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1.  Cloning and Comparative Analyses of the Zebrafish Ugt Repertoire Reveal Its Evolutionary Diversity 
PLoS ONE  2010;5(2):e9144.
UDP-glucuronosyltransferases (Ugts) are a supergene family of phase II drug-metabolizing enzymes that catalyze the conjugation of numerous hydrophobic small molecules with the UDP-glucuronic acid, converting them into hydrophilic molecules. Here, we report the identification and cloning of the complete zebrafish Ugt gene repertoire. We found that the zebrafish genome contains 45 Ugt genes that can be divided into three families: Ugt1, Ugt2, and Ugt5. Both Ugt1 and Ugt2 have two unlinked clusters: a and b. The Ugt1a, Ugt1b, Ugt2a, and Ugt2b clusters each contain variable and constant regions, similar to that of the protocadherin (Pcdh), immunoglobulin (Ig), and T-cell receptor (Tcr) clusters. Cloning the full-length coding sequences confirmed that each of the variable exons is separately spliced to the set of constant exons within each zebrafish Ugt cluster. Comparative analyses showed that both a and b clusters of the zebrafish Ugt1 and Ugt2 genes have orthologs in other teleosts, suggesting that they may be resulted from the “fish-specific” whole-genome duplication event. The Ugt5 genes are a novel family of Ugt genes that exist in teleosts and amphibians. Their entire open reading frames are encoded by single large exons. The zebrafish Ugt1, Ugt2, and Ugt5 genes can generate additional transcript diversity through alternative splicing. Based on phylogenetic analyses, we propose that the ancestral tetrapod and teleost Ugt1 clusters contained multiple Ugt1 paralogs. After speciation, these ancestral Ugt1 clusters underwent lineage-specific gene loss and duplication. The ancestral vertebrate Ugt2 cluster also underwent lineage-specific duplication. The intronless Ugt5 open reading frames may be derived from retrotransposition followed by gene duplication. They have been expanded dramatically in teleosts and have become the most abundant Ugt family in these lineages. These findings have interesting implications regarding the molecular evolution of genes with diversified variable exons in vertebrates.
doi:10.1371/journal.pone.0009144
PMCID: PMC2819257  PMID: 20161780
2.  Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi 
PLoS ONE  2014;9(1):e86683.
Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes.
doi:10.1371/journal.pone.0086683
PMCID: PMC3899305  PMID: 24466198
3.  Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG) 
BMC Plant Biology  2014;14:82.
Background
Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.
Results
Five UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner.
Conclusion
We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.
doi:10.1186/1471-2229-14-82
PMCID: PMC3986616  PMID: 24678929
Flax; Lignan; UGTs; SDG; Secoisolariciresinol; Glucosylation; Glycosyltranferases
4.  Functional diversification of two UGT80 enzymes required for steryl glucoside synthesis in Arabidopsis  
Journal of Experimental Botany  2014;66(1):189-201.
Summary
Plants have two UDP-glc:sterol glucosyltransferase enzymes that synthesize sugar-conjugated forms of sterols known as steryl glucosides. We provide evidence for their specialized roles in the production of diverse sterol-derived compounds.
Steryl glucosides (SG) are abundant steroid conjugates in plant membranes. Beyond structural roles in lipid bilayers, functions in sugar transport, storage, and/or signalling are predicted. UDP-glucose:sterol glucosyltransferase 80A2 (UGT80A2) and UGT80B1, which share similarity to fungal counterparts, are implicated in SG synthesis in Arabidopsis thaliana. A third related enzyme, which seems specific to the plant lineage, is encoded by UGT713B1/At5g24750. Genetic and biochemical approaches were employed to determine the role of each UGT gene in the production of specific SGs and acyl SGs (ASGs). Using direct infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS), SG and acyl SG (ASG) contents of ugt80 and ugt713 mutants, and triple and double mutants were profiled in seeds. In vitro enzyme assays were performed to assay substrate preferences. Both UGT80A2 and UGT80B1, but not UGT713B1 were shown to be coordinately down-regulated during seed imbibition when SG levels decline, consistent with similar functions as UGT80 enzymes. UGT80A2 was found to be required for normal levels of major SGs in seeds, whereas UGT80B1 is involved in accumulation of minor SG and ASG compounds. Although the results demonstrate specific activities for UGT80A2 and UGT80B1, a role for UGT713B1 in SG synthesis was not supported. The data show that UGT80A2, the more highly conserved enzyme, is responsible for the bulk production of SGs in seeds, whereas UGT80B1 plays a critical accessory role. This study extends our knowledge of UGT80 enzymes and provides evidence for specialized functions for distinct classes of SG and ASG molecules in plants.
doi:10.1093/jxb/eru410
PMCID: PMC4265157  PMID: 25316063
Arabidopsis; lipids; seed; steryl glycosides; UDP-glucose:sterol glucosyltransferase; UGT80A2; UGT80B1; UGT713B1.
5.  Evolution of a Major Drug Metabolizing Enzyme Defect in the Domestic Cat and Other Felidae: Phylogenetic Timing and the Role of Hypercarnivory 
PLoS ONE  2011;6(3):e18046.
The domestic cat (Felis catus) shows remarkable sensitivity to the adverse effects of phenolic drugs, including acetaminophen and aspirin, as well as structurally-related toxicants found in the diet and environment. This idiosyncrasy results from pseudogenization of the gene encoding UDP-glucuronosyltransferase (UGT) 1A6, the major species-conserved phenol detoxification enzyme. Here, we established the phylogenetic timing of disruptive UGT1A6 mutations and explored the hypothesis that gene inactivation in cats was enabled by minimal exposure to plant-derived toxicants. Fixation of the UGT1A6 pseudogene was estimated to have occurred between 35 and 11 million years ago with all extant Felidae having dysfunctional UGT1A6. Out of 22 additional taxa sampled, representative of most Carnivora families, only brown hyena (Parahyaena brunnea) and northern elephant seal (Mirounga angustirostris) showed inactivating UGT1A6 mutations. A comprehensive literature review of the natural diet of the sampled taxa indicated that all species with defective UGT1A6 were hypercarnivores (>70% dietary animal matter). Furthermore those species with UGT1A6 defects showed evidence for reduced amino acid constraint (increased dN/dS ratios approaching the neutral selection value of 1.0) as compared with species with intact UGT1A6. In contrast, there was no evidence for reduced amino acid constraint for these same species within UGT1A1, the gene encoding the enzyme responsible for detoxification of endogenously generated bilirubin. Our results provide the first evidence suggesting that diet may have played a permissive role in the devolution of a mammalian drug metabolizing enzyme. Further work is needed to establish whether these preliminary findings can be generalized to all Carnivora.
doi:10.1371/journal.pone.0018046
PMCID: PMC3065456  PMID: 21464924
6.  Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target 
PLoS ONE  2014;9(9):e107209.
Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.
doi:10.1371/journal.pone.0107209
PMCID: PMC4164535  PMID: 25222113
7.  Genome Wide Association Mapping for Arabinoxylan Content in a Collection of Tetraploid Wheats 
PLoS ONE  2015;10(7):e0132787.
Background
Arabinoxylans (AXs) are major components of plant cell walls in bread wheat and are important in bread-making and starch extraction. Furthermore, arabinoxylans are components of soluble dietary fibre that has potential health-promoting effects in human nutrition. Despite their high value for human health, few studies have been carried out on the genetics of AX content in durum wheat.
Results
The genetic variability of AX content was investigated in a set of 104 tetraploid wheat genotypes and regions attributable to AX content were identified through a genome wide association study (GWAS). The amount of arabinoxylan, expressed as percentage (w/w) of the dry weight of the kernel, ranged from 1.8% to 5.5% with a mean value of 4.0%. The GWAS revealed a total of 37 significant marker-trait associations (MTA), identifying 19 quantitative trait loci (QTL) associated with AX content. The highest number of MTAs was identified on chromosome 5A (seven), where three QTL regions were associated with AX content, while the lowest number of MTAs was detected on chromosomes 2B and 4B, where only one MTA identified a single locus. Conservation of synteny between SNP marker sequences and the annotated genes and proteins in Brachypodium distachyon, Oryza sativa and Sorghum bicolor allowed the identification of nine QTL coincident with candidate genes. These included a glycosyl hydrolase GH35, which encodes Gal7 and a glucosyltransferase GT31 on chromosome 1A; a cluster of GT1 genes on chromosome 2B that includes TaUGT1 and cisZog1; a glycosyl hydrolase that encodes a CelC gene on chromosome 3A; Ugt12887 and TaUGT1genes on chromosome 5A; a (1,3)-β-D-glucan synthase (Gsl12 gene) and a glucosyl hydrolase (Cel8 gene) on chromosome 7A.
Conclusions
This study identifies significant MTAs for the AX content in the grain of tetraploid wheat genotypes. We propose that these may be used for molecular breeding of durum wheat varieties with higher soluble fibre content.
doi:10.1371/journal.pone.0132787
PMCID: PMC4503733  PMID: 26176552
8.  CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design 
Nucleic Acids Research  2003;31(13):3763-3766.
We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3–4 highly conserved amino acids within a 3′ degenerate core. A longer 5′ non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).
PMCID: PMC168931  PMID: 12824413
9.  Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns 
BMC Genomics  2012;13:175.
Background
The glycosylation process, catalyzed by ubiquitous glycosyltransferase (GT) family enzymes, is a prevalent modification of plant secondary metabolites that regulates various functions such as hormone homeostasis, detoxification of xenobiotics and biosynthesis and storage of secondary metabolites. Flax (Linum usitatissimum L.) is a commercially grown oilseed crop, important because of its essential fatty acids and health promoting lignans. Identification and characterization of UDP glycosyltransferase (UGT) genes from flax could provide valuable basic information about this important gene family and help to explain the seed specific glycosylated metabolite accumulation and other processes in plants. Plant genome sequencing projects are useful to discover complexity within this gene family and also pave way for the development of functional genomics approaches.
Results
Taking advantage of the newly assembled draft genome sequence of flax, we identified 137 UDP glycosyltransferase (UGT) genes from flax using a conserved signature motif. Phylogenetic analysis of these protein sequences clustered them into 14 major groups (A-N). Expression patterns of these genes were investigated using publicly available expressed sequence tag (EST), microarray data and reverse transcription quantitative real time PCR (RT-qPCR). Seventy-three per cent of these genes (100 out of 137) showed expression evidence in 15 tissues examined and indicated varied expression profiles. The RT-qPCR results of 10 selected genes were also coherent with the digital expression analysis. Interestingly, five duplicated UGT genes were identified, which showed differential expression in various tissues. Of the seven intron loss/gain positions detected, two intron positions were conserved among most of the UGTs, although a clear relationship about the evolution of these genes could not be established. Comparison of the flax UGTs with orthologs from four other sequenced dicot genomes indicated that seven UGTs were flax diverged.
Conclusions
Flax has a large number of UGT genes including few flax diverged ones. Phylogenetic analysis and expression profiles of these genes identified tissue and condition specific repertoire of UGT genes from this crop. This study would facilitate precise selection of candidate genes and their further characterization of substrate specificities and in planta functions.
doi:10.1186/1471-2164-13-175
PMCID: PMC3412749  PMID: 22568875
10.  Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom 
BMC Bioinformatics  2009;10(Suppl 11):S3.
Background
As a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement.
Results
We analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis.
Conclusion
The research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.
doi:10.1186/1471-2105-10-S11-S3
PMCID: PMC3226193  PMID: 19811687
11.  Adaptive evolution of multiple-variable exons and structural diversity of drug-metabolizing enzymes 
Background
The human genome contains a large number of gene clusters with multiple-variable-first exons, including the drug-metabolizing UDP glucuronosyltransferase (UGT1) and I-branching β-1,6-N-acetylglucosaminyltransferase (GCNT2, also known as IGNT) clusters, organized in a tandem array, similar to that of the protocadherin (PCDH), immunoglobulin (IG), and T-cell receptor (TCR) clusters. To gain insight into the evolutionary processes that may have shaped their diversity, we performed comprehensive comparative analyses for vertebrate multiple-variable-first-exon clusters.
Results
We found that there are species-specific variable-exon duplications and mutations in the vertebrate Ugt1, Gcnt2, and Ugt2a clusters and that their variable and constant genomic organizations are conserved and vertebrate-specific. In addition, analyzing the complete repertoires of closely-related Ugt2 clusters in humans, mice, and rats revealed extensive lineage-specific duplications. In contrast to the Pcdh gene clusters, gene conversion does not play a predominant role in the evolution of the vertebrate Ugt1, Gcnt2 and Ugt2 gene clusters. Thus, their tremendous diversity is achieved through "birth-and-death" evolution. Comparative analyses and homologous modeling demonstrated that vertebrate UGT proteins have similar three-dimensional structures each with N-terminal and C-terminal Rossmann-fold domains binding acceptor and donor substrates, respectively. Molecular docking experiments identified key residues in donor and acceptor recognition and provided insight into the catalytic mechanism of UGT glucuronidation, suggesting the human UGT1A1 residue histidine 39 (H39) as a general base and the residue aspartic acid 151 (D151) as an important electron-transfer helper. In addition, we identified four hypervariable regions in the N-terminal Rossmann domain that form an acceptor-binding pocket. Finally, analyzing patterns of nonsynonymous and synonymous nucleotide substitutions identified codon sites that are subject to positive Darwinian selection at the molecular level. These diversified residues likely play an important role in recognition of myriad xenobiotics and endobiotics.
Conclusion
Our results suggest that enormous diversity of vertebrate multiple variable first exons is achieved through birth-and-death evolution and that adaptive evolution of specific codon sites enhances vertebrate UGT diversity for defense against environmental agents. Our results also have interesting implications regarding the staggering molecular diversity required for chemical detoxification and drug clearance.
doi:10.1186/1471-2148-7-69
PMCID: PMC1885805  PMID: 17475008
12.  Impacts of the Glucuronidase Genotypes UGT1A4, UGT2B7, UGT2B15 and UGT2B17 on Tamoxifen Metabolism in Breast Cancer Patients 
PLoS ONE  2015;10(7):e0132269.
Tamoxifen is used to prevent and treat estrogen-dependent breast cancer. It is described as a prodrug since most of its antiestrogen effects are exerted through its hydroxylated metabolites 4-OH-tamoxifen and endoxifen. In prior work, we correlated optimal plasma levels of these metabolites with certain genotypes of CYP2D6 and SULT1A2. This descriptive study examines correlations between concentrations of tamoxifen's glucuronide metabolites and genotypes UGT1A4 Pro24Thr, UGT1A4 Leu48Val, UGT2B7 His268Tyr, UGT2B15 Asp85YTyr UGT2B15 Lys523Thr and UGT2B17del in 132 patients with estrogen receptor-positive breast cancer under treatment with tamoxifen. Patients were genotyped by real-time and conventional PCR-RFLP. The glucuronides 4-OH-tamoxifen-N-glucuronide, 4-OH-tamoxifen-O-glucuronide and endoxifen-O-glucuronide were isolated from blood plasma and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Individuals who were homozygous for UGT1A448VAL showed significantly lower mean concentrations of both glucuronide metabolites compared to subjects genotyped as wt/wt plus wt/48Val (p=0.037 and p=0.031, respectively). Women homozygous for UGT2B7268Tyr also showed mean substrate/product ratios of 4-OH-tamoxifen/4-OH-tamoxifen-O-glucuronide and 4-OH-tamoxifen/4-OH-tamoxifen-N-glucuronide indicative of reduced glucuronidase activity compared to wt homozygotes or to heterozygotes for the polymorphism (p=0.005 and p=0.003, respectively). In contrast, UGT2B15 Lys523Thr and UGT2B17del were associated with possibly increased enzyme activity. Patients with at least one variant allele UGT2B15523Thr showed significantly higher 4-OH-tamoxifen-O-glucuronide and endoxifen-glucuronide levels (p=0.023 and p=0.025, respectively) indicating a variant gene-dose effect. Higher 4-OH-tamoxifen-N-glucuronide levels observed in UGT2B17del genotypes (p=0.042) could be attributed to a mechanism that compensates for the greater expression of other genes in UGT2B17 del/del individuals. Our observations suggest that patients carrying mutations UGT1A448Val, UGT2B7268Tyr or with wt genotypes for UGT2B17nodel and UGT2B15523Lys could be the best candidates for a good response to tamoxifen therapy in terms of eliciting effective plasma active tamoxifen metabolite levels. However, additional studies examining the effects of UGT genotype on overall patient response to TAM are needed to further examine the role of UGT polymorphisms in the therapeutic efficacy of TAM.
doi:10.1371/journal.pone.0132269
PMCID: PMC4503404  PMID: 26176234
13.  Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis 
PLoS ONE  2013;8(12):e81938.
Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.
doi:10.1371/journal.pone.0081938
PMCID: PMC3862569  PMID: 24349155
14.  Genetic Variation in UGT Genes Modify the Associations of NSAIDs with Risk of Colorectal Cancer: Colon Cancer Family Registry 
Genes, chromosomes & cancer  2014;53(7):568-578.
The use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal neoplasia. Previous studies have reported that polymorphisms in NSAID-metabolizing enzymes central to NSAID metabolism including UDP-glucuronosyltransferases (UGT) and cytochrome P450 (CYP) 2C9 may modify this protective effect. We investigated whether 35 functionally relevant polymorphisms within CYP2C9 and UGT genes were associated with colorectal cancer risk or modified the protective effect of NSAIDs on colorectal cancer susceptibility, using 1,584 colorectal cancer cases and 2,516 unaffected sibling controls from the Colon Cancer Family Registry. A three-SNP genotype in UGT1A6 (G-A-A; Ala7-Thr181-Arg184) and the Asp85 variant in UGT2B15 increased the risk of colorectal cancer (OR 3.87; 95% CI 1.04-14.45 and OR 1.34; 95% CI 1.10-1.63, respectively). We observed interactions between UGT1A3 Thr78Thr (A>G) and NSAID use (p-interaction=0.02), a three-SNP genotype within UGT2B4 and ibuprofen use (p-interaction=0.0018), as well as UGT2B15 Tyr85Asp (T>G) and aspirin use (p-interaction=0.01). The interaction with the UGT2B4 and the UGT2B15 polymorphisms were noteworthy at the 25% FDR level. This study highlights the need for further pharmacogenetic studies to identify individuals who might benefit from NSAID use as part of developing effective strategies for prevention of colorectal neoplasia.
doi:10.1002/gcc.22167
PMCID: PMC4326223  PMID: 24677636
UGT; CYP2C9; colorectal cancer; ibuprofen; aspirin; non-steroidal anti-inflammatory drugs; NSAIDs
15.  Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) for the detection of novel viruses in non-human primates 
Methods (San Diego, Calif.)  2009;49(1):32-41.
Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) have proven to be a powerful tool for the identification of novel genes. CODEHOPs are designed from highly conserved regions of multiply-aligned protein sequences from members of a gene family and are used in PCR amplification to identify distantly-related genes. The CODEHOP approach has been used to identify novel pathogens by targeting amino acid motifs conserved in specific pathogen families. We initiated a program utilizing the CODEHOP approach to develop PCR-based assays targeting a variety of viral families that are pathogens in non-human primates. We have also developed and further improved a computer program and website to facilitate the design of CODEHOP PCR primers. Here, we detail the method for the development of pathogen-specific CODEHOP PCR assays using the papillomavirus family as a target. Papillomaviruses constitute a diverse virus family infecting a wide variety of mammalian species, including humans and non-human primates. We demonstrate that our pan-papillomavirus CODEHOP assay is broadly reactive with all major branches of the virus family and show its utility in identifying a novel non-human primate papillomavirus in cynomolgus macaques.
doi:10.1016/j.ymeth.2009.05.011
PMCID: PMC2751581  PMID: 19477279
polymerase chain reaction; consensus; degenerate; oligonucleotide; primers; pathogens; virus; non-human primate; papillomavirus
16.  Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish 
BMC Genomics  2010;11:643.
Background
Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.
Results
Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.
Conclusions
Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.
doi:10.1186/1471-2164-11-643
PMCID: PMC3012610  PMID: 21087487
17.  Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana 
BMC Plant Biology  2011;11:51.
Background
Brassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development. In contrast to other plant hormones BRs act locally, close to the sites of their synthesis, and thus homeostatic mechanisms must operate at the cellular level to equilibrate BR concentrations. Whilst it is recognized that levels of bioactive BRs are likely adjusted by controlling the relative rates of biosynthesis and by catabolism, few factors, which participate in these regulatory events, have as yet been identified. Previously we have shown that the UDP-glycosyltransferase UGT73C5 of Arabidopsis thaliana catalyzes 23-O-glucosylation of BRs and that glucosylation renders BRs inactive. This study identifies the closest homologue of UGT73C5, UGT73C6, as an enzyme that is also able to glucosylate BRs in planta.
Results
In a candidate gene approach, in which homologues of UGT73C5 were screened for their potential to induce BR deficiency when over-expressed in plants, UGT73C6 was identified as an enzyme that can glucosylate the BRs CS and BL at their 23-O-positions in planta. GUS reporter analysis indicates that UGT73C6 shows over-lapping, but also distinct expression patterns with UGT73C5 and YFP reporter data suggests that at the cellular level, both UGTs localize to the cytoplasm and to the nucleus. A liquid chromatography high-resolution mass spectrometry method for BR metabolite analysis was developed and applied to determine the kinetics of formation and the catabolic fate of BR-23-O-glucosides in wild type and UGT73C5 and UGT73C6 over-expression lines. This approach identified novel BR catabolites, which are considered to be BR-malonylglucosides, and provided first evidence indicating that glucosylation protects BRs from cellular removal. The physiological significance of BR glucosylation, and the possible role of UGT73C6 as a regulatory factor in this process are discussed in light of the results presented.
Conclusion
The present study generates essential knowledge and molecular and biochemical tools, that will allow for the verification of a potential physiological role of UGT73C6 in BR glucosylation and will facilitate the investigation of the functional significance of BR glucoside formation in plants.
doi:10.1186/1471-2229-11-51
PMCID: PMC3073898  PMID: 21429230
arabidopsis; brassinosteroids; glycosylation; homeostasis; malonylation; steroids
18.  Convergent evolution in biosynthesis of cyanogenic defence compounds in plants and insects 
Nature Communications  2011;2:273-.
For more than 420 million years, plants, insects and their predators have co-evolved based on a chemical arms race including deployment of refined chemical defence systems by each player. Cyanogenic glucosides are produced by numerous plants and by some specialized insects and serve an important role as defence compounds in these intimate interactions. Burnet moth larvae are able to sequester cyanogenic glucosides from their food plant as well as to carry out de novo biosynthesis. Here we show that three genes (CYP405A2, CYP332A3 and UGT33A1) encode the entire biosynthetic pathway of cyanogenic glucosides in the Burnet moth Zygaena filipendulae. In both plants and insects, convergent evolution has led to two multifunctional P450 enzymes each catalysing unusual reactions and a glucosyl-transferase acting in sequence to catalyse cyanogenic glucoside formation. Thus, plants and insects have independently found a way to package a cyanide time bomb to fend off herbivores and predators.
Cyanide-releasing defence systems in plants and animals are important to the evolution of plant–herbivore interactions. The authors identify the enzymes responsible for biosynthesis of cyanogenic glucosides by Six-spot Burnet moth caterpillars, which have evolved independently from the known plant pathway.
doi:10.1038/ncomms1271
PMCID: PMC4354137  PMID: 21505429
19.  Delineating the structural, functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses 
BMC Plant Biology  2010;10:134.
Background
Sucrose phosphate synthase (SPS) is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII.
Results
The expression in bread wheat over the course of development of various sucrose biosynthesis genes monitored on an Affymetrix array showed that the SPS genes were regulated over time and space. SPSII homoeologue-specific assays were used to show that the three homoeologues contributed differentially to the global expression of SPSII. Genetic mapping placed the set of homoeoloci on the short arms of the homoeologous group 3 chromosomes. A resequencing of the A and B genome copies allowed the detection of four haplotypes at each locus. The 3B copy includes an unspliced intron. A comparison of the sequences of the wheat SPSII orthologues present in the diploid progenitors einkorn, goatgrass and Triticum speltoides, as well as in the more distantly related species barley, rice, sorghum and purple false brome demonstrated that intronic sequence was less well conserved than exonic. Comparative sequence and phylogenetic analysis of SPSII gene showed that false purple brome was more similar to Triticeae than to rice. Wheat - rice synteny was found to be perturbed at the SPS region.
Conclusion
The homoeologue-specific assays will be suitable to derive associations between SPS functionality and key phenotypic traits. The amplicon sequences derived from the homoeologue-specific primers are informative regarding the evolution of SPSII in a polyploid context.
doi:10.1186/1471-2229-10-134
PMCID: PMC3017794  PMID: 20591144
20.  Fungal Cytochrome P450 Monooxygenases: Their Distribution, Structure, Functions, Family Expansion, and Evolutionary Origin 
Genome Biology and Evolution  2014;6(7):1620-1634.
Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi.
doi:10.1093/gbe/evu132
PMCID: PMC4122930  PMID: 24966179
cytochrome P450; characteristic motif; fungi; evolution; duplication
21.  Genome-wide structural and evolutionary analysis of the P450 monooxygenase genes (P450ome) in the white rot fungus Phanerochaete chrysosporium : Evidence for gene duplications and extensive gene clustering 
BMC Genomics  2005;6:92.
Background
Phanerochaete chrysosporium, the model white rot basidiomycetous fungus, has the extraordinary ability to mineralize (to CO2) lignin and detoxify a variety of chemical pollutants. Its cytochrome P450 monooxygenases have recently been implied in several of these biotransformations. Our initial P450 cloning efforts in P. chrysosporium and its subsequent whole genome sequencing have revealed an extraordinary P450 repertoire ("P450ome") containing at least 150 P450 genes with yet unknown function. In order to understand the functional diversity and the evolutionary mechanisms and significance of these hemeproteins, here we report a genome-wide structural and evolutionary analysis of the P450ome of this fungus.
Results
Our analysis showed that P. chrysosporium P450ome could be classified into 12 families and 23 sub-families and is characterized by the presence of multigene families. A genome-level structural analysis revealed 16 organizationally homogeneous and heterogeneous clusters of tandem P450 genes. Analysis of our cloned cDNAs revealed structurally conserved characteristics (intron numbers and locations, and functional domains) among members of the two representative multigene P450 families CYP63 and CYP505 (P450foxy). Considering the unusually complex structural features of the P450 genes in this genome, including microexons (2–10 aa) and frequent small introns (45–55 bp), alternative splicing, as experimentally observed for CYP63, may be a more widespread event in the P450ome of this fungus. Clan-level phylogenetic comparison revealed that P. chrysosporium P450 families fall under 11 fungal clans and the majority of these multigene families appear to have evolved locally in this genome from their respective progenitor genes, as a result of extensive gene duplications and rearrangements.
Conclusion
P. chrysosporium P450ome, the largest known todate among fungi, is characterized by tandem gene clusters and multigene families. This enormous P450 gene diversity has evolved by extensive gene duplications and intragenomic recombinations of the progenitor genes presumably to meet the exceptionally high metabolic demand of this biodegradative group of basidiomycetous fungi in ecological niches. In this context, alternative splicing appears to further contribute to the evolution of functional diversity of the P450ome in this fungus. The evolved P450 diversity is consistent with the known vast biotransformation potential of P. chrysosporium. The presented analysis will help design future P450 functional studies to understand the underlying mechanisms of secondary metabolism and oxidative biotransformation pathways in this model white rot fungus.
doi:10.1186/1471-2164-6-92
PMCID: PMC1184071  PMID: 15955240
22.  Homozygous deletions of UGT2B17 modifies effects of smoking on TP53-mutations and relapse of head and neck carcinoma 
BMC Cancer  2015;15:205.
Background
Smoking induces oncogenic TP53-mutations in head and neck squamous cell carcinomas (HNSCCs). Disruptive mutations of TP53-gene and expression of p16 protein [p16 (+)] in tumor tissue associate with worse and better prognosis, respectively. UDP-glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17) detoxifies smoking-related metabolites. Differences among ethnic groups in UGT2B17 are extremely high. Homozygous deletions of UGT2B17 gene (UGT2B17-deletion) are a common copy number variant (CNV) among Japanese, but not a common CNV among Africans and Europeans. Thus, we examined Japanese patients with HNSCC to explore if UGT2B17-deletion and/or p16 (+) modify effects of smoking on TP53-mutations and affect relapse.
Methods
We conducted a posthoc analysis of a prospective cohort. Polymerase chain reaction, immunohistochemistry, and direct sequencing were used to determine UGT2B17-deletion, p16 (+), and detailed TP53-mutations, respectively.
Results
UGT2B17-deletion was observed in 80% of this study population. For this 80%, TP53-mutations were significantly more common among smokers than non-smokers (P = 0.0016), but this difference between smokers and nonsmokers was not significant for the 20% with UGT2B17. In patients with UGT2B17-deletion and p16 (+), simultaneously, TP53-mutations were much more common among smokers than among non-smokers (81% versus 17%; P = 0.0050). Patients with both UGT2B17-deletion and disruptive TP53-mutations had higher relapse rates than other patients (hazard ratio, 2.22; 95% confidence interval, 1.30 to 3.80, P = 0.004) in a stepwise method.
Conclusions
These results suggest that UGT2B17-deletion interacting with p16 (+) may modify effects of smoking on TP53-mutations and may further interact with the disruptive TP53-mutations to raise relapse rates among Japanese patients with HNSCC.
doi:10.1186/s12885-015-1220-2
PMCID: PMC4389803  PMID: 25886176
UGT2B17; TP53; HNSCC (head and neck squamous-cell carcinoma) and smoking
23.  Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae 
BMC Genomics  2013;14:457.
Background
Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression.
Results
Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength.
Conclusions
Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of green algae and higher plants. Protein domain structures and expression analyses in green alga H. pluvialis indicate that various chy genes are in different manners response to light. The knowledge of evolution of chy genes in photosynthetic eukaryotes provided information of gene cloning and functional investigation of chy genes in algae in the future.
doi:10.1186/1471-2164-14-457
PMCID: PMC3728230  PMID: 23834441
Carotenoid hydroxylase; Xanthophylls biosynthesis; Structure and evolution; Molecular cloning; Expression profiles; Algae
24.  The WRKY transcription factor family in Brachypodium distachyon 
BMC Genomics  2012;13:270.
Background
A complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement.
Results
We have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors.
Conclusions
The description of the WRKY transcription factor family in Brachypodium that we report here provides a framework for functional genomics studies in an important model system. Our database is a resource for both Brachypodium and wheat studies and ultimately projects aimed at improving wheat through manipulation of WRKY transcription factors.
doi:10.1186/1471-2164-13-270
PMCID: PMC3583182  PMID: 22726208
WRKY transcription factor; Brachypodium distachyon; Wheat; Comparative genomics; Database
25.  Molecular Evolution and Functional Divergence of the Cytochrome P450 3 (CYP3) Family in Actinopterygii (Ray-Finned Fish) 
PLoS ONE  2010;5(12):e14276.
Background
The cytochrome P450 (CYP) superfamily is a multifunctional hemethiolate enzyme that is widely distributed from Bacteria to Eukarya. The CYP3 family contains mainly the four subfamilies CYP3A, CYP3B, CYP3C and CYP3D in vertebrates; however, only the Actinopterygii (ray-finned fish) have all four subfamilies and detailed understanding of the evolutionary relationship of Actinopterygii CYP3 family members would be valuable.
Methods and Findings
Phylogenetic relationships were constructed to trace the evolutionary history of the Actinopterygii CYP3 family genes. Selection analysis, relative rate tests and functional divergence analysis were combined to interpret the relationship of the site-specific evolution and functional divergence in the Actinopterygii CYP3 family. The results showed that the four CYP3 subfamilies in Actinopterygii might be formed by gene duplication. The first gene duplication event was responsible for divergence of the CYP3B/C clusters from ancient CYP3 before the origin of the Actinopterygii, which corresponded to the fish-specific whole genome duplication (WGD). Tandem repeat duplication in each of the homologue clusters produced stable CYP3B, CYP3C, CYP3A and CYP3D subfamilies. Acceleration of asymmetric evolutionary rates and purifying selection together were the main force for the production of new subfamilies and functional divergence in the new subset after gene duplication, whereas positive selection was detected only in the retained CYP3A subfamily. Furthermore, nearly half of the functional divergence sites appear to be related to substrate recognition, which suggests that site-specific evolution is closely related with functional divergence in the Actinopterygii CYP3 family.
Conclusions
The split of fish-specific CYP3 subfamilies was related to the fish-specific WGD, and site-specific acceleration of asymmetric evolutionary rates and purifying selection was the main force for the origin of the new subfamilies and functional divergence in the new subset after gene duplication. Site-specific evolution in substrate recognition was related to functional divergence in the Actinopterygii CYP3 family.
doi:10.1371/journal.pone.0014276
PMCID: PMC3000819  PMID: 21170327

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