver. adh1b expression followed the typical pattern found for adh genes in vertebrates, as it was differentially expressed in the tissues analyzed, while adh was only detected in stomach. moreover, adh8b transcripts were detected by rt-pcr in x. laevis esophagus, stomach and intestine but not in liver, confirming the gastrointestinal location of this enzyme reported in r. perezi. the diffuse band of low molecular weight observed in the liver corresponds to an unspecific amplification product, as confirmed by the lack of nadp+-dependent activity observed in electrophoresed liver extracts. additional data on the expression profile of the identified adhs was obtained from xenopus est libraries. although est evidence is not quantitative, it can reflect very low transcript amounts, undetectable by less sensitive methods, but which may be physiologically relevant. table  combines the results of localization studies with data obtained from expression libraries of adult tissues and embryonic stages. chromosomal location and structure of x. tropicalis, a. carolinensis and p. sinensis adh genes a total of eleven loci encoding adh enzymes were identified in scaffolds gl and gl of the x. tropicalis genome assembly. scaffold gl comprises the overlapping scaffolds and from previous version, referred in prior publications. scaffold gl contains nine genes that constitute the main adh cluster and one isolated gene, while scaffold gl contains another single adh gene, probably located in a different chromosome. likewise, twelve adh loci were identified in scaffolds gl and gl of the a. carolinensis assembly anocar. scaffold gl contains eleven genes ascribed to adh and adh classes; and a single gene is located in scaffold gl. in the p. sinensis assembly pelsin_, four adh loci were identified in scaffolds jh, and jh. therefore, although no adh locus has been found in a. carolinensis, the analysis of the p. sinensis genome confirms the presence of class ii in reptiles. sequence data of x. laevis, x. tropicalis, a. carolinensis and p. sinensis adhs were deposited in the european molecular biology laboratory nucleotide sequence database, and accession numbers are provided in tables  and synteny of amphibian, reptile and human regions containing adh genes is shown in figure  scaffolds gl of x. tropicalis, gl of a. carolinensis, and jh and jh of p. sinensis are syntenic to human chromosome where all seven human adh genes are closely linked. on the other hand, x. tropicalis scaffold gl may be syntenic to human chromosome arrangement of several orthologous genes flanking the adh loci in x. tropicalis scaffold gl indicates a past inversion that isolated adh10a from the main adh cluster, presumably followed by other rearrangements in the same region. in contrast, reptiles show a single cluster although adh genes are found in both orientations in the a. carolinensis genome. all x. tropicalis genes contain nine exons and eight introns and the insertion points are those conserved in animal adhs. comparison of the gene structures shows a wide range of intron sizes; therefore this parameter has not been conserved, even among close genes. the fact that no stop codons or reading frame alterations were detected in the coding sequences, together with evidence from x. tropicalis est collections, support the idea that all these genes are transcriptionally active. moreover, all the x. tropicalis ests found could be assigned to an adh genomic sequence. the eleven genes were phylogenetically classified in seven classes: adh,, and. furthermore, sequences from uniprot provided supporting evidence and sometimes complementary information, like the first exon of adh missing in the assembly but present in sequence q5m7k. promoter analysis of x. tropicalis adh genes regions including 600 bp upstream of the translation start codon were screened for proximal regulatory elements. putative tata boxes were established for all genes except for adh upstream tata box has been described for most adh genes, such as those encoding human and mouse class i and human class ii; whereas the promoters of human and mouse class iii contain gc boxes clustered near the start site. putative transcription factor binding sites were also predicted, although they should be functionally validated in vivo since competition, chromatin structure and other influences are as important as binding affinity. promoters of adh1a and adh1b contain putative sites for hnf3beta, gata- overlapping half-sites for estrogen and retinoid receptors, three ap sites in adh1a and one site for sp in adh1b. positive regulation of the human adh1a gene was reported to be influenced by gata- while differences in hnf3beta binding could be related with tissue specificity of adh. in addition, ap1-responsive genes are susceptible to be negatively regulated by retinoic acid. adh1c promoter has a single site for oct and reverse sites for hnf3beta and sp the adh promoter contains one nf1-binding site in forward orientation and two reverse sites for gata- nf and ap and one for gfi the adh tata-less promoter shows single sites for oct c-myb and hnf3beta, a reverse ccaat box and reverse sites for usf, gata- and ap both adh promoters exhibit a ccaat box, and gata-, xfd and hnf3beta sites. their differential traits are cre-bp and ap sites in adh8a, and overlapping half-sites for estrogen and retinoid receptors in adh8b. the adh promoter has also a ccaat box, and gata- and xfd sites, in addition to a site for oct finally, adh10a promoter has one usf, one chop:c/ebpalpha and three hnf3beta sites, while adh10b shows a putative site for estrogen receptor, and gata- and ap1-binding sites. furthermore, single polyadenylation signals were found within the first 650 bp of the 3′-non-coding region of the three adh genes, adh adh and adh8b; two signals were observed in the case of adh adh8a, adh and adh10b, and up to five signals were located at the 3′-end of adh10a. sequence analysis and evolutionary relationships available amino acid sequences of r. perezi, x. laevis and x. tropicalis were aligned and key residues for substrate and coenzyme binding are summarized in table  all available sequences, full-length and partial, are considered. sequence annotation consists of source organism abbreviation followed by the assigned class number. position numbering is based on horse adh1e. residues at positions 223– are involved in the interaction with the extra phosphate group of nadp+ in adh and determine preference for this coenzyme. xenopus orthologs are closely related phylogenetically, with an intraclass identity of 85-97%. evidence of x. laevis genome duplication was exclusively found for x. tropicalis adh1a, which corresponds to x. laevis adh1a and adh1a genes. although only partial sequences were found for x. laevis adh1a identical key residues in x. tropicalis adh1a, which are not found in x. laevis adh1a suggest that the two former sequences would be functionally closer. the percentage of identity between x. laevis adh1a and adh1a is % in residues. when rana and xenopus class i sequences were compared, r. perezi adh showed the highest identity with xenopus adh1b forms and identical residues at most important positions. for adh the percent identity between the r. perezi and xenopus forms is 71-73% and all possess ser ser phe gly and leu amphibian sequences were also compared to other vertebrate adhs, including those identified in a. carolinensis and p. sinensis, in unrooted phylogenetic trees. molecular phylogenetic analysis on the deduced protein sequences using nj and ml estimations produced similar topologies. for each tree construction, among-site rate heterogeneity was taken into account and confidence in each node was assessed by and bootstrap replicates, respectively. in the tree of figure  xenopus adh adh adh adh and adh sequences cluster with other extant members of their classes, whereas adh branches separately. the tree topology pictures the constant nature of class iii, in contrast to the other adh classes. related to class i, amphibian adh adh adh and adh form a protein cluster presumably derived from a common ancestor. an important radiation would have occurred in amphibians from a primitive adh gene, originating the four classes mentioned above, although the order and genes involved in each duplicatory event cannot be ascertained. likewise, the presence of eight adh and three adh forms in a. carolinensis suggests that specific duplications could have occurred in lizards but not in turtles, as these organisms belong to different reptilian lineages. percent identity within anole class i sequences ranges from % to %, and adh1a and adh1b show the highest identity with uromastyx adh1a and adh1b, respectively. within anole adh sequences, the percent identity is around 71-78%. interestingly, multiple alignments reveal that all class i enzymes from reptiles to humans, as well as other classes derived from amniote adh such as adh and adh show a deletion at position with respect to amphibian adh proteins and the remaining adh classes. phylogenetic data supporting the results of this study are available from the treebase repository. discussion a total of eleven adh genes with a conserved structure have been identified in the x. tropicalis genome, and grouped in seven enzyme classes: adh,, and. these loci are distributed in two scaffolds, one containing the main discontinuous adh cluster, syntenic to human 4q21- but broken by several rearrangements, and the other showing the single adh1a locus. the amphibian adh system represents a unique organization among tetrapods since sequencing data and comparative analysis of genomes describe single clusters in human, rat, mouse and chicken. genes similar to those of x. tropicalis have been identified in x. laevis, indicating that the multiplicity of adh forms was present prior to the divergence of the two species. duplication of the x. laevis genome affected a great number of gene families, such as those of globin or α-actin, but the only adh duplicates found to date correspond to the x. tropicalis adh1a gene, which were named adh1a and adh1a in x. laevis. this suggests that many adh duplicated loci could have been lost. nevertheless, further identification of other gene duplicates in x. laevis should not be discarded. in this regard, activity staining of hepatic adhs revealed the existence of two polymorphic genes coding for ethanol dehydrogenase subunits that did not heterodimerize and were placed in separate genetic linkage groups. in the following description of the amphibian adh properties, we include functional features of forms not yet characterized, predicted from the wide information of the structure/function relationships available for the adh family. however, the proposed functions have to be confirmed by the expression and kinetic characterization of the novel enzymes, especially adh and adh ancient forms of vertebrate adh classes in amphibians adh1 amphibian class adh clusters with the novel amphibian classes adh adh and adh since all of them derive from a primitive adh gene ancestor, also common to the amniote class i line. later, adh duplications generated adh1a, 1b and 1c; adh8a and 8b; and adh10a and 10b from their corresponding ancestors. xenopus adh1a is the most similar to other vertebrate adh enzymes, whereas xenopus adh1b shows the highest identity with r. perezi adh xenopus adh1a and adh1b show arg his phe and phe typical class i residues that are associated with ethanol dehydrogenase activity. the substrate-binding pocket of r. perezi adh1b is extremely hydrophobic and space-restricted, resulting in low km values for aliphatic alcohols, it has wide substrate specificity and is moderately active with retinoids. in adh1a, smaller substrate-binding residues val and val anticipate higher km values for this isozyme, and substitution by his suggests an increased rate of nad+ dissociation and higher kcat values. x. laevis adh1a has many atypical residues, such as gly or thr which suggest an alternative proton-relay pathway in comparison with all the other class i enzymes, showing his moreover, voluminous residues phe met and met would increase hydrophobicity and would narrow the substrate cleft even more than in adh1b. these residue exchanges predict different substrate specificity and suggest that adh1a may have acquired a new function after gene duplication, while adh1a would have maintained the original one. adh1c has unique features among substrate-binding residues. at position the lack of an aromatic ring expands the substrate cleft and permits the accommodation of large substrates, as in human adh1a and chicken adh. in contrast, an unusual phe would narrow the entrance, although still may allow productive binding of retinoids as occurs in x. laevis adh8b. these features predict that adh1c binds large alcohol substrates better than ethanol. substitutions in the coenzyme-binding site, in relation to amniote class i, are his instead of arg, asp and asn/thr which could weaken the coenzyme binding and increase kcat values. expression pattern of amphibian class i in adult and embryonic tissues resembles that of other vertebrates, and transcripts of adh1b are abundant in the developing tadpole. regarding reptiles, specific duplications of the adh gene occurred in lizards. anole adh1a is the ortholog of uromastyx adh1a and clusters with other known reptilian and avian class i enzymes, while anole adh1b is the ortholog of uromastyx adh1b and clusters with the rest of anole class i forms. thus, a. carolinensis adh1c-1h genes may have arisen from further tandem duplications of adh1b in this organism, although the existence of additional adh genes in uromastyx cannot be discarded. interestingly, a. carolinensis adh1d, adh1e and adh1g share the residues gln-arg-ser instead of the typical class i triad asp-ile-gln at positions 223– which interact with the adenosine moiety of the coenzyme. similar residues are found in nadp+-dependent mdr enzymes such as sulfolobus solfataricus glucose dehydrogenase, xilella fastidiosa cinnamyl alcohol dehydrogenase or saccharomyces cerevisiae adh, among others, thus suggesting a higher preference for this coenzyme. given the phylogenetic distance between these adh forms and amphibian class adh this suggests that the nadp+ specificity could have arisen at least twice during the vertebrate adh evolution. adh2 amphibians are the most ancient organisms that possess a class ii enzyme. therefore, the duplicatory event that generated adh from adh can be placed between fish/tetrapod and amphibian/amniote splits, to mya. moreover, x. tropicalis adh already has the four-residue insertion, considered as the most distinctive trait of class ii enzymes. the phylogenetic proximity of amphibian and avian adh enzymes, in spite of the overall variability of this class, predicts similar structure and kinetic behaviour. the ostrich enzyme has been described as a mixed-class, structurally similar to mammalian class ii but resembling class-i kinetic properties, since it is notably active with short-chain alcohol substrates such as ethanol. ostrich adh shares %, % and % identity with turtle, frog and human adh respectively. these four sequences show ser/thr phe tyr leu phe val ile/leu and phe in the substrate-binding site; and also ser and ser which distinguish human class ii from class i. all the residues involved in the substrate interaction are almost identical in frog, turtle and ostrich adh moreover, the three enzymes show arg ser/thr his and ile at the coenzyme-binding site, the same residues which are found in human adh1b concluding that the primitive class ii forms may share common kinetic properties with class i enzymes. human adh1b is included for comparison with all available adh enzymes. the four-residue insertion of class ii enzymes is also shown. residues of the coenzyme-binding site are common to human adh1b residues at positions 223-224- are d-i-n in all cases. *human adh is polymorphic at position and shows ser or thr. class ii can be divided in two structurally and functionally distinct subgroups. the first one exhibits a low activity with ethanol and comprises mouse and rat adh both showing pro and rabbit adh2b, which lacks a his residue at both positions or. in contrast, the second group is constituted by rabbit adh2a and amphibian, reptilian and avian adh all of them possessing his51; and human, marmoset and bovine adh which show his. these forms may share not only the ethanol dehydrogenase activity but also the ability of metabolizing retinoids, as reported for human adh. adh3 x. laevis and x. tropicalis adh sequences show the functionally important residues strictly conserved in class iii enzymes from reptiles to mammals. adh is a glutathione-dependent formaldehyde dehydrogenase, as seen spectrophotometrically for the purified enzyme of r. perezi and by activity staining of electrophoresed tissue homogenates from r. perezi and x. laevis. expression of amphibian adh is detected in every stage and tissue studied, although it is more abundant in some organs such as the ovary, suggesting that oocytes may store large amounts of maternal adh for its later use during the embryonic development. the maternal origin of adh mrna has been previously described in drosophila and zebrafish embryos. adh7 the novel reptilian genomic sequences supported the class assignment of x. tropicalis adh sharing identity percentages of % and % with turtle adh and anole adh7b, respectively. as listed in table  all adh enzymes show thr involved in the stereospecificity for secondary alcohols; a small residue such as cys at position, indicative of high km values for ethanol and correct positioning of steroid substrates; phe and leu; and similar coenzyme-binding residues. positions to are almost identical, and his and trp are common for all adh enzymes. these two positions were reported to affect the conformation of the loop 112– widening the entrance of the substrate-binding site and conferring to adh the ability to oxidize large hydrophobic alcohols. residues and involved in the rearrangement of the loop 112– at the entrance of the substrate-binding pocket and present in all adh forms, are also included. residues at positions 223-224- are d-i-n in all cases. among the three forms identified in a. carolinensis, adh7b is the most similar to turtle, chicken and frog adh with identity percentages of %, % and %, respectively. already present in amphibians, adh appeared between the tetrapod/fish and the amniote/amphibian splits, 450– mya. on the other hand, the common position of turtle and chicken adh and vertebrate adh5-adh loci within the adh cluster, together with the fact that they do not coexist in any organism, suggests that the loss of adh might have occurred close to the origin of adh and adh or even in the same genetic event. novel adh classes in amphibians adh8 a total of five members are now known from the nadp+-dependent class viii: adh from r. perezi, and adh8a and adh8b from both x. laevis and x. tropicalis. they show the conserved triad gly223-ser/thr224-gln/his that interacts with the 2′-phosphate of the adenosine moiety of nadp+. despite their different substrate-binding sites, especially concerning large hydrophobic residues, both x. laevis adh8b and r. perezi adh reduce retinaldehyde and medium-chain aldehydes. one major difference is the substitution of phe by cys that correlates with the poor ethanol oxidizing activity of x. laevis adh8b. residues gly ser and ser would determine the proposed catalytic mechanism and proton-relay pathway of r. perezi adh. while gly is conserved in all adh members, ser and ser also found in adh8a, are substituted by thr and ala in adh8b enzymes. remarkably, several deletions exist in adh with respect to amphibian class i. the first one is found at position of r. perezi adh and both xenopus adh8b enzymes, and it may account to some extent for the wide substrate pocket observed in r. perezi adh. the second one, at position is common for all adh sequences. and the third, located at position is only found in xenopus adh8b enzymes. regarding coenzyme-interacting positions, the presence of gly and the lack of a typical hydrophobic residue in position usually val or ile, suggest a lower affinity for the coenzyme and increased kcat values for adh enzymes. adult expression pattern of adh includes the gastrointestinal tract and skin, where it may participate in cell differentiation through regulation of retinoic acid levels acting as a retinaldehyde reductase. in embryos, adh expression is also observed after neurulation, although est evidences are too scarce to suggest a possible function. adh8a and adh8b promoters are closely related, both exhibiting a putative ccaat box, and common hnf-3beta, xfd and gata-binding sites. adh9 x. laevis and x. tropicalis adh are the only members known from this novel class, and none of them has been characterized at the protein level. these enzymes share a percentage of amino acid identity lower than 60% with any other adh, and show many special sequence features, such as cys met phe val met and phe318; together with his residues at both positions and his indicates the same proton-relay pathway as in class i enzymes, while his suggests rapid coenzyme dissociation. asp at the cofactor-binding site indicates nad+-dependence. cys phe met and phe predict a substrate pocked enlarged at its inner part, but narrow and hydrophobic at the middle and the entrance, similar to that of adh substrate preferences may be large substrates rather than ethanol, but probably not steroids. northern blot studies detect adh transcripts in adult stomach, esophagus and skin, while discard the expression of the enzyme at embryonic stages. colocalization with adh in spite of their sequence divergence and different cofactor specificity, may obey to the common regulatory elements found in their gene promoters, and it is consistent with the adjacent chromosomal location of their genes. adh was initially described as an adh4-like form in x. laevis, likely because its tissue localization. based on phylogenetic analyses and class-specific sequence signatures, it is now clear that adh constitutes a separate class and thus adh is not present in amphibians. the absence of adh forms in reptiles and birds, and its presence in marsupials supports its emergence at the origin of mammals. adh10 two isoenzymatic forms occur in this class, adh10a and adh10b, which are closely related to adh common residues of adh and adh enzymes are ser phe met and leu for substrate interaction; and arg his and leu for coenzyme binding. val and his are particular of adh substitution of typical ile by smaller val can affect the strength of coenzyme interaction, and substitution of arg by his was suggested to increase kcat values in human adh. three residue exchanges characterize adh10b: val phe and arg val results in a wider bottom of the substrate pocket and it has not been found in any other adh. phe present in most class ii enzymes, narrows the middle region and increases its hydrophobicity, but hydrophilic arg should compensate for this fact. adh is the only class with a charged residue at position also interesting is the basic residue found at position which, similarly to arg in class iii enzymes, could contribute to a substrate-binding site with a wider entrance and higher volume. a deletion at position of x. tropicalis adh10b could also participate in this rearrangement. the substrate pocket of adh10b with a widened entrance and inner part could accommodate large substrates, such as steroids, provided that phe was not a major steric constraint. ser can also be found in horse adh1s and human adh1c, both able to oxidize steroids. adrenal and gonadal steroids are therefore proposed as adh substrates, in agreement with its predominant expression in xenopus mesonephric kidney and testis, and the presence of a putative site for estrogen receptor in the adh10b promoter.  in conclusion, the complex xenopus adh system is composed of the vertebrate classes adh adh adh and adh together with novel class i-derived enzymes, adh adh and adh exclusively found in amphibians. adh is not present in amphibians and reptiles. the study of the ancient forms of adh and adh also found in reptiles, led to significant conclusions about the evolutionary history of the adh family, whereas the special features showed by the novel forms described herein point to the acquisition of new functions following the adh gene family expansion occurred in amphibians. availability of supporting data the data sets supporting the results of this article are included within the article and its additional files. abbreviations adh: alcohol dehydrogenase; ap1: activator protein 1; camp: cyclic adenosine monphosphate; c/ebpalpha: ccaat-enhancer binding protein alpha; chop: c/ebp homologous protein; c-myb: mieloblastosis viral oncogene homolog; cre-bp1: camp-responsive element binding protein; est: expressed sequence tag; fdh: formaldehyde dehydrogenase; gata-1: gata-binding protein 1; gfi1: growth factor independence 1; hnf3beta: hepatic nuclear factor beta; mdr: medium-chain dehydrogenase/reductase; nad: nicotinamide adenine dinucleotide; nf1: nuclear factor 1; oct1: octamer-binding factor 1; rt-pcr: reverse transcription-polymerase chain reaction; sds: sodium dodecyl sulfate; usf: upstream stimulating factor; ssp1: stimulating protein 1; xfd: xenopus fork head domain. competing interests the authors declare that they have no competing interests. authors’ contributions eb carried out the cloning and detection of x. laevis adh forms, identified adh sequences in genomic and expression databases, performed comparative analyses, constructed nj trees, and drafted the manuscript. ra calculated evolutionary parameters of vertebrate adh, constructed ml trees, and participated in drafting of the manuscript. gd provided partial sequences of x. laevis adh8b and adh cdnas, and critically reviewed the manuscript. xp participated in the design and coordination of the study. jf conceived the study, participated in its design and coordination, and provided critical review of the manuscript. all authors have read and approved the final manuscript. supplementary material additional file 1 percentage of amino acid identity between xenopus tropicalis and representative vertebrate adh sequences. click here for file additional file 2 percentage of amino acid identity between amphibian adh sequences. click here for file additional file 3 xenopus tropicalis adh1a cdna sequence. the sequence includes the translated coding exons, intron flanking regions, proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 4 xenopus tropicalis adh1b cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 5 xenopus tropicalis adh1c cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 6 xenopus tropicalis adh cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 7 xenopus tropicalis adh cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 8 xenopus tropicalis adh cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. click here for file additional file 9 xenopus tropicalis adh8a cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 10 xenopus tropicalis adh8b cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 11 xenopus tropicalis adh cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 12 xenopus tropicalis adh10a cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 13 xenopus tropicalis adh10b cdna sequence. the sequence includes the translated coding exons, intron flanking regions, and the proximal promoter and 3′-untranslated region with predicted regulatory elements. putative tata boxes and polyadenylation signals are in bold and underlined. putative transcription factor binding sites are underlined, with the core sequence of the matrix in bold and italics; and the orientation is given in parentheses. click here for file additional file 14 alignment of amphibian adh amino acid sequences. click here for file additional file 15 alignment of vertebrate adhs included in the phylogenetic tree. click here for file acknowledgements this work was supported by grants from the spanish ministerio de economía y competitividad and generalitat de catalunya. we thank dr. ines hoffmann for her preliminary work on x. laevis adh1b and adh we are also grateful to dr. salvador bartolomé, from the laboratori de luminiscència i espectroscòpia de biomolècules of the universitat autònoma de barcelona, for image capture and processing.
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. 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. class-specific signatures have been assigned to adh and ancestral adh is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian adh but with class-i kinetic properties. the study of the ancient forms of adh and adh 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. xenopus cdna and genomic sequences along with expressed sequence tags were used in phylogenetic analyses and structure-function correlations of amphibian adhs. novel adh sequences identified in the genomes of anolis carolinensis and pelodiscus sinensis were also included in these studies. 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. 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.
 the alcohol dehydrogenase 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 adh 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.  xenopus cdna and genomic sequences along with expressed sequence tags were used in phylogenetic analyses and structure-function correlations of amphibian adhs. novel adh sequences identified in the genomes of anolis carolinensis and pelodiscus sinensis 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 adh and the novel adh1-derived forms adh and adh in addition, they exhibit adh adh adh and adh also present in reptiles and birds. class-specific signatures have been assigned to adh and ancestral adh is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian adh but with class-i kinetic properties. remarkably, many adh and adh forms are observed in the lizard, probably due to lineage-specific duplications. adh is not present in amphibians and reptiles.  the study of the ancient forms of adh and adh 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. alcohol dehydrogenaseenzymogenesisgene familyvertebrate evolution 