To determine susceptibility of chickens, turkeys, and mice to pandemic (H1N1) 2009 virus, we conducted contact exposure and inoculation experiments. We demonstrated that chickens were refractory to infection. However, oculo-oronasally inoculated turkeys and intranasally inoculated mice seroconverted without clinical signs of infection. Pandemic (H1N1) 2009 virus causes only mild disease compared with pandemic influenza viruses from the 20th century. However, this characteristic might change if pandemic (H1N1) 2009 viruses acquire virulence markers by reassorting with influenza viruses that cause severe disease in humans, such as highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype. Such reassortment might occur in humans but appears more likely in so-called mixing vessels. Pigs, which had been described as potential mixing vessels (6 50% tissue culture infectious dose (TCID50) of virus A/Regensburg/D6/09/H1N1 on the cornea, nares, and oropharynx. Ten chickens  were inoculated intravenously with 104 TCID50. Five chickens  were housed (permanent contact behind bars and daily direct contact for 30 min) with pigs infected with pandemic (H1N1) 2009 virus . Five chickens  were inoculated oculo-oronasally by dripping a 106 TCID50) directly into the left air sac. Six fattening turkeys (16 weeks of age) from a local flock were inoculated oculo-oronasally with 106 TCID50 of A/Regensburg/D6/09 (H1N1) virus. Six fattening turkeys were inoculated oculo-oronasally with 108 TCID50 of the more recently isolated pandemic (H1N1) 2009 A/Bayern/74/2009 virus. Cloacal and oropharyngeal swab samples from poultry infected oculo-oronasally, through the air sac, or by contact with pigs were collected daily. Samples were tested by using a real-time reverse transcription–PCR with subtype H1N1–specific primers (http://offlu.net) . In addition, 28 chickens (1 week of age) and 28 turkeys (1 week of age) were inoculated with 200 μL of virus suspension (10flu.net) .2–105 TCID50/animal (30 μL), and 4 mice were inoculated intraperitoneally with 105 TCID50/animal (30 μL).Additional studies were conducted to determine the 50% lethal dose of strain A/Regensburg/D6/2009 pandemic (H1N1) 2009 virus for mice. Sixteen BALB/c mice (6 weeks of age) were inoculated intranasally with 10>26 (contact experiment with chickens) or >17 (contact experiment with turkeys). Virus RNA was detected  in swab samples obtained 1–6 days postinoculation from the oropharynx of turkeys inoculated with 108 TCID50 of the A/Bayern/74/2009 (H1N1) strain. Small amounts of virus RNA also were detected in the lung and left air sac from a few chicks and poults early after infection through the air sac  2009 virus. Transmission of swine influenza viruses to poultry, particularly to turkeys, has been demonstrated .,On the basis of our experiments, risk for transmission of pandemic (H1N1) 2009 virus strain to chicken and turkeys and subsequent possible reassortment with other avian influenza viruses should be low. However, we observed a slightly higher susceptibility of older turkeys to high doses of pandemic (H1N1) 2009 virus, which is consistent with recent reports of infected layer turkey flocks in Chile and Canada  2009 virus in mice without prior adaptation was indicated by seroconversion, at least for the higher infectious doses (As in other studies (us doses . IntrapeOur results demonstrate lack of susceptibility and absence of serologic responses of chickens and turkeys to contact infection with an early human-origin isolate of pandemic (H1N1) 2009 virus. However, direct inoculation of fattening turkeys resulted in seroconversion and detection of virus RNA in oropharyngeal swab samples. Generation of new variants and reassortants caused by double infections with other influenza A viruses and pandemic (H1N1) 2009 virus in poultry other than turkeys appears unlikely. However, further adaptation of pandemic (H1N1) virus strains in turkeys cannot be excluded.


Picrorhiza kurrooa is a highly reputed medicinal herb known for its hepato-protective properties which are attributed to a novel group of iridoid glycosides known as picrosides. Although the plant is well studied in terms of its pharmacological properties, very little is known about the biosynthesis of these important secondary metabolites. In this study, we identified two family-1 glucosyltransferases from P. kurrooa. The full length cDNAs of UGT94F4 and UGT86C4 contained open reading frames of 1455 and 1422 nucleotides, encoding polypeptides of 484 and 473 amino acids respectively. UGT94F2 and UGT86C4 showed differential expression pattern in leaves, rhizomes and inflorescence. To elucidate whether the differential expression pattern of the two Picrorhiza UGTs correlate with transcriptional regulation via their promoters and to identify elements that could be recognized by known iridoid-specific transcription factors, upstream regions of each gene were isolated and scanned for putative cis-regulatory elements. Interestingly, the presence of cis-regulatory elements within the promoter regions of each gene correlated positively with their expression profiles in response to different phytohormones. HPLC analysis of picrosides extracted from different tissues and elicitor-treated samples showed a significant increase in picroside levels, corroborating well with the expression profile of UGT94F2 possibly indicating its implication in picroside biosynthesis. Using homology modeling and molecular docking studies, we provide an insight into the donor and acceptor specificities of both UGTs identified in this study. UGT94F2 was predicted to be an iridoid-specific glucosyltransferase having maximum binding affinity towards 7-deoxyloganetin while as UGT86C4 was predicted to be a kaempferol-specific glucosyltransferase. These are the first UGTs being reported from P. kurrooa.Uridine diphosphate glycosyltransferases (UGTs) are pivotal in the process of glycosylation for decorating natural products with sugars. It is one of the versatile mechanisms in determining chemical complexity and diversity for the production of suite of pharmacologically active plant natural products.  Glycosylation is a common modification reaction in plant metabolism and is invariably associated with the production of large array of secondary metabolites. The enzymes that lead to glycoside formation are known as uridine diphosphate glycosyltransferases (UGTs), members of family-1 glycosyltransferases superfamily. It contains over 80 families of enzymes Picrorhiza kurrooa Royle ex. Benth. (Plantaginaceae) is a perennial medicinal herb, also known as ‘Kutki’ or ‘Kadu’, endemic to north western alpine Himalayas, found at an altitude of 2800 m to 4800 m. It is a reputed herb in the Ayurvedic system of medicine and has been used traditionally to treat liver ailments, dyspepsia, chronic diarrhoea and upper respiratory tract ailments. In modern medicine, it is being used in hepatic disorders, gastric troubles, anaemia, asthma and pregnancy related problems. Most of its pharmacological activities are attributed to novel monoterpene derived iridoid glycosides known as picrosides which include picroside-I, picroside-II and other metabolites like picroside-III, picroside-IV, apocynin, androsin, catechol, kutkoside, etc. P. kurrooa in which picroside-I is the major ingredient and therefore, makes this compound a highly valued secondary metabolite of P. kurrooa. Studies have shown that picroliv exhibits hepato-protective effect against aflatoxin O-glucosylation step in the picroside biosynthetic pathway has been poorly characterized and genes for enzymes relevant to the glucosylation step in the picroside biosynthesis are yet to be characterized.Picrosides are essentially iridoid glycosides (cyclopenta-[c]-pyran monoterpenoids), distributed in numerous plant families, usually as glucosides P. kurrooa is that it figures prominently in endangered list of plant species, primarily because of its indiscriminate collection from natural habitats. Owing to its immense therapeutic importance, low content of picrosides and dwindling natural populations, it becomes all the more important to develop elite lines with improved phytochemical content either by classical breeding approaches or through biotechnological interventions for commercial and conservation purposes.An additional constraint with P. kurrooa as 1) it showed maximum binding affinity for iridotrial class of compounds, particularly 7-deoxyloganetin, 2) the expression pattern of UGT94F2 corroborated well with the picroside levels, 3) the binding sites for important transcription factors including MYB, MYC and WRKY, known to be involved in regulating iridoid expression in Catharunthus roseus, were found in the cis-elements of Picrorhiza UGT94F2, and 4) low temperature response elements identified in upstream region of UGT94F2 provided additional support for UGT94F2 to be an iridoid-specific glucosyltransferase, since picroside content has been shown to be higher at low temperatures P. kurrooa correlated well with picroside abundance and elicitors like methyl jasmonate (MeJA), salicylic acid (SA) and 2, 4-dichlorophenoxyacetic acid  were found to alter the expression pattern of UGT94F2 and UGT86C4 in a time dependant manner. Furthermore, our results also show possible relationship between specific cis-elements identified within promoter sequences of UGT86C4 and UGT94F2, and their corroboration with the expression pattern of UGT86C4 and UGT94F2 in relation to exogenous application of different phytohormones. We anticipate that the present investigation leading to identification of two UGTs may enable to manipulate the production of picrosides in P. kurrooa for pharmacological applications.Considerable effort has been focused on understanding the biosynthesis of bioactive picrosides and enhancing their production. Chemosynthesis and manipulation of cell and tissue cultures for picrosides production has also been investigated P. Kurrooa were originally collected from a private property (Hotel Snowland Resorts), Sonamarg area  after seeking proper permission from the owner of the property. P. kurrooa plants are cultivated therein for aesthetic purposes. In vitro cultures were initiated and established from field grown plants at Indian Institute of Integrative Medicine, Srinagar  Cultivated plants of P. kurrooa, total RNA was extracted from the leaf tissues using TRIzol reagent following the manufacturer’s instructions . Absorbance of RNA samples was determined using a NanoDrop® ND-1000 spectrophotometer . RNA quality was assessed by the optical density (OD) ratios at 260 nm/280 nm and by separation on 1% agarose gels followed by visual evaluation of band integrity. Total RNA was treated with DNase I  to eliminate genomic DNA present in the samples. For cDNA synthesis, 2 µg of DNase I treated total RNA was reverse transcribed using Revert-aid premium reverse transcription kit with an modified Adapter-oligo dT primer in a total volume of 20 µL for 1 h at 42°C as described in the instruction manual . The resultant reaction mixture was used as a template for PCR amplification.As a first step in identifying glucosyltransferase genes involved in iridoid O-glucosylation in pTZ57R/T cloning vector . Randomly selected cloned inserts were sequenced using a BigDye terminator cycle sequencing kit  with an ABI PRISM® 3130XL genetic analyser .Cloning of glucosyltransferase cDNAs was performed as described earlier by Nagatoshi et al. pTZ57R/T cloning vector . The ligated DNA was transformed in E. coli cells . The clones (25 colonies) were picked individually and grown in 10 mL of Luria-Bertani (LB) medium at 37°C overnight. The plasmid DNA from each clone was extracted using a DNA plasmid Miniprep kit  and sequenced using M13 primers. DNA sequencing was performed with ABI PRISM 3130XL Genetic Analyser and BigDye Terminator v.3.1 Cycle Sequencing Kit .The 5′ ends of two putative UGTs were obtained using the SMART™ RACE cDNA amplification kit . The 5′ RACE-ready first-strand cDNA was synthesized following the protocol provided by the manufacturer. For the first PCR amplification of UGT86C4, UGT1-5race-out and UPM  and subcloned into chemically competent E. coli cells . Primers used in the study are listed in By aligning 3′ RACE and 5′ RACE product sequences, the full-length cDNAs of UGT94F2 and UGT86C4 were obtained. The open reading frame (ORF) of both UGT94F2 and UGT86C4 were amplified using single-stranded cDNA template and gene-specific primers (http://www.expasy.ch/tools/dna.html) and the properties of deduced amino acid sequences were estimated using ProtParam (http://www.expasy.ch/tools/protparam.html) and Phobius (http://www.ebi.ac.uk/Tools/pfa/phobius/) programs.The full length nucleotide sequences obtained were translated using Translate tool (http://www.ebi.ac.uk) using default parameters. Neighbour-joining tree was constructed with MEGA 5.10 software Protein sequences were retrieved from the GenBank through BLASTp algorithm at the National Centre for Biotechnology Information (NCBI) using UGT94F2and UGT86C4 sequences as query and several UGT orthologs of different families from various plant species were selected. Sequences were aligned using the ClustalW program  (http://www.sbg.bio.ic.ac.uk/phyre2/html/) using the crystal structure of Arabidopsis thaliana UGT72B1 (Protein Data Bank (PDB) Id. 2VCH-A) as template for modeling of both the proteins. Protein refinements of UGT86C4 and UGT94F2 models were performed using Protein Preparation Wizard of the Schrodinger Suite 2012 (http://www.schrodinger.com/). The stereo chemical analysis of the modeled proteins was carried out using Ramachandran plot obtained from Procheck module of the SAVES server (http://services.mbi.ucla.edu/SAVES/). Ligand binding site was predicted using 3DLigandSite (http://www.sbg.bio.ic.ac.uk/3dligandsite/). Structurally, evolutionary, and functionally important regions were identified in deduced protein sequences by ConSurf (http://consurf.tau.ac.il/). Topology of the modeled UGT86C4 and UGT94F2 proteins was analysed using PDBsum (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html).The three-dimensional structure of UGT86C4 and UGT94F2 were performed using PHYRE2 server (Medicago truncatula (PDB Id. 3HBF). The ligand coordinates at the acceptor site of 3HBF were copied to UGT86C4 and UGT94F2 modeled structures. The grid was generated around the copied ligand at the acceptor sites of UGT86C4 and UGT94F2. The UGT86C4 and UGT94F2 proteins were prepared for docking calculations using the Protein Preparation Workflow . Ligand docking was carried out for both proteins UGT86C4 and UGT94F2, using Glide XP precision mode. Ligands used for the study included iridotrial, 7-deoxyloganetic acid, 7-deoxyloganetin, apigenin, kaempferol and naringenin.To gain an understanding of the molecular structure governing acceptor as well as donor specificity of UGT86C4 and UGT94F2, structural alignment of both the proteins was carried out with the crystal structure of UGT78G1 from P. kurrooa was isolated from fresh leaves using DNeasy Plant mini kit  according to manufacturer’s protocol with minor modifications DraI, EcoRV, PvuI and StuI respectively and recovered using a PCR purification kit . PCR purified digested DNA were then ligated separately to the AP adaptor (provided in the kit) to construct the genomic walking libraries. For isolation of UGT86C4 gene promoter, the primary PCR was performed using AP1 (provided with the kit) and gwUGT1-out as primers, and constructed libraries as template via the following protocol: 5 cycles at 94°C for 25 s and 70°C for 3 min; 30 cycles at 94°C for 25 s, 65°C for 3 min; and at 67°C for 7 min. The product was diluted 10-fold and used in nested PCR, which was performed using AP2 (provided with the kit) and gwUGT1-in as nested primers under the following conditions: 5 cycles at 94°C for 25 s and 70°C for 3 min; 30 cycles at 94°C for 25 s and 67°C for 3 min, and followed by 67°C for 7 min. For UGT94F2, gwUGT2-out and gwUGT2-in were used as primers for primary and nested PCR respectively, keeping rest of the conditions same as that for UGT86C4 promoter. The PCR products were purified and cloned into the pTZ57R/T cloning vector, and subsequently sequenced. Putative cis-acting elements upstream to start codon of the both UGTs were identified by searching PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE (http://www.dna.affrc. go.jp/PLACE/) databases.GenomeWalker DNA libraries were constructed following the user manual provided by the manufacturer . Genomic DNA of 260:OD280 nm absorption ratio (1.8–2.0) and the integrity of the preparations was ascertained by electrophoresis using 1.2% agarose gel containing formaldehyde. About 2 µg of total RNA was used to synthesize first strand cDNA primed with OligodT in a 20 µL reaction mix using Revert-aid Premium M-MuLV reverse transcriptase  following manufacturer’s instructions. Real-time PCR reactions were performed in triplicates using SYBR Premix Ex Taq  in 48-well optical plates using ABI StepOne Real-time qPCR system . The PCR reaction (20 µL) included 0.2 µL cDNA template, 200 nM each of the primers, and 10 µL SYBR Premix Ex Taq. The cycling parameters were 95°C for 20 s, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The specificity of each primer pair was validated by a dissociation curve (a single peak was observed for each primer pair). The primers used for real-time PCR analysis were designed with the help of Primer Express Version 3.0. . A constitutive active gene, β-actin was used as endogenous control (RtActinF and RtActinR).Total RNA from leaf, rhizome and inflorescence tissues were extracted as described above. DNA contamination was removed from the RNA samples using DNase I . RNA concentration was measured at 260 nm by a spectrophotometer . The quality and purity of the preparations were determined at ODANOVA). The real-time PCR amplification data were exported into Microsoft Excel and gene expression levels were calculated based on the comparative Ct method To study the modulation of expression of UGTs by MeJA, SA and 2, 4-D, leaves were excised at different time intervals and analysed for UGT86C4 and UGT94F2 gene expression. The experiments were repeated thrice and the data were analysed statistically (P. kurrooa were completely dried under gentle air stream (temperature 25±2°C and relative humidity 65±5%). The leaves, inflorescence as well as the rhizomes of the dried samples were ground separately into fine powder with a mortar and pestle. The powdered samples (10 g of each sample) were serially extracted with ethanol:water (1∶1) for 3 h at 25±2°C. The aqueous ethanolic extracts were filtered and dried by evaporation at reduced pressure using rotary evaporator at 40°C. The crude extracts of all the samples were dissolved in the methanol (20 mg/mL) and were filter sterilized with 0.22 µm membrane filters . Stock solutions of the pure reference compounds (1 mg/mL) were prepared in HPLC grade water and stored in a refrigerator at 4°C. From the stock solutions, working solutions for each reference compound were prepared by dilution with HPLC grade water. These working solutions of both the reference compounds (5 µL of each) were mixed together in equal volumes for further analysis. A 10 µL aliquot of this solution was taken for the calibration curve. The HPLC (Shimadzu CLASS-VP V 6.14 SPI model) equipped with RP-18e column , a photo-diode array detector (SPD-M10A VP model) and a pump (LC-10AT VP model) was used for analysis of picrosides. The analysis was carried out using a mobile phase of methanol:water (2∶3) which was delivered at a flow rate of 0.7 mL/min. The detection wavelength was set at 270 nm. Injection volume of the sample was 10 µL and the column temperature was set at 30°C.Plant samples of BamHI restriction site and reverse primers NOTUGT1R/NOTUGT2R that included a NotI site. PCR was performed using Phusion high fidelity amplification system . The resulting PCR products were digested with BamHI and NotI, gel purified, and ligated into the BamHI and NotI digested pGEX4T-2 vector . The construct was sequenced to confirm that the gene was in the correct reading frame. The constructs, pGEX-UGT94F2 and pGEX-UGT86C4 were transformed into the E. coli strain BL21 (DE3) according to the supplier’s recommendations . After transformation, colonies were selected on LB medium containing 100 µg/mL ampicillin. Individual positive colonies were grown overnight in 5 mL LB and 1% of overnight cultures were used to inoculate fresh LB and cells were grown at 37°C until the O.D of 0.5–0.6 was reached. 1 mM IPTG  was added to the exponentially growing cells to induce the gene expression and 1 mL of induced cultures were harvested after every 2 h interval for 8 h. The harvested 1 mL culture samples were centrifuged at 13000 g for 5 min at 4°C. The cells were re-suspended in 3 mL of extraction buffer  and disrupted by sonication on ice for 15–20 s burst time period. The lysate was centrifuged at 13000 g for 20 min at 4°C and the supernatant was loaded on 10% SDS-PAGE gel after heating the sample with 2 X SDS loading dye at 99°C for 10 min. Protein concentration was determined by Bradford assay using serum albumin as a standard.The entire open reading frames of UGT86C4 and UGT94F2 were amplified using first-strand cDNA as the template with forward primers BAMUGT1F/BAMUGT2F harbouring a P. kurrooa, a homology based strategy was employed taking advantage of the highly conserved amino acid motif among plant secondary product glycosyltransferases (PSPG box) located in the C-terminal region. Two forward degenerate primers were designed based on amino acid sequences F(L/V)(T/S)HCGWN and CGWNS(T/V)LE respectively in the conserved PSPG box of plant glucosyltransferases. Since there is only one conserved region in UGTs, we employed 3′ RACE strategy using a modified adapter-oligo-dT primer for the first strand cDNA synthesis. Partial cDNA fragments were amplified using the degenerate primers along with 3′ RACE adapter primer, and the fragments were cloned into pTZ57R/T cloning vector and analysed. Homology search using BLASTX algorithm revealed two partial cDNA fragments with their deduced amino acid sequences similar to the C-terminal sequences of various PSPGs in the database. Primers were designed using these two partial cDNA sequences and the full length cDNAs of both the glucosyltransferases were obtained using 5′ RACE PCR. The sequence information from 5′ and 3′ RACE allowed the design of gene specific primers to obtain the full-length transcripts.To identify glucosyltransferases involved in the iridoid O-glucosylation from http://www.flinders.edu.au/medicine/sites/clinical-pharmacology/ugt-homepage.cfm).The nucleotide sequence of full length cDNAs of both genes were submitted to NCBI GenBank under accession numbers JQ996408 and JQ996409. The amino acid sequences of both the cDNAs were named UGT86C4 (JQ996408) and UGT94F2 (JQ996409) as per the UGT nomenclature (P. kurrooa based on the conservation of amino acid domains of plant UDP-glycosyltransferases associated with secondary metabolism The full length cDNA of UGT86C4 (1641 bp) contains an open reading frame (ORF) of 1422 bp corresponding to a protein of 473 amino acids -A with aAnalysis of UGT86C4 and UGT94F2 sequence for N-terminal targeting signal or C-terminal membrane anchor signal using the Phobius Web-based program predicted trans membrane N-terminal domain for UGT94F2 , whereasArabidopsis thaliana AAM91353 (47%), Linum usitatissimum AFJ53011 (47%), Dianthus caryophyllus BAF75888 (46%), Lycium barbarum BAG80548 (44%), Gardenia jasminoides BAK55737 (33%), Catharunthus roseus BAK55749 (32%) and Medicago truncatula XP_003615832 (32%). UGT94F2 showed the highest amino acid sequence identity to other orthologs from Veronica persica BAI44133 (53%), G. jasminoides BAM28984 (47%), C. roseus BAH80312.1 (42%), Prunus persica AEJ88222.1 (41%) and Panax notoginseng AED99884.1 (40%). In case of glycosyltransferases, the phylogenetic relationships are not always an indicative of the possible role of these enzymes since the relationship between the degree of amino acid sequence identity and substrate specificity of the plant UGTs is highly complex. It has been observed that appreciably diverse UGTs often share the same substrates. It is amply illustrated by the two Dorotheanthus bellidiformis UGTs, UGT73A5 and UGT71F2. There is 20% amino acid sequence identity between these two UGTs Allium cepa UGTs designated AcUGT73G1 and AcUGT73J1 belong to the same UGT family but with different range of specificities. AcUGT73G1 enzyme exhibits very broad acceptor specificity whereas AcUGT71J1 has a very narrow specificity M. truncatula UGT71G1 and Arabidopsis UGT71C1 have glucosylation activity towards triterpene and coumarin in vitro, respectively Arabidopsis UGT71B proteins are able to glucosylate a range of different compounds such as abscisic acid and hydroxybenzoic acid Pairwise alignment of deduced primary structures of UGT94F2 and UGT86C4 showed that these are highly diverse, with only 22% identical at amino acid level and 47.8% identical at the nucleotide level. To ascertain the degree of evolutionary relatedness, Neighbour-joining phylogenetic tree was constructed with MEGA 5.10 software from the ClustalW alignment of UGT94F2 and UGT86C4 with number of UGT orthologs from different plants retrieved from the NCBI GenBank database. As shown in phylogenetic tree , UGT86C4Because sequence homology is far from being a definitive argument to describe the precise enzymatic activity of glycosyltransferase enzymes M. truncatula UGT71G1 Vitis vinifera VvGT1 A. thaliana UGT72B1 Arabidopsis UGT72B1 (pdb id. 2VCH-A) as the template  and assigned PMDB IDs, i.e., PM0078442 and PM0078441 for UGT86C4 and UGT94F2 respectively. Successfully modeled structures of UGT86C4 and UGT94F2 (PM0078442 and PM0078441) were subjected to PDBsum server for structural motif analysis. The presence of α-helix, β-sheets, turns, and coil were frequently observed in both the UGT proteins. UGT86C4 consists of 3 β-sheets, 6 β-α-β units, 1 β-hairpin, 12 strands, 21 helices, 25 helix-helix interacs, 46 β-turns and 12 γ-turns. UGT94F2 consists of 2 β-sheets, 8 β-α-β units, 1 β-bulge, 12 strands, 20 helices, 24 helix-helix interacs, 46 β-turns and 15 γ-turns  A. thaliana AtUGT72B1 (Pro-22), G. jasmonides UGT85A24 (Pro-25) and C. roseus UGT85A23 (Pro-28), all known to use UDP-glucose as sugar donor and act as UDP-glucosyltransferases  . Noguchisferases . Therefocis-regulatory region of the important pathway genes. In Arabidopsis, there is clear evidence that R2R3-MYB transcription factors are able to activate glycosyltransferase expression Picrorhiza UGT genes correlates with transcriptional regulation via their promoters, upstream region of both the UGT genes were isolated and scanned for putative cis-regulatory elements using the PlantCARE and PLACE databases.Variation in the picroside accumulation pattern in different organs could be mainly because of the differences in the cis-elements including eight potential TATA box sequences within the promoter region of both the genes . Another consensus eukaryotic cis-element CAAT box, was also observed at positions −9, −166, −68, −448, −166, −68 and −448 in case of UGT86C4, while in case of UGT94F2, CAAT box was observed at −188, −405, −257, +477, −223 and +258. Scanning of promoter boxes also resulted in the identification of several cis-elements including numerous light-responsive elements, some sugar-responsive elements, hormone-responsive elements, stress-responsive elements, and a meristem-specific activation element  correlated well with the 5′ RACE analysis and is located at 45 bp upstream in case of UGT86C4 and 80 bp upstream in case of UGT94F2, with respect to the ATG start codon . Both th element ; Table 2P. kurrooa, total RNA was isolated from rhizomes, leaves and inflorescence and subjected to qPCR analysis using gene specific primers  is induced in a 2, 4-D-free medium and can be further increased by the addition of MeJA C. roseus ORCA3, a transcription factor controlling many MIA biosynthetic genes MeJA is known to induce the biosynthesis of many secondary metabolites that play important role in the adaptation of plants to biotic stress P. kurrooa, 2, 4-D increased the picroside levels, although the increase was less as compared to the effect of MeJA or SA.With SA, our study shows differential transient increase in accumulation of UGT86C4 and UGT94F2. UGT94F2 showed maximum increase of 10-fold after 12 h of induction which corroborated well with the picroside accumulation. While as UGT86C4 showed 16-fold increase in the expression profile 6 h post induction . The resP. kurrooa including rhizomes, leaves and inflorescence were analysed for picroside accumulation using high performance liquid chromatography (To corroborate the expression pattern of UGT86C4 and UGT94F2 with the level of picroside accumulation in different tissues of tography . The picTo comprehend the role of glucosyltransferases in the biosynthesis of picrosides and to establish a correlation between the change in picroside flux and the change in gene expression of glucosyltransferases (UGT86C4 and UGT94F2), the effect of MeJA, SA and 2, 4-D treatments on the biosynthesis of a key picrosides was analysed. These elicitors have been found to modulate the important secondary metabolic pathways P. kurrooa. These results are further endorsed by the molecular docking studies, wherein iridoid moieties especially 7-deoxyloganetin presented high binding affinity of towards UGT94F2.Interestingly, the change in picroside flux upon elicitor treatment corroborated well with the change in expression profile of UGT94F2 . All theBamHI and NotI restriction enzymes and inserted into vector pGEX4T-2 yielding pGEX4T2-UGT86C4 and pGEX4T2-UGT94F2 respectively. The expression constructs were checked for in-frame fusion by DNA sequencing. The gene constructs were transformed into competent BL21 cells and their expressions were induced by the addition of 0.2 mM IPTG at approximately OD600 = ∼0.5. This resulted in the appearance of a new fusion polypeptide with an expected molecular mass of approximately 68 kDa when resolved on SDS-PAGE. Time-course study revealed that the optimum expression of UGT86C4 as examined from SDS-PAGE profile was obtained after 6 h of induction at 37°C  and UGT94F2 (b). 5′ UTR and 3′ UTR are underlined. The dash marks the translation termination codon.(TIF)Click here for additional data file.Figure S2Multiple sequence alignment of deduced amino acid sequences of UGT86C4 and UGT94F2. Multiple sequence alignment of UGTs identified from P. kurrooa  with other plant orthologs using ClustalW2 multiple alignment tool. Arabidopsis thaliana (AtUGT72B1), Medicago tranculata , Gardenia jasmonoides (GjUGT85A24), Catharunthus roseus (CrUGT85A23) and Vitis vinifera (VvGT1). The highly conserved PSPG box is shaded in dark grey. Catalytically important amino acid residues are also shown.(TIF)Click here for additional data file.Figure S3Transmembrane domain prediction of UGT86C4 (A) and UGT94F2 (B) using Phobious web server.(TIF)Click here for additional data file.Figure S4Prediction of putative glycosylation sites on UGT86C4 (A) and UGT94F2 (B) using NetNGlyc 1.0 server.(TIF)Click here for additional data file.Figure S5Structural alignment of template (2vch-a) with the predicted three dimensional structures of UGT86C4 and UGT94F2. Template (2vch-a) is shown in cyan color and UGT86C4 is shown in purple color while as UGT94F2 is shown in brown color.(TIF)Click here for additional data file.Figure S6Ramachandran plot of two UGTs. Ramachandran plot of UGT86C4 (A) and UGT94F2 (B). The plot calculations on the 3D models of Picrorhiza UGT proteins were computed with the PROCHECK server. Most favoured regions are coloured red , additional allowed , generously allowed , and disallowed regions are indicated as yellow, light yellow and white regions, respectively.(TIF)Click here for additional data file.Figure S7Secondary structure analysis of UGT86C4 and UGT94F2 by linear depiction of UGT86C4 (A) and UGT94F2 (B) secondary structure.(TIF)Click here for additional data file.Figure S8Chemical structures of flavonoid and iridoid aglycones used as the glucosyl acceptors to examine the substrate specificity of UGT86C4 and UGT94F2 using molecular modeling and docking analysis.(TIF)Click here for additional data file.Figure S9Time-course heterologous expression of UGT86C4 (A) and UGT94F2 (B) in E. coli. Total protein extracts of E. coli cells were used for detection by SDS-PAGE and stained with coomassie blue. Lane M, Protein Molecular Weight Marker. Lane C, induction of control (vector only), expression of recombinant protein after 0 h (U), 2 h, 4 h and 6 h after induction with 0.2 mM IPTG at 37°C.(TIF)Click here for additional data file.Table S1List of primers used in the study.(DOCX)Click here for additional data file.


Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking.dN/dS ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding.Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance. Influenza virus belongs to the viral family Orthomyxoviridae and has a segmented negative-sense RNA genome in an enveloped virion Genetic mutation is considered one of the most important molecular mechanisms in the evolution of influenza virus Influenza virus has also shown the propensity to escape immunity because of continuous antigenic drift, i.e., mutation at the epitope positions of HA and NA segments Each of the influenza viral genes is thought to be important in viral replication and interaction with host cells; therefore, understanding the evolutionary tempo and mode of each viral gene can provide new insight into the epidemiology of influenza viruses −3 substitution/site/year) in previous studies Influenza A viral neuraminidases are classified into nine subtypes (N1–N9) according to their antigenic properties, whereas influenza B neuraminidases are classified into two lineages The Maximum Likelihood (ML) and MCMC Bayesian analyses demonstrate that the influenza NA gene diverged first into A and B (Group I and Group II), followed by the division of influenza A subtypes , File S1A total of 23 lineages, two to three lineages for each subtype, were identified within influenza A viruses, while two lineages were classified within influenza B . LineageThree lineages, 1A, 1B and 1C, were identified based upon strong bootstrap support values (100%) of the phylogenetic tree, which was generated from 4,146 sequences . The genSublineage 1A.1 originated from the recent highly pathogenic H5N1 avian influenza epizootic that started in Asia around 1996 and has spread throughout the Eastern Hemisphere. The viruses in 1A.1 are mostly from birds , but some are from humans (n = 164), swine (n = 8), tigers (n = 2) and mink (n = 1). Sublineage 1A.2 is composed of mostly Eurasian avian influenza viruses (n = 230), whereas some human highly pathogenic H5N1 influenza viruses (n = 24) sampled in 1997 in Hong Kong were also found in 1A.2. Sublineage 1A.4 consists of Eurasian swine influenza viruses which were originally derived from Eurasian avian viruses and first detected in Belgium in 1979. Not surprisingly, 1A.3 (Pandemic H1N1 2009) is grouped together with Eurasian swine, which confirms previous findings that the NA segment of pandemic H1N1 2009 viruses originated from the Eurasian swine influenza viruses. Sublineage 1A.5 is composed of viruses mainly from North American avian species (n = 162), with a few exceptions: 1 viral sequence from human and 3 from environmental samples.Lineage 1B consists of mainly North American swine influenza viruses, while 1C is a human lineage, consisting mainly of H1N1 human influenza viruses. The viruses in 1B correspond mostly to the classical H1N1 isolates from swine (n = 126), but include 9 isolates from humans and 9 from birds, indicating sporadic interspecies transmissions of influenza viruses from swine to humans or birds. Lineage 1C consists predominantly of human viruses (n = 1204), with a few exceptions, namely, swine (4 isolates) and birds (2 isolates). Within the influenza A N1 subtype, avian influenza viruses include sequences from multiple HA subtypes , whereas human and swine viruses have limited HA subtypes .The N2 sequences  were classified into two major lineages, 2A and 2B . The genThe 2A.1 is a subtype-specific sublineage consisting of mainly H9N2 avian influenza viruses, with the majority from birds (n = 412), but with 24 sequences from swine and 4 from humans, which indicates the occurrence of interspecies transmissions. The 2A.2 and 2A.3 correspond to Eurasian and North American avian viruses, respectively. The viruses of 2A.2 are mainly from birds (n = 342), but a few are from swine (n = 7) and humans (n = 2). A similar result was also found in 2A.3, which includes 291 avian viruses, 1 H7N2 human virus, and 29 viruses isolated from environmental samples.Within 2B, most of the influenza viruses are from human H2N2 and H3N2 influenza viruses  and swine H3N2 and H1N2 viruses (n = 214). However, avian influenza H3N2 viruses (n = 11) were also found in this lineage. Interestingly, there were five major clades of swine influenza viruses scattered within lineage 2B, suggesting these viruses originate from human viruses through either genome reassortment or direct transmission events. It is also noted that the branch lengths of the swine clusters are much longer as compared to those of the closely related human viruses, indicating extensive evolution of the N2 gene in swine viruses after transmission from humans to swine.Three lineages, 3A, 3B, and 3C, were found in N3, with genetic distances between lineages ranging from 0.173 to 0.349 . LineageThe N4, N5 and N6 subtypes were each classified into two lineages, one corresponding to North American avian  and the other Eurasian/Oceanian avian  . The genThree lineages were identified in N7 and N8, which correspond to North American avian , equine  and Eurasian/Oceanian avian , respectively -D. For NThe NA genes of influenza B viruses were divided into two distinct lineages, B/Victoria/2/87-like (Vic87) and B/Yamagata/16/88-like (Yam88) . All infOutliers were identified and removed before the estimation of substitution rate and tMRCA for each lineage . The mea−3 subs/site/year (−3) found in earlier branches (blue) and a high rate (4.20×10−3) in later branches (red)  in earlier branches (red) and a low rate (0.9×10−3) in later branches (blue) (−3) during 1918–1957 (blue) and a high rate (2.9×10−3) after 1977 (red)  -A. In cos (blue) -B. It is77 (red) -C.−3 (−3 in the majority of branches (red) and a low rate (2.6×10−3) in a small number of branches (blue) (−3) to late branches (blue) (1.6×10−3) and averaged at 2.65×10−3 (−3 (−3 in the majority of branches (red) and a rate of 1.5×10−3 in a small number of branches (blue)  -D. The s.65×10−3 -E. The i10−3  -F. DiffeThe time of most recent common ancestor (tMRCA) varies from lineage to lineage . The tMRdN) to synonymous (dS) substitutions per site (dN/dS) (N/dSd ratio was observed in 2B - human N2 lineage (0.313), which was slightly higher than that of 8B - equine N8 lineage (0.281), 1C - human N1 lineage (0.261), 1A.1 - H5N1 (0.274) and 2A.1- H9N2 (0.252), most likely reflecting host immune selection pressure, as a result of continuous circulation within the respective hosts and/or vaccination. The lineages under the most purifying selection were lineage 9C (0.068), 4B (0.062) and 5B (0.078). In comparison, the N/dSd ratios for influenza B lineages were comparable: 0.259 for Yam88 and 0.257 for Vic87.Different selection pressures were revealed in different lineages as indicated by the ratio of non-synonymous (N/dS) . Within Human lineages were found to have the largest numbers of positively selected sites, with 16 sites for the human N2 lineage (2B), 9 sites for human H1N1 lineage (1C), and 8 sites for Yam88 lineage . In addiProtein structure analyses revealed all the positively selected sites were located at the surface of the NA protein and pertained to antibody binding and/or interactions with the sugar molecules of host cells , S16. In2+ ion interacts with positions 379, 389, 387, 382, and 381, forming H-bonds with position 385 and position 383. These interactions are crucial in protein folding to create the appropriate tertiary structure for sialic acid binding  or for NA inhibitor binding.In the human H1N1 lineage (1C), amino acid positions 151, 222 and 344 were found to be under a strong positive selection, and the amino acids in these appear to interact with the NA inhibitor – zanamivir, a drug molecule according to the NA structure -A. In adWith regard to another human lineage (2B), positions 126 and 127 were found to be within the binding pocket of influenza A virus -B. TheseFor human influenza B, positions 42, 65, 248, 345, 373, 389, 395, and 436 were found to be under positive selection . The cryThe ML and Bayesian MCMC analyses revealed that the divergence of influenza A and B NA genes occurred earlier than the divergence of influenza A NA subtypes. Similar findings were reported for the hemagglutinin (HA) genes In this study, 23 NA lineages were determined within influenza A based upon both theoretical  and empirical criteria . The majority of lineages were found to be specific in hosts, or geographical locations, with a genetic distance around 0.2, ranging from 0.117 to 0.349. These results are generally consistent with previous findings Classification and designation of the lineages and sublineages within the influenza A virus are essential for studies of viral evolution, ecology and epidemiology. However, how to accurately identify an evolutionary lineage of influenza A viruses is challenging. Whether the naming system will be accepted and used by influenza researchers is even more challenging. To trace the evolutionary change of highly pathogenic avian influenza (HPAI) viruses, a hierarchical nomenclature system for HPAI hemagglutinin clades and sub-clades has been implemented by the WHO/OIE/FAO H5N1 Evolution Working Group and widely adapted by the research community It is notable that substitution rates are not the same across all branches within a phylogenetic tree. The relaxed clock model was developed to cope with this issue. An average rate across all branches in the tree is estimated under relaxed clock model in BEAST with 95% HPDs summarized from average rates, which are estimated from the sampled trees This study demonstrated that human influenza viruses were shown to have little geographical restriction, indicating that human viruses were transmitted globally and probably rapidly as well dN/dS) ratios , suggesting strong immune selection in viruses persistently circulating in humans Lineage 2B includes human influenza viruses isolated from two different subtypes, H2N2 between 1957 and 1968 and H3N2 after 1968, which share the same N2 gene maintained in human influenza virus after the antigenic shift from H2 to H3 occurred in 1968 dN/dS ratio of the NA genes of pandemic H1N1 2009 was 0.226, which was higher than the ratio of the closely related swine NA genes (0.100) In addition to the above discussed human lineages, pandemic H1N1 2009 influenza viruses are believed to have arisen from a reassortment between North American and Eurasian swine lineages, and as expected, the pandemic H1N1 2009 viruses grouped with the Eurasian swine lineage dN/dS ratios. Similar observations were also described in a previous study Influenza viruses circulating in non-human species have evolved in association with their various hosts on different continents for extended periods of time The two subtype-specific avian sublineages, 1A.1 for H5N1 and 2A.1 for H9N2, are considered to have pandemic potential and were found to evolve relatively faster compared with other avian lineages from multiple subtypes . In addiTwo lineages, H7N7 (7C) and H3N8 (8B), were revealed in equine influenza viruses. The H7N7 equine influenza viruses have not been detected since the late 1970s dN/dS ratios were observed in both swine lineages, suggesting comparable selection pressures occurred in both lineages Two major swine virus groups, Eurasian (avian-like) swine (1A.4) and North American swine (1B), were found within N1 . Our obsComplicated evolutionary dynamics were observed in lineage 2B. Within this major human lineage, five separate sub-clusters of swine viruses occurred in North America and Eurasia, suggesting that human-origin N2 genes were transmitted to swine in at least five separate instances -B and 6.In addition to the complexity found in Eurasian swine N2 viruses, similarly, in North America in 1998 there were outbreaks of influenza observed in swine herds in Minnesota, Iowa, and Texas. The outbreaks were caused by a triple-reassortant H3N2 virus which contained genes from human , swine , and avian (PB2 and PA) influenza viruses In summary, we analyzed 14,328 influenza A and B NA sequences and studied the evolutionary history and phylodynamics of the influenza NA gene. The divergence of influenza NA into influenza A and B NA occurred first, and nine NA subtypes further diverged within influenza A, with two to three lineages identified within each NA subtype. The analyses of substitution rates, dN/dS ratio, selection sites and protein structures revealed important associations between mutations and antiviral drug resistance/vaccine escape. Further analyses of other influenza segments are needed in order to obtain a comprehensive understanding of influenza virus evolution, which will facilitate influenza surveillance and control.http://sysbio.harvard.edu) was used to remove identical sequences, which resulted in 10,679 NA sequences, including 10,001 influenza A and 678 influenza B sequences. The influenza A NA sequences were divided into nine datasets (one for each subtype) that consist of 4146, 3754, 351, 85, 128, 488, 189, 684, and 176 sequences respectively for N1–N9.A total of 14,328 neuraminidase (NA) nucleotide sequences longer than 1330 nts, excluding laboratory recombinant sequences, were downloaded from the Influenza Virus Resource at NCBI Homologous gene recombination was identified using the 3SEQ algorithm under RDP3 The SeqMat program was used to collapse similar sequences from the same location and the same year, which results in ∼1500 representative sequences, respectively, for N1 and N2 Influenza A and B NA sequences are remotely related with around 40% nucleotide sequence similarity. We thus conducted both protein and nucleotide sequence alignments using Expresso - a program based upon protein structural information for alignment and TranslatorX - a program referring to the corresponding protein sequence alignment to align nucleotide sequences, respectively http://hcc.unl.edu/main/index.php). The trees were visualized and color-coded using FigTree (version 1.3.1) (http://tree.bio.ed.ac.uk/software/figtree/) to demonstrate tree topologies and corresponding hosts, subtypes and geographic locations.Phylogenetic analysis was conducted using the Maximum-likelihood (ML) method in RAxML Lineages were determined based upon the topology of phylogenetic trees and strong bootstrap support values (100 for influenza A and approximately 90 for influenza B). The genetics distances between lineages were calculated using the Kimura-2-Parameter (K2P) distance matric under MEGA 5.0 The substitution rate and the time of most recent common ancestor (tMRCA) were estimated for each lineage/sublineage using the Bayesian Markov Chain Monte Carlo (MCMC) method available in the BEAST package Three clock models were compared statistically for each dataset using a Bayes factor test in the Tracer program dN) to synonymous (dS) substitutions per site (ratio dN/dS) were estimated using the single likelihood ancestor counting (SLAC) method available in the HYPHY package dN/dS estimates were based on Maximum-likelihood trees under the GTR + Г substitution model. Protein structures of template NAs used in structural analyses were downloaded from the Protein Data Bank (www.pdb.org). Positively selected sites were mapped on the structure of the protein using Molecular Operating Environment (MOE) The ratio of non-synonymous  tree of influenza A N3 genes. Three lineages, denoted 3A, 3B and 3C, were classified. The bootstrap values supporting the corresponding lineages are shown on the major nodes. The scale bars indicate the numbers of nucleotide substitutions per site.(TIF)Click here for additional data file.Figure S2Maximum-likelihood (ML) tree of influenza A N4 genes. Two lineages, denoted 4A and 4B, were classified. The bootstrap values supporting the corresponding lineages are shown on the major nodes. The scale bars indicate the numbers of nucleotide substitutions per site.(TIF)Click here for additional data file.Figure S3Maximum-likelihood (ML) tree of influenza A N6 genes. Two lineages, denoted 6A and 6B, were classified. The bootstrap values supporting the corresponding lineages are shown on the major nodes. The scale bars indicate the number of nucleotide substitutions per site.(TIF)Click here for additional data file.Figure S4Maximum-likelihood (ML) tree of influenza A N7 genes. Three lineages, denoted 7A, 7B and 7C, were classified. The bootstrap values supporting the corresponding lineages are shown on the major nodes. The scale bars indicate the number of nucleotide substitutions per site.(TIF)Click here for additional data file.Figure S5Maximum-likelihood (ML) tree of influenza A N9 genes. Three lineages, denoted 9A, 9B and 9C, were classified. The bootstrap values supporting the corresponding lineages are shown on the major nodes. The scale bars indicate the number of nucleotide substitutions per site.(TIF)Click here for additional data file.Figure S6The structure of 1A.1 influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S7The structure of 1A.2influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S8The structure of 1A.3 influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S9The structure of 1A.4 influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S10The structure of 1A.5 influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S11The structure of 1B influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S12The structure of 2A.1 influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S13The structure of 2A.3 influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S14The structure of 5A influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S15The structure of 6A influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Figure S16The structure of 8A influenza neuraminidase, with positive selection sites denoted as green balls.(TIF)Click here for additional data file.Table S1The number of sequences of each lineage and the number of outliers identified by residual analysis.(DOCX)Click here for additional data file.File S1The phylogenetic tree of influenza A and B neuraminidase sequences.(TREE)Click here for additional data file.File S2The alignment of influenza A and B neuraminidase sequences. The quality of the alignment is indicated by different colors.(DOCX)Click here for additional data file.


Oikopleura dioica, a member of the sister group to vertebrates, exhibits profound modification of this ancestral architecture.Histone variants alter the composition of nucleosomes and play crucial roles in transcription, chromosome segregation, DNA repair, and sperm compaction. Modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates, but the urochordate, O. dioica lacks homologs of macroH2A and H2AX. The genome encodes many H2A and H2B variants and the repertoire of H2A.Z isoforms is expanded through alternative splicing, incrementally regulating the number of acetylatable lysine residues in the functionally important N-terminal "charge patch". Mass spectrometry identified 40 acetylation, methylation and ubiquitylation posttranslational modifications (PTMs) and showed that hallmark PTMs of "active" and "repressive" chromatin were present in O. dioica. No obvious reduction in silent heterochromatic marks was observed despite high gene density in this extraordinarily compacted chordate genome.We show that a histone complement of 47 gene loci encodes 31 histone variants, grouped in distinct sets of developmental expression profiles throughout the life cycle. A particularly diverse array of 15 male-specific histone variants was uncovered, including a testes-specific H4t, the first metazoan H4 sequence variant reported. Universal histone variants H3.3, CenH3, and H2A.Z are present but These results show that histone gene complements and their organization differ considerably even over modest phylogenetic distances. Substantial innovation among all core and linker histone variants has evolved in concert with adaptation of specific life history traits in this rapidly evolving chordate lineage. In eukaryotes, chromatin is the interface through which genetically encoded information is read to orchestrate a diversity of cellular and organismal functions. Chromosomal packaging of nuclear DNA is achieved through small basic histone proteins. The highly conserved core histones, H4, H3, H2A and H2B form an octamer of two H2A/H2B dimers flanking a central H3/H4 tetramer that wrap ~147 bp of DNA in 1.7 turns to form the nucleosome core particle (NCP) , the depMain lineages of canonical core histones, constituting the bulk of histone proteins, are assembled into chromatin during DNA replication. These replication dependent (RD) genes lack introns, are typically organized in gene clusters and their mRNAs possess a conserved stem-loop (SL) in the 3'UTR coupling gene expression to DNA replication. In contrast, histone variants are often transcribed from orphan genes that contain introns, lack the SL and their expression is not restricted to S-phase. Consequently, they are referred to as replacement or replication-independent (RI) variants. From an evolutionary perspective, histone variants are classified as universal or lineage-specific variants, where the term variant refers to non-allelic sequence variation. Universal variants have ancient functions common to eukaryotic cells, whereas lineage-specific variants appear specialized to certain organismal requirements. Core histones clearly differ in their evolutionary propensity to diversify as distinct variants. H4 is highly constrained as it makes contacts with the other 3 core histones and its N-terminal tail residues are subject to extensive PTMs. This is reflected in its nearly invariant stature. No H4 amino acid sequence variants have been reported in any multicellular organism to date and diversified H4s are known only in trypanosomes and ciliates ,11. DespL1) domains. Specializations have evolved in the H3 lineage with at least two H3 variants present in most eukaryotes. The replacement variant H3.3 can undergo RD or RI assembly and deposition is primarily in transcribed regions of euchromatin [The two H3 molecules interact with each other in the nucleosome, as do the two H2A molecules, whereas H4 and H2B do not. Two-fold nucleosomal symmetry is organized along the C-terminal four-helix bundle dimerization interface of the two H3s . Interachromatin ,14. H3.3hromatin . All eukhromatin .C. elegans and protozoan parasites, Plasmodium and trypanosomes. H2A.Z had a single evolutionary origin, and has remained distinct from canonical H2A [Among core histones, H2As have evolved the greatest variant diversity. There are two nearly universal variants, H2AX and H2A.Z. H2AX plays an important role in the maintenance of eukaryotic genome integrity by participating in double-strand DNA-break (DSB) repair by nonhomologous end joining (NHEJ). H2AX has arisen multiple times during evolution but similar constraints have led to convergent acquisition of the H2AX-specific phosphorylation motif SQE/DΦ (Φ = hydrophobic residue) . Phosphoical H2A . It is eical H2A -20 and iical H2A . A commoical H2A  combinedLinker H1 variants form a complex family and subtypes are classified as canonical RD (H1.1-H1.5 in human) or RI linker histones (H1x and H1.0 in human). They display tissue and developmental specificity, with vertebrates commonly expressing testes-specific  and oocyte-specific (H1oo) variants . StructuOikopleura dioica (Od), a member of the closest sister group to vertebrates, demonstrates that this ancestral genome architecture can be profoundly modified [O. dioica over a short, 6-day life cycle is achieved principally through endoreduplication [The above picture is derived from histone sequence comparisons in a wide array of eukaryotes, but to date, very few full histone complements have been analyzed more comprehensively and information is principally restricted to universal variants as well as some lineage-specific variants in mammals and plants. Modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates  but the modified . In thislication . Here, wO. dioica (Od) genome with most in clusters up to quintets. In total, 47 histone genes  encoding 31 different histone proteins  were identified  contains a high number (~130) of histone genes partly organized in large clusters, whereas the C. intestinalis genome (~156 Mb) encoded only 27 histone genes, mostly interspersed among non-histone genes. A locus within the C. savignyi genome was found where histone genes are arranged similarly to the tandem-arrays of sea urchin and Drosophila. Table To compare the  genome ~80 Mb conO. dioica histone genes were co-regulated in clusters and most genes belonging to the same locus showed the same developmental expression profiles  exclus Figure , 3) males Figure , 4) exprs Figure , 5) exprs Figure , 6) exprs Figure  and 7) ts Figure . Histones Figure , from 1 s Figure  or afters Figure .To improve resolution of germline-specific expression of different histone proteins, maturing D5/D6 animals were dissected and cDNAs prepared separately from testes, ovaries and trunks. Expression ratios were up to 4300-fold higher in testes and 330-fold higher in ovaries for certain isoforms  but do not show significant similarities to testes-specific H2BFWT of mammals, which also shows reduced sequence identity (45%) to mammalian somatic H2B [No replacement variants have been identified among H2Bs and all OdH2B genes are intron-less and contain the SL in their 3'UTR  highly enriched in testes . We did not find similar H3t genes within the genomes of the two Ciona species, suggesting these isoforms might be specific to the appendicularian lineage. A notable substitution within the N-terminus of H3t.3 is the change of A31 to a phosphorylatable T residue. The same position in H3.3 is S, which is phosphorylated in mitosis and meiosis and is the only known H3.3-specific modification [O. dioica canonical H3s  and α-helix 3, I119 and I124, which make contact with loop 1 (L1) and α-helix 3 of H4, respectively, are replaced by V119 and E124.Within chordates, testes-specific H3 variants have only been reported from mammals. OdH3ts, however, are more divergent than mammalian H3ts when compared to canonical H3.1/H3.2. OdH3t.3 has three residue substitutions in the N-terminal tail and ten in the histone fold, whereas mammalian H3ts exhibit only one and two substitutions, respectively. OdH3ts and mammalian H3ts share no common residue changes and phylogenetic analyses indicate that the H3ts of fication ,31. Withs Figure . FurtherO. dioica centromeric H3. OdCenH3 shows characteristic CenH3 features: a very divergent N-terminal tail, its L1 loop is extended and F84 in L2, which interacts with the L1 of H4, is replaced by W. When expressed as an eGFP fusion protein, OdCenH3 specifically localized to the centromeres of O. dioica chromosomes [Another intron-containing H3 gene lacking the SL encodes a divergent H3 replacement variant that we identified as the omosomes .O. dioica. H2A.1, encoded by 5 genes, exhibits the highest identity to canonical vertebrate H2As. H2A.1 and H2A.2, both expressed throughout development, differ by a single amino acid change from A to S at position 13 (confirmed by ESTs). O. dioica also expresses six male-specific isoforms  and two divergent replacement variants, H2A.3 and H2A.4, lacking the SL  contain a putative phosphorylation SQ-motif, adjacent to the C-terminal alpha helix, as a result of a residue change from A104 to S . OdH2A.Z differs from consensus H2A.Z sequences at several residues that are invariant in higher eukaryotes and similar in position to residue changes in Tetrahymena H2A.Z  or two (H2A.Zc) of the first three GK-pairs , genome size, and phylogenetic relationship do not appear to be overriding determinants of histone gene organization.Analyses of histone complements of higher eukaryotes have been published for metazoans such as osophila ,35, humaosophila , chickenosophila , C. eleg elegans Xenopus [[Xenopus  and sea [Xenopus . ComprehO. dioica histones. Whereas unicellular eukaryotes, such as yeast, Tetrahymena and Plasmodium principally exhibit modifications associated with transcriptional activation, mammals also employ a wider range of marks involved in gene silencing [Tetrahymena [O. dioica genome (one gene per 4-5 kb) and the short intergenic regions, we also expect the proportion of the O. dioica genome that is permanently silenced to be relatively low compared to that of human. However, our analysis revealed no obvious lack of silent heterochromatic marks in O. dioica, since methylation of H3K27 -repair pathway in ng γH2AX  is centrng γH2AX . Neverthng γH2AX  and the ng γH2AX . HR effing γH2AX . The chach locus  are avai pathway , includiTetrahymena revealed that acetylation of the N-terminus works to modulate an essential charge patch [Tetrahymena, in contrast to a site-specific histone code, is to alter the charge of the N-terminal domain. It is therefore intriguing that O. dioica uses alternative splicing to control the number of acetylatable K residues in the N-terminus of H2A.Z. Deployment of H2A.Z splice variants with altered numbers of acetylatable K residues would seem less dynamic than simply modulating the acetylation status of a fixed number of K-residues. However, in the endoreduplicative cell cycle variant used predominantly for growth in O. dioica, genes are disrupted transiently by passage of the replication fork but do not undergo the extensive condensation cycles occurring during the proliferative mitotic cell cycles characteristic of growth in most chordates. On the other hand, a challenge during non-polytene endocycles is to coordinately regulate hundreds of copies of a given gene dispersed in a single interphase nucleus [O. dioica.H2A.Z is one of the best studied universal core histone variants and, with the exception of CenH3, the only variant found to be essential in several species -20. H2A.ge patch  and viab nucleus . In this nucleus  displayi nucleus  suggestsO. dioica has a particularly high number of histone isoforms expressed exclusively or highly enriched in testes, including 6 H2At, 5 H2Bt, 3 H3t and 1 H4t. Testes-specific histones are poorly studied but are thought to substitute for canonical histones in meiotic and post-meiotic cells prior to replacement of histones by protamines [O. dioica histone isoforms exclusively expressed in testes  might affect intranucleosomal interactions. O. dioica H2At and H2Bt histones exhibit alterations in regions that promote stabilization of interactions with the H3-H4 tetramer and within the H2A-H2B dimer. Furthermore, several of the OdH2Bts have shortened N-terminal tails, lacking the first 14-21 N-terminal residues , predicted to form an α-helix in vertebrate H2Bs (residues 10-21) [otamines . Analyseotamines ,60 and Hotamines  indicates 10-21) . The N-ts 10-21)  and are O. dioica tiling array data confirm expression of a bromodomain-containing testes-specific factor (BRDT) [The massive synthesis of histone variants, including testes-specific members, and the stage-specific posttranslational modification of histones during spermiogenesis have led to proposal of a testes-specific "histone code" generated by combining both histone variants and PTMs . In zebrr (BRDT)  that indr (BRDT) , possiblO. dioica. In H3t.3, not only have the surrounding residues been modified, but K79 has been replaced by R, raising the possibility that the OdH3t isoforms lack K79 methylation. These residues are nearly invariant across species, though substitutions are present in pollen-specific H3s of Arabidopsis and rice, C. elegans H3.3 variants, and all H3s identified in Plasmodium and Tetrahymena  is found in euchromatin  but the bidopsis ,72 and Pasmodium ,73 may sasmodium ). A singO. dioica males express a H4 sequence variant appears unique. Expression of a male-specific H4 gene has been reported in rats [3 affects internucleosomal interactions resulting in more compact higher order chromatin structure [O. dioica H4.1 genes, a neutral function of these mutations is questionable.No H4 sequence variant has been described in higher metazoans and the fact that  in rats , but thitructure . In viewThe traditional view of histones as slow evolving proteins has changed considerably within the last decade. It is now clear that histone variants have diversified in many species to assume crucial roles in transcriptional activation, DNA repair, chromosome segregation and other processes. Recent data from more exotic model organisms such as trypanosomes  and rotiOikopleura genome [Oikopleura core histone fold domain sequences or linker H1 globular domain sequences [Xenopus macro H2A sequence, respectively. Sequences were confirmed by ESTs, RACE or sequencing from qRT-PCR products as indicated on the Oikopleura histone webpage [Oikopleura histone gene families are given in the legend of Additional File Oikopleura histone variants obtained from I-tasser [Xenopus nucleosome specified as a template . PDB files of Oikopleura histone variants were viewed and aligned to PDB files of nucleosome crystal structures (PDB files 1kx5 and 1f66) in Pymol . Databases and browsers used for analyzing histone genes: Ensembl [Ciona savignyi database [Ciona intestinalis JGI [Strongylocentrotus purpuratus [BLAST searches of the a genome ,80 were equences  and H1 t webpage . MethodoI-tasser  with the Ensembl , Histone Ensembl , Ciona sdatabase , Ciona ialis JGI  and Ghosalis JGI , and Strrpuratus .O. dioica culture was performed as described [in vitro fertilizations, females were collected in watch glasses, washed with artificial seawater  and left to spawn. Sperm from 3-5 males was checked for viability and used for fertilization. Embryos were left to develop at room temperature (RT) and subsequently frozen in liquid nitrogen. D2 - D6 animals were removed from houses and anaesthetized in seawater containing 3-aminobenzoic acid ethyl ester . For qRT-PCR at all developmental stages, animals from the same population were used. Two replicate populations were characterized. For preparation of histones, 4000 D4 and 1000 D6 animals were poured against a glass plate to separate them from their houses and collected in 1 L glass beakers. Sea water was replaced two times by 500 ml of seawater-MS222 and beakers left on ice for 10 minutes to sediment and harvest the animals. Mature sperm was collected by allowing 200 males to spawn in Petri-dishes and the sperm-sea water suspension was collected in 5 ml falcon tubes. Sperm was sedimented in a Sorvall centrifuge (RT 6000 D), 3000 rpm at 4°C for 10 min. For testes and ovary-specific samples, animals were selected and dissected as in [escribed . For in ed as in .RNA extraction and first strand cDNA synthesis was performed as previously . cDNAs fPCR was performed with CC212 forward (5' GGCGCGCAACTGAGAGAAATC 3') and CC213 reverse (5'CCAGCACTTCCGGCACC 3') primers, flanking the alternative splice sites with 5 μl D6 cDNA as template. After initial denaturation for 5 min at 95°C, 35 cycles of 95°C for 20 sec, 56°C for 15 sec and 72°C for 15 sec were conducted, with a final extension for 3 min at 72°C. PCR products were run on a 15% acrylamid gel with a 10 bp ladder (Promega). Bands were excised and acrylamid slices crushed in 400 μl TE, 8.2 μl 5M NaCl with subsequent nucleic acid precipitation by adding 1/10 vol 3M NaAc and 2.5 vol cold 100% EtOH. Precipitated PCR fragments were cloned into the Topo vector with the TOPO TA Cloning kit (Invitrogen) according to manufacturer's instructions.Histones were extracted using a histone purification kit  according to manufacturer's instructions and by the acid extraction method. For acid extraction, Lysis buffer [1 × PBS, 0.5% Triton X 100 Sigma, 5 mM PMSF, 100 mM NaCl, 10 mM Sodiumbutyrate, 1× Phosphatase inhibitor cocktail  was added to frozen sperm or D4/D6 animals. D4/D6 animals were dounced  in 1.5 ml eppendorf tubes and sperm was sonicated in the lysis buffer . HCl was added to 0.2 N and tubes were shaken vigorously for 2 h (D4/D6) at room temperature or overnight (sperm) at 4°C. Insoluble material was removed by centrifugation at 13000 rpm at 4°C for 15 min and Trichloracetic acid was added to 25% (vol/vol) to precipitate histones for 30 min at 4°C. Precipitates were collected by centrifugation at 14000 rpm for 15 min at 4°C and the supernatant was saved for later analysis. Histone pellets were washed twice with 500 μl acetone containing 0.1% HCl and dissolved in Laemmli loading buffer for subsequent mass-spectromery and silver gel analyses. Purified histones were loaded on 15% SDS-PAGE gels and stained with the Silver Stain Plus kit (Bio-Rad # 161-0461).Oikopleura. Initial search criteria were: Trypsin as enzyme; peptide charge 1+, 2+, and 3+ with a peptide tolerance of 10 parts per million for the parental peptide and 0.6 Da for fragmentation spectra. Fixed modifications were set to fixed carbamidomethyl modification for cysteines. Oxidation of methionine, deamidation (Q and N), acetylation (K), mono- and dimethylation (K and R), trimethylation (K), phosphorylation , and ubiquitinylation (K) were searched as variable modifications. Parent ion masses were internally calibrated by MSQuant [Histones were loaded on 15% SDS-PAGE gels, excised, reduced and alkylated . Digests MSQuant  to obtaiThe authors declare that they have no competing interests.AM, EMT and CC identified the histone complement, AM and MR performed the developmental expression profiles, PWTCJ and HGS performed the mass spectrometry, CC and CN assessed H2A.Z alternative splicing, AM, CC and EMT designed experiments and analyzed the data, AM and EMT wrote the manuscript. All authors approved the final manuscript.Additional Tables S1-S3 and additional Figures S1-S5. Table S1. Oikopleura dioica histone genes: stem-loop sequences and expression profiles. Table S2. Histone variants retained in mature Oikopleura dioica sperm. Table S3. Primer pairs used for quantitative RT-PCR. Figure S1. Phylogenetic relationships among Oikopleura dioica histone genes. Figure S2. Histones extracted from D4 and D6 animals. Figure S3. Spectra of O. dioica histone modifications obtained by LC-MS/MS. Figure S4. Alignment of mammalian somatic and testes-specific H2B variants with the canonical H2B of O. dioica. Figure S5. Histone H3 isoforms of species with substitutions surrounding the methylation site at K79. Additional Data References.Click here for file


In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32.  Signals initiated by transmembrane receptors at the cell surface are transmitted through the activity of guanine nucleotide exchange factors (GEFs) responsible for activation of small GTPases. Through their action on multiple effector molecules, GTPases enable regulation of diverse cellular functions like proliferation, actin reorganization, adhesion, motility, apoptosis and differentiation. The ubiquitously expressed GEF, C3G  functions in signaling pathways initiated by growth factors, integrins, T and B-cell receptors, cytokines, mechanical force etc and is known to target Rap1, 2, R-Ras and TC-10 C3G is a 140 kDa protein that primarily possesses a catalytic domain at the extreme C-terminus responsible for guanine nucleotide exchange and a central region comprising of multiple proline-rich sequences involved in protein interaction Knockout mice lacking C3G are embryonic lethal and show defects in multiple systems like vascular maturation and neural cortical development The T-cell protein tyrosine phosphatase (TC-PTP) is an intracellular PTPase expressed as two alternately spliced isoforms TC48 and TC45, which differ only in their C-termini 2. Cells were grown as monolayers in dishes for lysate preparation or on coverslips for immunofluorescence. Transfection of IMR-32 cells and Cos-1 cells were performed as described IMR-32, human neuroblastoma cells, HEK293, human embryonic kidney cells and Cos-1, monkey kidney epithelial cells, obtained from ATCC were maintained in DMEM containing 10% FCS at 37°C in a humidified incubator with 5% COPlasmids for expression of full length C3G, Y504FC3G, GST-CBR, GFP-c-Src, c-Src, GFP-TC48, GFP-TC45, GFP-TC48-C-66, and GFP-p23 have been described earlier Antibodies against C3G (C-19); pC3G (pY504-C3G), c-Src, Cdk2, p-Tyr and GFP were from Santa Cruz. TC-PTP antibody which recognizes both human isoforms was from Calbiochem. ERGIC-53 antibody was from Axxora, and giantin antibody was from Covance.in vitro binding assays were performed as described in vitro binding. Western blotting was carried out using standard protocols. Conditions for Western blotting of pC3G have been described Immunoprecipitations were carried out essentially as described Expression and localization of overexpressed or endogenous protein was determined in cells grown on coverslips. Essentially, cells were fixed in 3.7% formaldehyde in PBS for 10 mins and processed for staining as described Effect of TC-PTP on neurite outgrowth (NOG) in IMR-32 cells was determined by transfecting GFP-TC48 or mTC48 plasmids and inducing differentiation by 25 µM forskolin treatment for 72 hrs. NOG was quantitated as described by determining the number of cells with extensions greater than 2 cell diameters among the GFP expressing and non-expressing cells in vivo. We have earlier shown that cellular C3G is phosphorylated on Y504 upon treatment of cells with pervanadate (PV) As a first step towards exploring the functional regulation of C3G by TC-PTP, possible interaction between C3G and TC48 was tested by examining the ability of C3G to co-precipitate with a catalytically inactive substrate trap mutant, mTC48 (D182A). Mutant enzymes have been used to study interaction between tyrosine phosphatases and their substrates TC48 has earlier been shown to localize to the Golgi and ER in vitro pull down assay using a fusion protein of GST coupled to the Crk binding region of C3G encompassing 268-648 residues of C3G (GST-CBR), shown in in vitro pull down assays, we observed that C-terminal residues of TC48 (350-416) are sufficient for interaction with C3G  is known to induce the activity of cellular tyrosine phosphatases to modulate signaling pathways phatases . Examinaphatases .To examine the contribution of endogenous TC48, we tested the presence of pC3G in c-Src expressing cells co-transfected with mTC48. Catalytically inactive TC-PTP constructs have been earlier shown to act as dominant negatives to inhibit the function of endogenous TC48 We have earlier shown that C3G is phosphorylated at the Golgi dependent on endogenous SFK activation upon treatment of IMR-32 cells with Fsk or NGF Results from this study provide evidence for the identification of C3G as an interacting partner and substrate of TC48. The ability of TC-PTP to dephosphorylate C3G is dependent on its sub-cellular localization since expression of the nuclear isoform, TC45 did not dephosphorylate C3G. Distinct sub-cellular localization to defined cellular compartments often defines the role of PTPases in specific biological functions and this is the first report of a Golgi localized substrate for TC48. Evidence for C3G being a substrate of TC48 has been provided based on the following findings: (1) Better interaction between C3G and catalytically inactive TC48 which functions as a substrate trapping mutant, compared to WT TC48 within mammalian cells. (2) Inhibition of interaction between C3G and mTC48 by the phosphotyrosine mimetic, sodium vanadate. (3) TC48 expression causes dephosphorylation of endogenous C3G, but does not affect overall tyrosine phosphorylation in cells. (4) Inhibition of cellular TC48 function using a dominant negative mutant disables IGF mediated dephosphorylation of C3G. (5) Vanadate treatment inhibits TC48 mediated dephosphorylation of C3G.in vivo or that phophorylated C3G is targeted to the Golgi. Effectors of C3G function like Rap1 are known to localize to the Golgi and have also been shown to be specifically activated in distinct sub-cellular compartments C3G and TC48 appear to occupy distinct compartments, with C3G showing a more diffused staining with higher levels in the sub-cortical region and TC48 localizing in the nuclear membrane, Golgi and ER. Nevertheless, C3G and TC48 complexes form in the cellular context. Though C3G is distributed throughout the cytoplasm, PV or Fsk induced pC3G staining is seen in the core of the Golgi, indicating that either Golgi localized tyrosine kinases mediate C3G phosphorylation in vitro binding assays, we have identified that the C-terminal sequences of TC-PTP (350-381 amino acids) are sufficient for direct interaction with Crk binding region of C3G. This region of C3G is known to interact with SH3 domain containing molecules. Our study shows that it may also be capable of other types of previously undefined interactions. That stable interaction between full length TC-PTP and C3G was partially dependent on the catalytic domain was indicated by results showing that: 1) wild type TC48 differed in interaction with C3G compared to its catalytically inactive mutant, and 2) reduced interaction between mTC48 and phosphorylated C3G in the presence of vanadate. It therefore appears that the catalytic domain of TC-PTP may aid in the stability of interaction with C3G, primarily mediated through its non-catalytic sequences. This was evident from the fact that full length TC-PTP isoforms interacted better with C3G-CBR in in-vitro assays compared with the deletion constructs with only C-terminal sequences.Many TC-PTP substrates have been identified using substrate-trap approaches, wherein interaction between the phosphatase and its target is mediated through the catalytic domain and is dependent on substrate phosphorylation. Proteins like p97, p23, p25 and integrin α1, known to associate with non-catalytic sequences of TC-PTP, function in regulating its activity and localization Neurotransmitter induced neurite growth involves complex and coordinated participation of a number of signaling proteins These results describe the identification of the first intracellular tyrosine phosphatase in the regulation of C3G. Interaction with, and dephosphorylation by TC48 represents a novel mechanism of C3G regulation that is physiologically significant in the context of neurite growth in human neuroblastoma cells.Figure S1in vitro interaction assay. GST and GST-CBR coupled to glutathione agarose beads were incubated with lysates of HEK-293 cells expressing GFP-TC45 or GFP-C66. The WCL and washed beads were subjected to western blotting for GFP. The recombinant protein levels are shown in the Ponceau stained blot. M, protein molecular weight markers. * indicates position of GFP-TC45 and the GFP-C66 fusion proteins.C3G Crk binding region interacts with C-terminal regulatory sequences of TC-PTP. TC45 and a deletion construct of TC48, GFP-C66 (schematic shown in (TIF)Click here for additional data file.Figure S2TC48 localizes predominantly to the ER & Golgi in IMR-32 cells. IMR-32 cells expressing GFP-TC48 were stained for Giantin (Golgi marker) (A) or ERGIC-53 (ER-Golgi intermediate compartment marker) (B). Panels show images of a single section captured using 63X objective of Leica Confocal Microscope (A) or using 40X Objective of Carl Zeiss, Axioimager Z1 upright microscope. Bar, 10 µm.(TIF)Click here for additional data file.Figure S3in situ by indirect immunofluorescence. IMR-32 cells were transfected with C3G or Y504F mutant and left untreated (Utr) or subjected to PV treatment  and stained for C3G and pC3G. Endogenous pC3G is seen in the Golgi and membrane of all PV treated cells. WT C3G expressing cells show enhanced pC3G staining in pervanadate (PV) treated cells whereas Y504F mutant expressing cells do not. C3G and Y504F expressing cells are indicated by arrows. No pC3G signal is seen in untreated cells. Y504F mutant expressing cells show slightly weaker staining for endogenous pC3G by virtue of its ability to function as a dominant negative to inhibit phosphorylation of endogenous C3G. Bar 10 µm.pY504-C3G antibody specifically recognizes C3G phosphorylated on Y504. (A) Lysate of HEK293 cells expressing GFP-Src with either WT C3G or Y504F mutant were subjected to immunoprecipitation using control IgG or C3G antibody. The immunoprecipitates were subjected to Western blotting along with cell lysates (WCL) using the antibodies against pY504 C3G, C3G and pTyr. The pC3G antibody recognizes WT-C3G from cells co-expressing GFP-Src. No reaction is seen with Y504F mutant, which is recognized by pTyr antibodies indicating that this mutant is phosphorylated on tyrosine residues other than Y504. A dotted line has been added in the pTyr blot to indicate that probing of the blots corresponding to C3G and Src were carried out seperately. Level of C3G is shown for comparison. (B) Specificity of pC3G antibody to detect pY504 C3G (TIF)Click here for additional data file.Figure S4pC3G is dephosphorylated by cellular tyrosine phosphatases. IMR-32 cells were treated with PV  and left in normal medium (cDMEM) or medium containing 50 µM sodium vanadate for 4 hrs. Cells were fixed and stained for pC3G. Cells left in cDMEM show loss of pC3G whereas enhanced levels of pC3G were seen upon exposure to medium containing VO4. Bar, 10 µm. Utr: untreated cells.(TIF)Click here for additional data file.Figure S5TC48 inhibits PV induced pC3G in IMR32 and Cos-1 cells: (A) IMR32 cells expressing HA-TC45 or HA-TC48 were left untreated (Utr) or subjected to PV treatment and stained for pC3G. TC45 expression has no effect on pC3G while TC48 expressing cells showed decreased pC3G staining. Bar, 10 µm.  Cos-1 cells expressing GFP-TC45 or GFP-TC48 were left untreated (Utr) or subjected to PV treatment and stained for pC3G (B) and p-Tyr (C). While TC45 expression has no effect on either pC3G or pTyr staining in cells, TC48 expressing cells showed significantly less pC3G, but no difference in pTyr staining.(TIF)Click here for additional data file.Figure S6TC-PTP expression does not affect c-Src induced cellular pTyr. IMR-32 cells were transfected with c-Src along with either GFP, GFP-TC48, GFP-mTC48, or GFP-TC45. Fixed cells were stained to visualize expression of Src and pTyr. Images show fields captured using a fluorescence microscope. Bar, 10 µm.(TIF)Click here for additional data file.Figure S7IGF treatment does not affect Src induced pTyr on most cellular proteins. IMR-32 cells expressing GFP-Src were either left untreated or subject to 100 nM IGF treatment prior to preparation of whole cell lysates. Cell lysates were subject to Western blotting to detect total cellular pTyr levels. GFP blotting was performed to indicate expression of GFP-Src and CDK2 as loading control. UT, untransfected. pTyr band corresponding to C3G is indicated.(TIF)Click here for additional data file.


G6PC3 gene encodes the ubiquitously expressed glucose-6-phosphatase enzyme (G-6-Pase β or G-6-Pase 3 or G6PC3). Bi-allelic G6PC3 mutations cause a multi-system autosomal recessive disorder of G6PC3 deficiency . To date, at least 57 patients with G6PC3 deficiency have been described in the literature.The G6PC3 mutations in result in reduced enzyme activity, endoplasmic reticulum stress response, increased rates of apoptosis of affected cells and dysfunction of neutrophil activity.G6PC3 deficiency is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defects and uro-genital anomalies. The phenotypic spectrum of the condition is wide and includes rare manifestations such as maturation arrest of the myeloid lineage, a normocellular bone marrow, myelokathexis, lymphopaenia, thymic hypoplasia, inflammatory bowel disease, primary pulmonary hypertension, endocrine abnormalities, growth retardation, minor facial dysmorphism, skeletal and integument anomalies amongst others. Dursun syndrome is part of this extended spectrum. G6PC3 deficiency can also result in isolated non-syndromic severe neutropenia. G6PC3 gene. A number of G6PC3 founder mutations are known in various populations and a possible genotype-phenotype relationship also exists. G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia.In this review we demonstrate that loss of function in missense G6PC3 mutations likely results from decreased enzyme stability. The condition can be diagnosed by sequencing the Treatment with G-CSF leads to improvement in neutrophil numbers, prevents infections and improves quality of life. Mildly affected patients can be managed with prophylactic antibiotics. Untreated G6PC3 deficiency can be fatal. Echocardiogram, renal and pelvic ultrasound scans should be performed in all cases of suspected or confirmed G6PC3 deficiency. Routine assessment should include biochemical profile, growth profile and monitoring for development of varicose veins or venous ulcers. G6PC1, G6PC2 and G6PC3. No. No30]. −/− mice.g6pc3−/− mice that a specific CXCR4 antagonist, AMD3100, rapidly reversed neutropenia.Some patients with G6PC3 deficiency paradoxically demonstrate normal or hypercellularity of the bone marrow. McDermott et al. studied such a sibling pair and showed increased expression of CXCR4 in neutrophils. They prThe immune problems of G6PC3 deficiency extend beyond neutrophil dysfunction. Jun et al. demonstrated impaired macrophage respiratory burst, chemotaxis, calcium flux and phagocytic activities with down-regulation of reduced NADPH oxidase subunits and membrane translocation of p47phox,.G6PC3 maps to 17q21.31, consists of six exons and encodes the G6PC3 protein that is comprised of 346 residues and is anchored in the ER by nine transmembrane helices in a way that keeps the active site inside the ER lumen[6-RP-(X12-54)-PSGH-(X31-54)-SR-X5-H-X3-D, spans between residues 66 and 171 of G6PC3.ER lumen,6. HistiER lumen. The sigG6PC3 missense mutations resulting in amino acid substitutions in nine different codons have been identified. These missense mutations are spread all across the gene but no missense mutation has been described in exon 2. The largest number of mutated missense alleles have been described in exon 6. The other 48/114 mutated G6PC3 alleles result from four nonsense mutations, seven frameshift deletions or insertions and four splice-site mutations.G6PC3 deficiency can be diagnosed by sequencing all the six exons of the gene NM_138387.3). There are now at least 57 patients with genetically proven G6PC3 deficiency described in the literature ,27. HoweG6PC3 mutations have been identified more frequently in particular ethnic groups. The homozygous p.P44S mutation has been described in three unrelated Pakistani patients and in two of these patients it was confirmed to be present on a common haplotype[To date, a number of aplotype. Ten patG6PC3 missense mutations lead to loss of enzyme activity remains unclear. Notably, the protein structure of G6PC3 has not been defined. G6PC3 is a paralog of G6PC and the two genes, along with G6PC2, are thought to have arisen from ancient duplication events[G6PC) is better characterized[The mechanism by which n events. The strcterized. Due to cterized,6. The GG6PC3 mutants with reduced enzyme activity that have not been demonstrated in patients, but were created in the laboratory , also affect fully conserved sites[G6PC3 mutations are fully conserved in orthologs till the level of Xenopus   demonstrate complete conservation between G6PC3 and G6PC amino acid sequences . Hence, it is likely that the protein structure, the mutation spectrum and the molecular mechanisms underlying missense mutations in the two diseases are comparable.In GSD1a, 54 reported. Out of G6PC missense mutations have been classified into those affecting active site, helical and non-helical depending on their predicted topological position[position. StudiesG6PC3 mutations on the predicted protein structure shows that all nine missense mutations are predicted to be helical  have been reported in five patients with G6PC3 deficiency  have been described to date  onhttp://www.orpha.net.ER: Endoplasmic reticulum; IBD: Inflammatory bowel disease; G6PC3: Ubiquitous glucose-6-phosphatase; G-CSF: Granulocyte-colony stimulating factor; NADPH: Nicotinamide adenosine dinucleotide phosphate; PHT: Pulmonary hypertension.The authors have no competing interests to declare.Both authors planned the study. SB compiled and reviewed the data and wrote the manuscript. WGN reviewed the manuscript. Both authors read and approved the final manuscript.aG6PC3 deficiencyUbiquitous glucose-6-phosphatase deficiencySevere congenital neutropenia type 4Dursun syndrome (ORPHA178503)