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1.  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
2.  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
3.  CODEHOP-mediated PCR – A powerful technique for the identification and characterization of viral genomes 
Virology Journal  2005;2:20.
Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) PCR primers derived from amino acid sequence motifs which are highly conserved between members of a protein family have proven to be highly effective in the identification and characterization of distantly related family members. Here, the use of the CODEHOP strategy to identify novel viruses and obtain sequence information for phylogenetic characterization, gene structure determination and genome analysis is reviewed. While this review describes techniques for the identification of members of the herpesvirus family of DNA viruses, the same methodology and approach is applicable to other virus families.
doi:10.1186/1743-422X-2-20
PMCID: PMC1079958  PMID: 15769292
4.  Ectoparasitic growth of Magnaporthe on barley triggers expression of the putative barley wax biosynthesis gene CYP96B22 which is involved in penetration resistance 
BMC Plant Biology  2014;14:26.
Background
Head blast caused by the fungal plant pathogen Magnaporthe oryzae is an upcoming threat for wheat and barley cultivation. We investigated the nonhost response of barley to an isolate of the Magnaporthe species complex which is pathogenic on Pennisetum spp. as a potential source for novel resistance traits.
Results
Array experiments identified a barley gene encoding a putative cytochrome P450 monooxygenase whose transcripts accumulate to a higher concentration in the nonhost as compared to the host interaction. The gene clusters within the CYP96 clade of the P450 plant gene family and is designated as CYP96B22. Expression of CYP96B22 was triggered during the ectoparasitic growth of the pathogen on the outside of the leaf. Usage of a fungicidal treatment and a Magnaporthe mutant confirmed that penetration was not necessary for this early activation of CYP96B22. Transcriptional silencing of CYP96B22 using Barley stripe mosaic virus led to a decrease in penetration resistance of barley plants to Magnaporthe host and nonhost isolates. This phenotype seems to be specific for the barley-Magnaporthe interaction, since penetration of the adapted barley powdery mildew fungus was not altered in similarly treated plants.
Conclusion
Taken together our results suggest a cross-talk between barley and Magnaporthe isolates across the plant surface. Since members of the plant CYP96 family are known to be involved in synthesis of epicuticular waxes, these substances or their derivatives might act as signal components. We propose a functional overlap of CYP96B22 in the execution of penetration resistance during basal and nonhost resistance of barley against different Magnaporthe species.
doi:10.1186/1471-2229-14-26
PMCID: PMC3897914  PMID: 24423145
Nonhost resistance; Magnaporthe oryzae; Head blast; Cytochrome P450; Wax; Cuticle; Penetration; BSMV-VIGS
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.  Origins of P450 diversity 
The P450 enzymes maintain a conserved P450 fold despite a considerable variation in sequence. The P450 family even includes proteins that lack the single conserved cysteine and are therefore no longer haem-thiolate proteins. The mechanisms of successive gene duplications leading to large families in plants and animals are well established. Comparisons of P450 CYP gene clusters in related species illustrate the rapid changes in CYPome sizes. Examples of CYP copy number variation with effects on fitness are emerging, and these provide an opportunity to study the proximal causes of duplication or pseudogenization. Birth and death models can explain the proliferation of CYP genes that is amply illustrated by the sequence of every new genome. Thus, the distribution of P450 diversity within the CYPome of plants and animals, a few families with many genes (P450 blooms) and many families with few genes, follows similar power laws in both groups. A closer look at some families with few genes shows that these, often single member families, are not stable during evolution. The enzymatic prowess of P450 may predispose them to switch back and forth between metabolism of critical structural or signal molecules and metabolism dedicated to environmental response.
doi:10.1098/rstb.2012.0428
PMCID: PMC3538418  PMID: 23297351
P450 sequence diversity; copy number variation; duplication; gene clusters
7.  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
8.  Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. 
Nucleic Acids Research  1998;26(7):1628-1635.
We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein sequences. Each primer consists of a short 3' degenerate core region and a longer 5' consensus clamp region. Only 3-4 highly conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptase-like genes in a human genome, and by detection of C5DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with a set of related protein sequences.
PMCID: PMC147464  PMID: 9512532
9.  Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group 
A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS−) and EPS-producing (EPS+) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS+ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS+ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.
doi:10.1128/AEM.69.6.3299-3307.2003
PMCID: PMC161484  PMID: 12788729
10.  iCODEHOP: a new interactive program for designing COnsensus-DEgenerate Hybrid Oligonucleotide Primers from multiply aligned protein sequences 
Nucleic Acids Research  2009;37(Web Server issue):W222-W228.
PCR amplification using COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) has proven to be highly effective for identifying unknown pathogens and characterizing novel genes. We describe iCODEHOP; a new interactive web application that simplifies the process of designing and selecting CODEHOPs from multiply-aligned protein sequences. iCODEHOP intelligently guides the user through the degenerate primer design process including uploading sequences, creating a multiple alignment, deriving CODEHOPs and calculating their annealing temperatures. The user can quickly scan over an entire set of degenerate primers designed by the program to assess their relative quality and select individual primers for further analysis. The program displays phylogenetic information for input sequences and allows the user to easily design new primers from selected sequence sub-clades. It also allows the user to bias primer design to favor specific clades or sequences using sequence weights. iCODEHOP is freely available to all interested researchers at https://icodehop.cphi.washington.edu/i-codehop-context/Welcome.
doi:10.1093/nar/gkp379
PMCID: PMC2703993  PMID: 19443442
11.  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
12.  Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion 
Journal of Experimental Botany  2008;60(2):509-521.
Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.
doi:10.1093/jxb/ern290
PMCID: PMC2651457  PMID: 19039100
Cell autofluorescence; cell wall apposition; cereal; methylated monolignol
13.  The UDP-glucosyltransferase multigene family in Bombyx mori 
BMC Genomics  2008;9:563.
Background
Glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. A class of UDP-glycosyltransferases (UGTs) is involved in this process. Insect UGTs play important roles in several processes, including detoxication of substrates such as plant allelochemicals, cuticle formation, pigmentation, and olfaction. Identification and characterization of Bombyx mori UGT genes could provide valuable basic information for this important family and explain the detoxication mechanism and other processes in insects.
Results
Taking advantage of the newly assembled genome sequence, we performed a genome-wide analysis of the candidate UGT family in the silkworm, B. mori. Based on UGT signature and their similarity to UGT homologs from other organisms, we identified 42 putative silkworm UGT genes. Most of them are clustered on the silkworm chromosomes, with two major clusters on chromosomes 7 and 28, respectively. The phylogenetic analysis of these identified 42 UGT protein sequences revealed five major groups. A comparison of the silkworm UGTs with homologs from other sequenced insect genomes indicated that some UGTs are silkworm-specific genes. The expression patterns of these candidate genes were investigated with known expressed sequence tags (ESTs), microarray data, and RT-PCR method. In total, 36 genes were expressed in tissues examined and showed different patterns of expression profile, indicating that these UGT genes might have different functions.
Conclusion
B. mori possesses a largest insect UGT gene family characterized to date, including 42 genes. Phylogenetic analysis, genomic organization and expression profiles provide an overview for the silkworm UGTs and facilitate their functional studies in future.
doi:10.1186/1471-2164-9-563
PMCID: PMC2633020  PMID: 19038024
14.  Gene structure of the novel cytochrome P4501D1 genes in stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes) 
Marine environmental research  2008;66(1):19-20.
The cytochrome P450 1 (CYP1) family has expanded with the addition of the CYP1B and CYP1C subfamilies. We recently identified a new CYP1 subfamily in zebrafish, CYP1D, with a single gene, CYP1D1. Here we examined sequences found in other fish genomes, i.e., stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes), for similarities among fish CYP1D1 genes. The full-length deduced amino acid sequences for CPY1D1 in these two species averaged about 43% identity to the CYP1As, but nearly 50% when sequence alignment ambiguities were masked. CYP1D1 has seven exons, similar in size and position to the exons in CYP1D1 and CYP1A in zebrafish. However, the intronic distances were substantially smaller in the medaka and stickleback. There also were differing numbers of xenobiotic response elements in the CYP1D1 of the various species. Whether the stickleback or medaka genes are inducible by aryl hydrocarbon receptor (AHR) agonists is yet to be determined.
doi:10.1016/j.marenvres.2008.02.011
PMCID: PMC3529149  PMID: 18400289
Fish; isozymes; microsomal oxidases; CYP1A; bioinformatics; ortholog
15.  The Role of bZIP Transcription Factors in Green Plant Evolution: Adaptive Features Emerging from Four Founder Genes 
PLoS ONE  2008;3(8):e2944.
Background
Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are regulators of many central developmental and physiological processes including photomorphogenesis, leaf and seed formation, energy homeostasis, and abiotic and biotic stress responses. Here we performed a comprehensive phylogenetic analysis of bZIP genes from algae, mosses, ferns, gymnosperms and angiosperms.
Methodology/Principal Findings
We identified 13 groups of bZIP homologues in angiosperms, three more than known before, that represent 34 Possible Groups of Orthologues (PoGOs). The 34 PoGOs may correspond to the complete set of ancestral angiosperm bZIP genes that participated in the diversification of flowering plants. Homologous genes dedicated to seed-related processes and ABA-mediated stress responses originated in the common ancestor of seed plants, and three groups of homologues emerged in the angiosperm lineage, of which one group plays a role in optimizing the use of energy.
Conclusions/Significance
Our data suggest that the ancestor of green plants possessed four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments.
doi:10.1371/journal.pone.0002944
PMCID: PMC2492810  PMID: 18698409
16.  Identification of CYP genes in Mytilus (mussel) and Crassostrea (oyster) species: First approach to the full complement of cytochrome P450 genes in bivalves 
Marine environmental research  2009;69S1:S1-S3.
Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochrome P450 (CYP) genes, which thus far are poorly known in bivalve mollusks. We searched for CYP sequences in EST databases for Mytilus and Crassostrea species, lophotrochozoan representatives of the protostomes. From ESTs averaging ca. 924 bp, we identified 58 CYP genes in Mytilus californianus and 39 CYP genes in Crassostrea gigas. The sequences fell in all known animal CYP clans, and collectively they clustered in phylogenetic analysis with vertebrate CYP families 1, 2, 3, 4, 17, 20, 26 and 27. As in deuterostomes, a majority of the sequences fell in Clan 2. The CYP sequences found thus far in bivalves suggest a diversity consistent with that found in many other animal species. The present description of mollusk genes provides the overall framework for classification of any additional bivalve sequences. The sequences identified also will be useful in obtaining full-length sequences and in designing primers for analysis of expression of mussel and oyster CYP genes, or for recombinant protein expression to identify potential substrates for the bivalve CYP proteins, and understand their roles in xenobiotic detoxification and physiology of bivalves.
doi:10.1016/j.marenvres.2009.10.013
PMCID: PMC2891413  PMID: 19926125
P450; CYP; Mytilus californianus; Crassostrea gigas; bivalve; pollution; biomarker
17.  Identification and expression analysis of the Glycine max CYP707A gene family in response to drought and salt stresses 
Annals of Botany  2012;110(3):743-756.
Background and Aims
Abscisic acid (ABA) plays crucial roles in plants' responses to abiotic stresses. ABA 8′-hydroxylation controlled by CYP707A genes has been well studied in Arabidopsis and rice, but not in legumes. The aims of the present study were to identify and functionally analyse the soybean CYP707A gene family, and to explore their expression patterns under dehydration and salt stresses.
Methods
A complementation experiment was employed to verify the function of soybean CYP707A1a in ABA catabolism. Genomic and cDNA sequences of other soybean CYP707A genes were isolated from the Phytozome database based on soybean CYP707A1a. The structure and phylogenetic relationship of this gene family was further analysed. The expression patterns of soybean CYP707A genes under dehydration and salt stress were analysed via quantitative real-time PCR.
Key Results
Over-expression of GmCYP707A1a in the atcyp707a2 T-DNA insertion mutant decreased its sensitivity to ABA, indicating that GmCYP707A1a indeed functions as an ABA 8′-hydroxylase in higher plants. The soybean genome contains ten CYP707A genes. Gene structure and phylogenetic analysis showed high conservation of ten GmCYP707A genes to the other CYP707A genes from monocots and dicots. Seed imbibition induced expression of A1a, A1b, A2a, A2b, A2c, A3a and A5 in embryo, and expression of A1a, A1b, A2a and A2b in cotyledon. Dehydration induced expression of A1a, A1b, A2b, A2c, A3a, A3b, A4a, A4b and A5 both in roots and in leaves, whereas rehydration stimulated transcription of A2a, A2b, A3b, A4a and A5 in roots, and only A3b and A5 in leaves. Expression of all soybean CYP707A genes was induced either by short- or by long-term salt stress.
Conclusions
The first biological evidence is provided that GmCYP7071a encodes an ABA 8′-hydroxylase through transgenic studies. Ten soybean GmCYP707A genes were identified, most of them expressed in multiple soybean tissues, and were induced by imbibition, dehydration and salinity.
doi:10.1093/aob/mcs133
PMCID: PMC3400457  PMID: 22751653
Abscisic acid; ABA catabolism; Glycine max; CYP707A gene family; drought; salt stress
18.  Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from Agelena orientalis 
Background
the use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then.
Results
here we report a novel and successful selection strategy for the design of hybrid partially degenerate primers for use with RT-PCR and RACE-PCR for the identification of unknown gene families. The technique (named PaBaLiS) has proven very effective as it allowed us to identify and clone a large group of mRNAs encoding neurotoxin-like polypeptide pools from the venom of Agelena orientalis species of spider. Our approach differs radically from the generally accepted CODEHOP principle first reported in 1998. Most importantly, our method has proven very efficient by performing better than an independently generated high throughput EST cloning programme. Our method yielded nearly 130 non-identical sequences from Agelena orientalis, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same Agelena material. In addition to the primer design approach reported here, which is almost universally applicable to any PCR cloning application, our results also indicate that venom of Agelena orientalis spider contains a much larger family of related toxin-like sequences than previously thought.
Conclusion
with upwards of 100,000 species of spider thought to exist, and a propensity for producing diverse peptide pools, many more peptides of pharmacological importance await discovery. We envisage that some of these peptides and their recombinant derivatives will provide a new range of tools for neuroscience research and could also facilitate the development of a new generation of analgesic drugs and insecticides.
doi:10.1186/1471-2199-8-32
PMCID: PMC1876241  PMID: 17498297
19.  UGT74D1 Catalyzes the Glucosylation of 2-Oxindole-3-Acetic Acid in the Auxin Metabolic Pathway in Arabidopsis 
Plant and Cell Physiology  2013;55(1):218-228.
IAA is a naturally occurring auxin that plays a crucial role in the regulation of plant growth and development. The endogenous concentration of IAA is spatiotemporally regulated by biosynthesis, transport and its inactivation in plants. Previous studies have shown that the metabolism of IAA to 2-oxindole-3-acetic acid (OxIAA) and OxIAA-glucoside (OxIAA-Glc) may play an important role in IAA homeostasis, but the genes involved in this metabolic pathway are still unknown. In this study, we show that UGT74D1 catalyzes the glucosylation of OxIAA in Arabidopsis. By screening yeasts transformed with Arabidopsis UDP-glycosyltransferase (UGT) genes, we found that OxIAA-Glc accumulates in the culture media of yeasts expressing UGT74D1 in the presence of OxIAA. Further, we showed that UGT74D1 expressed in Escherichia coli converts OxIAA to OxIAA-Glc. The endogenous concentration of OxIAA-Glc decreased by 85% while that of OxIAA increased 2.5-fold in ugt74d1-deficient mutants, indicating the major role of UGT74D1 in OxIAA metabolism. Moreover, the induction of UGT74D1 markedly increased the level of OxIAA-Glc and loss of root gravitropism. These results indicate that UGT74D1 catalyzes a committed step in the OxIAA-dependent IAA metabolic pathway in Arabidopsis.
doi:10.1093/pcp/pct173
PMCID: PMC3894777  PMID: 24285754
Arabidopsis; Auxin; Glucosyltransferase; Metabolism; Plant hormone
20.  Role of a respiratory burst oxidase of Lepidium sativum (cress) seedlings in root development and auxin signalling 
Journal of Experimental Botany  2012;63(18):6325-6334.
Reactive oxygen species are increasingly perceived as players in plant development and plant hormone signalling pathways. One of these species, superoxide, is produced in the apoplast by respiratory burst oxidase homologues (rbohs), a family of proteins that is conserved throughout the plant kingdom. Because of the availability of mutants, the focus of research into plant rbohs has been on Arabidopsis thaliana, mainly on AtrbohD and AtrbohF. This study investigates: (i) a different member of the Atrboh family, AtrbohB, and (ii) several rbohs from the close relative of A. thaliana, Lepidium sativum (‘cress’). Five cress rbohs (Lesarbohs) were sequenced and it was found that their expression patterns were similar to their Arabidopsis orthologues throughout the life cycle. Cress plants in which LesarbohB expression was knocked down showed a strong seedling root phenotype that resembles phenotypes associated with defective auxin-related genes. These transgenic plants further displayed altered expression of auxin marker genes including those encoding the auxin responsive proteins 14 and 5 (IAA14 and IAA5), and LBD16 (LATERAL ORGAN BOUNDARIES DOMAIN16), an auxin-responsive protein implicated in lateral root initiation. It is speculated that ROS produced by rbohs play a role in root development via auxin signalling.
doi:10.1093/jxb/ers284
PMCID: PMC3504488  PMID: 23095998
AtrbohB; auxin; Lepidium sativum; Rboh; reactive oxygen species; RNAi; root development; superoxide
21.  primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies 
Nucleic Acids Research  2009;37(Web Server issue):W95-W100.
Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira–Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface.
doi:10.1093/nar/gkp377
PMCID: PMC2703966  PMID: 19465390
22.  Polymorphisms of Aspirin-Metabolizing Enzymes CYP2C9, NAT2 and UGT1A6 in Aspirin-Intolerant Urticaria 
Acetyl salicylic acid (ASA) is metabolized by UDP-glucuronosyltransferase 1A6 (UGT1A6), cytochrome P4502C9 (CYP2C9), and N-acetyl transferase 2 (NAT2). Variations in the activities of these enzymes may modulate adverse ASA-related symptoms such as urticaria. We examined whether polymorphisms in the UGT1A6, CYP2C9, and NAT2 genes are related to ASA-intolerant urticaria (AIU). The genotypes of 148 subjects with AIU (AIU group) and 260 normal healthy control subjects (NC group) were analyzed with respect to the following single nucleotide polymorphisms: CYP2C9 -1188T>C and CYP2C9*3A1075C; UGT1A6 T181A A>G and UGT1A6 R184S A>C; and NAT2 9796A>T, NAT2 197G>A, NAT2 286G>A, NAT2 9601A>G, and NAT2 9306A>G. There were significant differences in the allele frequencies for the CYP2C9 polymorphisms between the two groups. The frequency of the minor allele CYP2C9 -1188T>C was significantly higher in the AIU group than in the NC group (P=0.005). The frequency of the variant genotype CC was higher in the AIU group compared with the controls in both the co-dominant (P=0.007) and recessive models (P=0.012). The frequency of haplotype 2 [CA] was also significantly higher in the AIU group in both the co-dominant (P=0.006) and dominant models (P=0.012). There was no significant difference in genotype frequencies for any of the UGT1A6 or NAT2 polymorphisms between the two groups. Clinical parameters did not differ according to genotype. These results suggest that the C allele of CYP2C9 -1188T>C may be associated with AIU.
doi:10.4168/aair.2011.3.4.273
PMCID: PMC3178826  PMID: 21966608
Aspirin; Cytochrome P4502C9; Metabolizing enzyme; Urticaria
23.  Selective Detoxification of Phenols by Pichia pastoris and Arabidopsis thaliana Heterologously Expressing the PtUGT72B1 from Populus trichocarpa 
PLoS ONE  2013;8(6):e66878.
Phenols are present in the environment and commonly in contact with humans and animals because of their wide applications in many industries. In a previous study, we reported that uridine diphosphate-glucose-dependent glucosyltransferase PtUGT72B1 from Populus trichocarpa has high activity in detoxifying trichlorophenol by conjugating glucose. In this study, more experiments were performed to determine the substrate specificity of PtUGT72B1 towards phenolic compounds. Among seven phenols tested, three were glucosylated by PtUGT72B1 including phenol, hydroquinone, and catechol. Transgenic Arabidopsis plants expressing the enzyme PtUGT72B1 showed higher resistance to hydroquinone and catechol but more sensitivity to phenol than wild type plants. Transgenic Pichia pastoris expressing PtUGT72B1 showed enhanced resistance to all three phenols. Compared with wild type Arabidopsis plants, transgenic Arabidopsis plants showed higher removal efficiencies and exported more glucosides of phenol, phenyl β-D-glucopyranoside, to the medium after cultured with the three phenols. Protein extracts from transgenic Arabidopsis plants showed enhanced conjugating activity towards phenol, hydroquinone and catechol. PtUGT72B1 showed much higher expression level in Pichia pastoris than in Arabidopsis plants. Kinetic analysis of the PtUGT72B1 was also performed.
doi:10.1371/journal.pone.0066878
PMCID: PMC3694158  PMID: 23840543
24.  CYP98A22, a phenolic ester 3’-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens 
BMC Plant Biology  2012;12:152.
Background
Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis.
Results
In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn’t affect the synthesis of scopoletin.
Conclusions
The overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to phytophageous insects.
doi:10.1186/1471-2229-12-152
PMCID: PMC3493272  PMID: 22931486
25.  Cytochromes P450: a success story 
Genome Biology  2000;1(6):reviews3003.1-reviews3003.9.
Cytochrome P450 proteins are enzymes that function in diverse pathways, from carbon source assimilation to hormone biosynthesis. In all P450s, heme is bound in a structurally conserved protein core, allowing them to catalyze regioselective and stereoselective oxidation of hydrocarbons.
Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.
PMCID: PMC138896  PMID: 11178272

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