WorldWideScience

Sample records for synthase gene promoter

  1. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk

    2016-09-01

    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  2. Functional analysis of a cotton cellulose synthase A4 gene promoter in transgenic tobacco plants.

    Science.gov (United States)

    Wu, Ai-Min; Hu, John S; Liu, Jin-Yuan

    2009-10-01

    A 1,482-bp promoter sequence of the cotton cellulose synthase gene (GhCesA4) was isolated from Chinese cultivar CRI12 of Gossypium hirsutum, and transcriptionally fused to a beta-glucuronidase (GUS) reporter gene for investigation of important regions controlling gene expression in transgenic tobacco plants. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in the roots, hypocotyls, vascular tissues of stems, trichomes, the central leaf veins, as well as in the anthers and pollen. Quantitative measurements of GUS activity demonstrated that higher expression levels were detected in the stems, fully expanded leaves, and styles of flowers. A series of 5' progressive deletions of the promoter revealed the presence of a negative regulatory region (-767 to -424) for promoter activity and a 247-bp fragment (-247 to -1) with the vascular tissue specificity of the basic transcription activity in the GhCesA4 promoter. Exposure of the transgenic tobacco to various abiotic stresses showed that the full-length construct predominantly responded to NAA, kinetin, and sugar. Furthermore, the NAA-response region was found to be located in the -1,482/-1204 fragment, while the element(s) for the sucrose-responsive expression may be present in the -247/-1 region in the GhCesA4 promoter. These findings will not only contribute to an explanation of the molecular mechanisms by which GhCesA4 participates in secondary cell wall morphogenesis and stress responses, but will also provide a good candidate for expression or accumulation of foreign genes of interest whose products are preferentially required in vascular tissues and are inducible under auxin treatment.

  3. β-Glucan Synthase Gene Overexpression and β-Glucans Overproduction in Pleurotus ostreatus Using Promoter Swapping

    Science.gov (United States)

    Liu, Dongren; Qi, Yuancheng; Gao, Yuqian; Shen, Jinwen; Qiu, Liyou

    2013-01-01

    Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi. PMID:23637884

  4. β-Glucan synthase gene overexpression and β-glucans overproduction in Pleurotus ostreatus using promoter swapping.

    Directory of Open Access Journals (Sweden)

    Ran Chai

    Full Text Available Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR, and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

  5. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

    Science.gov (United States)

    Ginis, Olivia; Courdavault, Vincent; Melin, Céline; Lanoue, Arnaud; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courtois, Martine; Oudin, Audrey

    2012-05-01

    The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

  6. Genetic variants in promoters and coding regions of the muscle glycogen synthase and the insulin-responsive GLUT4 genes in NIDDM

    DEFF Research Database (Denmark)

    Bjørbaek, C; Echwald, Søren Morgenthaler; Hubricht, P

    1994-01-01

    regions and regions of importance for translation, as well as coding sequences of the two genes, were studied using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The genetic analyses were performed in subgroups of 52 Caucasian NIDDM patients and 25 age-matched healthy......To examine the hypothesis that variants in the regulatory or coding regions of the glycogen synthase (GS) and insulin-responsive glucose transporter (GLUT4) genes contribute to insulin-resistant glucose processing of muscle from non-insulin-dependent diabetes mellitus (NIDDM) patients, promoter......'-untranslated region, and the coding region of the GLUT4 gene showed four polymorphisms, all single nucleotide substitutions, positioned at -581, 1, 30, and 582. None of the three changes in the regulatory region of the gene had any major influence on expression of the GLUT4 gene in muscle. The variant at 582...

  7. A single nucleotide polymorphism in glycogen synthase kinase 3-beta promoter gene influences onset of illness in patients affected by bipolar disorder.

    Science.gov (United States)

    Benedetti, Francesco; Bernasconi, Alessandro; Lorenzi, Cristina; Pontiggia, Adriana; Serretti, Alessandro; Colombo, Cristina; Smeraldi, Enrico

    2004-01-23

    Genetic studies in medicine exploited age of onset as a criterion to delineate subgroups of illness. Bipolar patients stratified with this criterion were shown to share clinical characteristics and patterns of inheritance of illness. The molecular mechanisms driving the biological clock in the suprachiasmatic nucleus of the hypothalamus may play a role in mood disorders. A single nucleotide polymorphism (SNP) (-50 T/C) falling into the effective promoter region (nt -171 to +29) of the gene coding for glycogen synthase kinase 3-beta (GSK3-beta) has been identified. GSK3-beta codes for an enzyme which is a target for the action of lithium and which is also known to regulate circadian rhythms in Drosophila. We studied the effect of this polymorphism on the age at onset of bipolar disorder type I. A homogeneous sample of 185 Italian patients affected by bipolar disorder was genotyped. Age at onset was retrospectively ascertained with best estimation procedures. No association was detected between GSK3-beta -50 T/C SNP and the presence of bipolar illness. Homozygotes for the wild variant (T/T) showed an earlier age at onset than carriers of the mutant allele (F=5.53, d.f.=2,182, P=0.0047). Results warrant interest for the variants of genes pertaining to the molecular clock as possible endophenotypes of bipolar disorder, but caution ought to be taken in interpreting these preliminary results and future replication studies must be awaited.

  8. Identification and Heterologous Expression of the Topopyrone Nonaketide Synthase Gene from Phoma sp.

    Science.gov (United States)

    Kashiwa, Nobuyuki; Ebizuka, Yutaka; Fujii, Isao

    2016-01-01

    Non-reducing iterative type I polyketide synthase genes, pnk1 and pnk2, were cloned from the fungus Phoma sp. BAUA2861, which produces the topoisomerase I inhibitors, topopyrones A to D. Heterologous expression of these polyketide synthase genes under the α-amylase promoter in Aspergillus oryzae was carried out to identify their functions. The pnk2 transformant produced topopyrones C, D, and haematommone. Therefore, the pnk2 gene was found to encode for the topopyrone nonaketide synthase.

  9. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-24

    May 24, 2010 ... NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained from the ... Key words: Periodontal diseases, nitric oxide synthases gene, DNA, PCR. INTRODUCTION ... various diseases' pathogenesis because of its dual role. *Corresponding author.

  10. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  11. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  12. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  13. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  14. Identification of a 467 bp promoter of maize phosphatidylinositol synthase gene (ZmPIS which confers high–level gene expression and salinity or osmotic stress inducibility in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Hongli eZhang

    2016-02-01

    Full Text Available Salinity and drought often affect plant growth and crop yields. Cloning and identification of salinity and drought stress inducible promoters is of great significance for their use in the genetic improvement of crop resistance. Previous studies showed that phosphatidylinositol synthase (PIS is involved in plant salinity and drought stress responses but its promoter has not been characterized by far. In the study, the promoter (pZmPIS, 1834 bp upstream region of the translation initiation site was isolated from maize genome. To functionally validate the promoter, eight 5´ deletion fragments of pZmPIS in different lengths were fused to GUS to produce pZmPIS::GUS constructs and transformed into tobacco, namely PZ1-PZ8. The transcription activity and expression pattern obviously changed when the promoter was truncated. Previous studies have demonstrated that NaCl and PEG treatments are usually used to simulate salinity and drought treatments. The results showed that PZ1-PZ7 can respond well upon NaCl and PEG treatments, while PZ8 not. PZ7 (467 bp displayed the highest transcription activity in all tissues of transgenic tobacco amongst 5´ deleted promoter fragments, which corresponds to about 20% and 50% of CaMV35S under normal and NaCl or PEG treatment, respectively. This implied that PZ7 is the core region of pZmPIS which confers high-level gene expression and NaCl or PEG inducible nature. The 113 bp segment between PZ7 and PZ8 (-467 to -355 bp was considered as the key sequence for ZmPIS responding to NaCl or PEG treatment. GUS transient assay in tobacco leaves showed that this segment was sufficient for the NaCl or PEG stress response. Bioinformatic analysis revealed that the 113 bp sequence may contain new elements that are crucial for ZmPIS response to NaCl or PEG stress. These results promote our understanding on transcriptional regulation mechanism of ZmPIS and the characterized PZ7 promoter fragment would be an ideal candidate for the

  15. The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose

    Directory of Open Access Journals (Sweden)

    Madoka eYonekura

    2013-03-01

    Full Text Available Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter–luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light–dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both POsSPS1::LUC and POsSPS11::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the 2 OsSPS genes.

  16. POTATO GRANULE-BOUND STARCH SYNTHASE PROMOTER-CONTROLLED GUS EXPRESSION - REGULATION OF EXPRESSION AFTER TRANSIENT AND STABLE TRANSFORMATION

    NARCIS (Netherlands)

    VANDERSTEEGE, G; NIEBOER, M; SWAVING, J; TEMPELAAR, MJ

    1992-01-01

    Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the beta-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient

  17. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence

    Science.gov (United States)

    2014-01-01

    Background Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Methods Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Results Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Conclusions Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene. PMID:24716800

  18. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  19. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... Full Length Research Paper. Sequence analysis of cereal sucrose synthase genes and isolation of sorghum sucrose synthase gene fragment. T. Sivasudha1* and P. A. Kumar2. 1Department of Environmental Biotechnology, Bharathidasan University, Tiruchy-620 024, India. 2NRC on Plant Biotechnology, ...

  20. Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L.

    Directory of Open Access Journals (Sweden)

    Hongzhen Wang

    Full Text Available In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS, epi-cedrol (ECS and β-farnesene (FS synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS, a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

  1. Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

    Science.gov (United States)

    Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E.

    2013-01-01

    In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production. PMID:24278301

  2. High diversity of polyketide synthase genes and the melanin biosynthesis gene cluster in Penicillium marneffei.

    Science.gov (United States)

    Woo, Patrick C Y; Tam, Emily W T; Chong, Ken T K; Cai, James J; Tung, Edward T K; Ngan, Antonio H Y; Lau, Susanna K P; Yuen, Kwok-Yung

    2010-09-01

    Despite the unique phenotypic properties and clinical importance of Penicillium marneffei, the polyketide synthase genes in its genome have never been characterized. Twenty-three putative polyketide synthase genes and two putative polyketide synthase nonribosomal peptide-synthase hybrid genes were identified in the P. marneffei genome, a diversity much higher than found in other pathogenic thermal dimorphic fungi, such as Histoplasma capsulatum (one polyketide synthase gene) and Coccidioides immitis (10 polyketide synthase genes). These genes were evenly distributed on the phylogenetic tree with polyketide synthase genes of Aspergillus and other fungi, indicating that the high diversity was not a result of lineage-specific gene expansion through recent gene duplication. The melanin-biosynthesis gene cluster had gene order and orientations identical to those in the Talaromyces stipitatus (a teleomorph of Penicillium emmonsii) genome. Phylogenetically, all six genes of the melanin-biosynthesis gene cluster in P. marneffei were also most closely related to those in T. stipitatus, with high bootstrap supports. The polyketide synthase gene of the melanin-biosynthesis gene cluster (alb1) in P. marneffei was knocked down, which was accompanied by loss of melanin pigment production and reduced ornamentation in conidia. The survival of mice challenged with the alb1 knockdown mutant was significantly better than those challenged with wild-type P. marneffei (P melanin-biosynthesis gene cluster contributed to virulence through decreased susceptibility to killing by hydrogen peroxide. © 2010 The Authors Journal compilation © 2010 FEBS.

  3. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  4. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... comparative analysis of grass genomes and as a source of beneficial genes for agriculture. Recent studies have shown that ... sequencing of sucrose synthase gene fragment from sor- ghum using primers designed at their ... Sequencing was carried out by Sanger dideoxy DNA sequencing method. Results.

  5. Association of endothelial nitric oxide synthase promoter region (T-786C gene polymorphism with acute coronary syndrome and coronary heart disease

    Directory of Open Access Journals (Sweden)

    Kılıçgedik M

    2008-02-01

    Full Text Available Abstract Background Nitric oxide (NO is an endothelium derived relaxing factor (EDRF which has an important role for regulating the heart-vessel physiology. The objective of this study was to evaluate the effects of the eNOS T-786C polymorphism on lipid parameters and the development of acute coronary syndrome (ACS and coronary heart disease (CHD for the first time in a Turkish study group. We have analyzed the genotype frequencies of the T-786C polymorphism of the eNOS gene in 10 ACS patients (5 men, 5 women, 20 CHD patients (14 men, 6 women, and 31 controls (10 men, 21 women, who were angiographically proven to have normal coronaries. Results The demographic, biochemical and left ventricule systolic dysfunction data of the ACS, CHD patients and controls were analyzed as a function of eNOS T-786C genotypes. The eNOS gene T-786C polymorphism frequencies for T/T, C/T and C/C genotypes were respectively 10%, 40%, 50% in subjects with ACS; 75%, 20%, 5% in subjects with CHD and 67.7%, 25.8%, 6.5% in the control group. Significant difference was observed in genotype frequencies between the study groups for T-786C polymorphism (p = 0.001. The CC genotype frequency was found to be the most prevalent in ACS group in comparison to CHD and control groups (p = 0.001. TT was the most frequently observed genotype in both CHD patients and controls (p = 0.001. Left ventricule systolic dysfunction frequency was found to be highest in C/T genotype carriers (66.7% in patients (ACS+CHD. None of the patients with LVSD were carrying the normal genotype (T/T. The eNOS T-786C polymorphism was not found to be effective over any analyzed lipid variable in patients (ACS+CHD. The HDL-cholesterol levels were found to be lower in CHD group were compared to controls (p Conclusion The significantly high frequency of eNOS -786C/C genotype in ACS patients than in those of controls, indicate the genotype association with ACS. The finding of significantly high frequency of T

  6. Clinical and anamnestic characteristics of patients with clinical manifestations of peripheral artery disease as a function of Т(–786С polymorphism of the endothelial NO-synthase gene promoter

    Directory of Open Access Journals (Sweden)

    V.I. Tseluyko

    2016-09-01

    Full Text Available The aim – to determine the prevalence of Т(–786С polymorphic gene variants of endothelial NO-synthase (eNOs in patients with verified atherosclerosis of lower extremities arteries and their association with clinical manifestations of the disease. Materials and methods. The study involved 100 patients with verified peripheral artery disease (PAD of the lower extremities. The control group included 35 people without changes of coronary arteries and with negative result of exercise stress test. The examination, apart from clinical tests, included determination of ankle-brachial index, selective coronary angiography, Doppler ultrasound of the lower extremities and carotid arteries as well as the estimation of their intima – media thickness. The study of allelic polymorphism of eNOs gene promoter was performed by polymerase chain reaction. Results. According to the results of genotyping of Т(–786С gene polymorphism of eNOs the share of T/T genotypes in the control group (45.7 % and in patients with PAD (33 % was not different (р = 0.18, and C/T more frequently found in the control group (48.6 % vs 26 %; p = 0.01, whereas the prevalence of the C/C genotype in patients with PAD was significantly higher than in healthy individuals (41 % vs 5.7 %; p = 0.0001. The patients were divided into three groups: I group – 33 people with T/T genotype, II group – 26 people with C/T genotype and IІI group – 41 people with C/C genotype. The average age of the manifestation of PAD in patients with genotype C/C was significantly lower (50.15 ± 0.63 years than in the group with the T/T genotype (58.45 ± 1.17 years; p = 0.001 and С/T (53.53 ± 1.28 years; p = 0.01. It was found that in the ІІІ group patients have more severe clinical manifestations of arterial insufficiency of lower limbs (according to Fontaine classification. The patients with C/C genotype had more severe course of the coronary artery disease (CAD: myocardial infarction in

  7. Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

    Directory of Open Access Journals (Sweden)

    Wen-Qin Bai

    Full Text Available Bioactive gibberellins (GAs comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

  8. Cloning and expression analysis of an anthocyanidin synthase gene ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 2. Cloning and expression analysis of an anthocyanidin synthase gene homologue from Brassica carinata. Mingli Yan Suping Ding Lili Liu Xiaoming Yin Jiabin Shu. Research Note Volume 93 Issue 2 August 2014 pp 513-516 ...

  9. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  10. Role of Endothelial Nitric Oxide Synthase Gene Polymorphisms ...

    African Journals Online (AJOL)

    Background: Previous studies indicated an association between endothelial nitric oxide synthase (eNOS) activity and maintenance of pregnancy, but it is rather controversial whether polymorphisms of the gene encoding for eNOS are associated with recurrent spontaneous abortions (RSAs). Aim: The aim was to investigate ...

  11. Molecular and functional evolution of the fungal diterpene synthase genes.

    Science.gov (United States)

    Fischer, Marc J C; Rustenhloz, Camille; Leh-Louis, Véronique; Perrière, Guy

    2015-10-19

    Terpenes represent one of the largest and most diversified families of natural compounds and are used in numerous industrial applications. Terpene synthase (TPS) genes originated in bacteria as diterpene synthase (di-TPS) genes. They are also found in plant and fungal genomes. The recent availability of a large number of fungal genomes represents an opportunity to investigate how genes involved in diterpene synthesis were acquired by fungi, and to assess the consequences of this process on the fungal metabolism. In order to investigate the origin of fungal di-TPS, we implemented a search for potential fungal di-TPS genes and identified their presence in several unrelated Ascomycota and Basidiomycota species. The fungal di-TPS phylogenetic tree is function-related but is not associated with the phylogeny based on housekeeping genes. The lack of agreement between fungal and di-TPS-based phylogenies suggests the presence of Horizontal Gene Transfer (HGTs) events. Further evidence for HGT was provided by conservation of synteny of di-TPS and neighbouring genes in distantly related fungi. The results obtained here suggest that fungal di-TPSs originated from an ancient HGT event of a single di-TPS gene from a plant to a fungus in Ascomycota. In fungi, these di-TPSs allowed for the formation of clusters consisting in di-TPS, GGPPS and P450 genes to create functional clusters that were transferred between fungal species, producing diterpenes acting as hormones or toxins, thus affecting fungal development and pathogenicity.

  12. Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Ha, S B; Smith, A P; Howden, R; Dietrich, W M; Bugg, S; O'Connell, M J; Goldsbrough, P B; Cobbett, C S

    1999-06-01

    Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species.

  13. Eugenol synthase genes in floral scent variation in Gymnadenia species.

    Science.gov (United States)

    Gupta, Alok K; Schauvinhold, Ines; Pichersky, Eran; Schiestl, Florian P

    2014-12-01

    Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

  14. Comparative analysis of ADS gene promoter in seven Artemisia ...

    Indian Academy of Sciences (India)

    Artemisinin is the most effective antimalarial drug that is derived from Artemisia annua. Amorpha-4,11-diene synthase (ADS) controls the first committed step in artemisinin biosynthesis. The ADS gene expression is regulated by transcription factors which bind to the cis-acting elements on the ADS promoter and are probably ...

  15. A transcribed polyketide synthase gene from Xanthoria elegans.

    Science.gov (United States)

    Brunauer, Georg; Muggia, Lucia; Stocker-Wörgötter, Elfie; Grube, Martin

    2009-01-01

    We characterize the transcript of a polyketide synthase gene (PKS) from the cultured mycobiont of Xanthoria elegans (XePKS1) using SMART-rapid amplification of cDNA ends (RACE) cDNA synthesis. Sequence analysis of the cloned cDNA reveals an open reading frame of 2144 amino acid residues. It contains features of a non-reducing fungal type I PKS with an N-terminal starter unit: acyl carrier protein (ACP) transacetylase domain, ketosynthase, acyltransferase, two acyl carrier protein domains, and a thioesterase domain. XePKS1 was the only paralogue detected in the cDNA and the genomic DNA of the cultured X. elegans mycobiont by using a degenerate PCR approach targeted at the conserved regions of non-reducing type I PKS genes. The hypothetical protein is phylogenetically related to genes that are basal to a clade of dihydroxynaphthalene synthases (non-reducing clade II) and anthraquinone type synthases of non-lichenized fungi (non-reducing clade I). According to hplc and tlc analyses, the cultured mycobiont exclusively produced anthraquinones and its precursors. Therefore, we discuss whether the characterized paralogue is involved in anthraquinone production, which raises the possibility of a paraphyletic origin of lichen anthraquinone biosynthesis. The cDNA of XePKS1 was the first full-length coding sequence of a lichen PKS to be published. This proves SMART RACE to be a suitable tool for obtaining full-length coding sequences of genes from environmental samples and organisms, which are hardly amenable to standard molecular approaches or genomic sequencing.

  16. Identification and characterization of the Populus sucrose synthase gene family.

    Science.gov (United States)

    An, Xinmin; Chen, Zhong; Wang, Jingcheng; Ye, Meixia; Ji, Lexiang; Wang, Jia; Liao, Weihua; Ma, Huandi

    2014-04-10

    In this study, we indentified 15 sucrose synthase (SS) genes in Populus and the results of RT-qPCR revealed that their expression patterns were constitutive and partially overlapping but diverse. The release of the most recent Populus genomic data in Phytozome v9.1 has revealed the largest SS gene family described to date, comprising 15 distinct members. This information will now enable the analysis of transcript expression profiles for those that have not been previously reported. Here, we performed a comprehensive analysis of SS genes in Populus by describing the gene structure, chromosomal location and phylogenetic relationship of each family member. A total of 15 putative SS gene members were identified in the Populus trichocarpa (Torr. & Gray) genome using the SS domain and amino acid sequences from Arabidopsis thaliana as a probe. A phylogenetic analysis indicated that the 15 members could be classified into four groups that fall into three major categories: dicots, monocots & dicots 1 (M & D 1), and monocots & dicots 2 (M & D 2). In addition, the 15 SS genes were found to be unevenly distributed on seven chromosomes. The two conserved domains (sucrose synthase and glycosyl transferase) were found in this family. Meanwhile, the expression profiles of all 15 gene members in seven different organs were investigated in Populus tomentosa (Carr.) by using RT-qPCR. Additional analysis indicated that the poplar SS gene family is also involved in response to water-deficit. The current study provides basic information that will assist in elucidating the functions of poplar SS family. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Hydroxymethylbilane synthase: Complete genomic sequence and amplifiable polymorphisms in the human gene

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hanwook; Warner, C.A.; Chen, Chiahsiang; Desnick, R.J. (Mount Sinai School of Medicine, New York, NY (United States))

    1993-01-01

    Acute intermittent porphyria (AIP), an autosomal dominant inborn error of heme biosynthesis, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (HMB-synthase). Heterozygous individuals are prone to life-threatening acute neurologic attacks, which are precipitated by certain drugs and other metabolic, hormonal, and nutritional factors. Since the biochemical diagnosis of heterozygous individuals has been problematic, recent efforts have focused on the identification of mutations and diagnostically useful restriction fragment length polymorphisms (RFLPS) in the HMB-synthase gene. To facilitate these endeavors, the human HMB-synthase gene, including 1.1 kb of the 5[prime] flanking region, was isolated and completely sequenced in both orientations. The 10,024-bp gene contained 15 exons ranging in size from 39 to 438 bp and 14 introns ranging from 87 to 2913 bp. All intron/exon boundaries conformed to the GT/AG consensus rule. There were six Alu repetitive elements, one of the J and five of the Sa subfamilies. Analysis of the 1. I -kb 5[prime]flanking region revealed putative regulatory elements for the housekeeping promoter including AP1, AP4, SP1, TRE, ENH, and CAC. This region contained 10 HpaII sites and had an overall GC content of 54%. Three new polymorphic sites were identified by the single-strand conformation polymorphism (SSCP) technique, a common BsmAI site in intron 3 (3581 A/G), a common HinfI RFLP in intron 10 (7064 C/A), and a rare MnlI site in intron 14 (7998G/A). The allele frequencies of five previously known and the new polymorphic sites in a normal Caucasian population indicated that the intron 1 and intron 3 RFLPs were in linkage disequilibrium; however, the Hint I site segregated independently. 54 refs., 6 figs., 3 tabs.

  18. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

    DEFF Research Database (Denmark)

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara

    2004-01-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about...... in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim...... for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone....

  19. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

    Directory of Open Access Journals (Sweden)

    Roslyn D Noar

    Full Text Available Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that

  20. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

    Energy Technology Data Exchange (ETDEWEB)

    Miyata, Maiko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Sugiura, Kazumitsu [Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Furukawa, Koichi, E-mail: koichi@med.nagoya-u.ac.jp [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Furukawa, Keiko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan)

    2014-03-07

    Highlights: • Melanocytes showed low ST8SIA1 and high B3GALT4 levels in contrast with melanomas. • Direct UVB irradiation of melanocytes did not induce ganglioside synthase genes. • Culture supernatants of UVB-irradiated keratinocytes induced ST8SIA1 in melanocytes. • TNFα and IL-6 secreted from keratinocytes enhanced ST8SIA1 expression in melanocytes. • Inflammatory cytokines induced melanoma-related ST8SIA1 in melanocytes. - Abstract: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

  1. Isolation and characterization of chalcone synthase gene isolated from Dendrobium Sonia Earsakul.

    Science.gov (United States)

    Pitakdantham, W; Sutabutra, T; Chiemsombat, P; Pitaksutheepong, C

    2010-10-15

    To isolate and characterize chalcone synthase gene in anthocyanin biosynthetic pathway during flower development of Dendrobium Sonia Earsakul. The gene was isolated from floral tissues of the orchid by reverse transcriptase polymerase chain reaction. Characterization of the gene considered to its relatedness to chalcone synthase gene in other orchid plants elucidated by construction of a neighbor-joining phylogenetic tree. Gene expression pattern related to flower development and pigmentation was investigated by relative quantification real time polymerase chain reaction. A complete coding sequence was obtained and sequence analysis revealed that the gene of Dendrobium Sonia Earsakul consisted of 1,188 bp. Blast analysis and multiple alignments showed that the chalcone synthase gene of Dendrobium Sonia Earsakul shares high homology to chalcone synthase gene of Dendrobium genus particularly Dendrobium hybrid Uniwai prince. Phylogenetic tree revealed that chalcone synthase of Dendrobium genus are highly conserved. The chalcone synthase gene of Dendrobium Sonia Earsakul was highly expressed in young flower bud with no pigmentation and the expression was sharply decreased when young flower bud started accumulation of pigments. Expression of chalcone synthase gene was then maintained at the same level until young bud developed into fully opened flowers.

  2. Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene

    Energy Technology Data Exchange (ETDEWEB)

    Feinbaum, R.L.; Ausubel, F.M.

    1988-05-01

    The authors cloned an Arabiodpsis thaliana chalcone synthase (CHS) gene on the basis of cross-hybridization with a Petroselinum hortense CHS cDNA clone. The protein sequence deduced from the A. thaliana CHS DNA sequence is at least 85% homologous to the CHS sequences from P. hortense, Antirrhinum majus, and Petunia hybrida. Southern blot analysis indicated that CHS is a single-copy gene in A. thaliana. High-intensity light treatment of A. thaliana plants for 24 h caused a 50-fold increase in CHS enzyme activity and an accumulation of visibly detectable levels of anthocyanin pigments in the vegetative structures of these plants. A corresponding increase in the steady-state level of CHS mRNA was detected after high-intensity light treatment for the same period of time. The accumulation of CHS mRNA in response to high-intensity light was due, at least in part, to an increased rate of transcription of the CHS gene as demonstrated by nuclear runoff experiment.

  3. Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

    Science.gov (United States)

    Mizuno, Kouhei; Kihara, Takahiro; Tsuge, Takeharu; Lundgren, Benjamin R; Sarwar, Zaara; Pinto, Atahualpa; Nomura, Christopher T

    2017-01-01

    Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

  4. Callose Synthase Family Genes Involved in the Grapevine Defense Response to Downy Mildew Disease.

    Science.gov (United States)

    Yu, Ying; Jiao, Li; Fu, Shufang; Yin, Ling; Zhang, Yali; Lu, Jiang

    2016-01-01

    The deposition of callose is a common plant defense response to intruding pathogens and part of the plant's innate immunity. In this study, eight grapevine callose synthase (CalS) genes were identified and characterized. To investigate biological function of CalS in grapevine against the infection of Plasmopara viticola, expression patterns of grapevine CalS family genes were analyzed among resistant/susceptible cultivars. After P. viticola infection, expression of CalS1, 3, 7, 8, 9, 10, and 11 were significantly modified among the grapevine cultivars. For example, the expression of CalS1 and CalS10 were greatly increased in downy mildew (DM)-immune Muscadinia rotundifolia 'Carlos' and 'Noble'. Transient expression assay with promoters of the CalS1 and CalS10 genes confirmed that they were regulated by the oomycete pathogen P. viticola. CalS1 promoter activity was also significantly up-regulated by ABA in DM-immune M. rotundifolia 'Noble', but down-regulated in DM-susceptible Vitis vinifera 'Chardonnay'. The CalS1 promoter, however, was also down-regulated by GA in 'Chardonnay', but not affected in 'Noble'. The promoter activity of CalS10 was significantly up-regulated by GA in 'Chardonnay', but not regulated by ABA at all. It is proposed that CalS1 and CalS10 were involved in grapevine defense against DM disease.

  5. A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

    Science.gov (United States)

    Pereira, Luiz Miguel; Baroni, Luciana; Yatsuda, Ana Patrícia

    2014-03-01

    Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii. Copyright © 2014. Published by Elsevier Inc.

  6. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    -transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  7. Expression and developmental function of the 3-ketoacyl-ACP synthase2 gene in Arabidopsis thaliana.

    Science.gov (United States)

    Hakozaki, Hirokazu; Park, Jong-In; Endo, Makoto; Takada, Yoshinobu; Kazama, Tomohiko; Takeda, Yoshimitsu; Suzuki, Go; Kawagishi-Kobayashi, Makiko; Watanabe, Masao

    2008-04-01

    The 3-ketoacyl-ACP synthase (KAS) II is a fatty-acid-related enzyme which catalyzes the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. The fatty acid biosynthesis 1-1 (fab1-1) mutant of Arabidopsis thaliana is partially deficient in its activity of Arabidopsis thaliana 3-ketoacyl-ACP synthase 2 (AtKAS2), and its phenotype has been intensively studied in connection with the chilling resistance and fatty acid composition. In this study, we used the T-DNA insertion mutant of AtKAS2 to examine its possible role in plant development. Reverse transcription (RT)-PCR showed that the AtKAS2 gene was expressed in various plant organs, except for roots, and was highly expressed in siliques. The fusion of beta-glucuronidase (GUS) to the AtKAS2 promoter demonstrated that the promoter was active in various tissues such as embryos, stomatal guard cells, inflorescences and pollen grains. We were not able to identify atkas2 homozygous mutant adult plants in heterozygous mutant progeny. Phenotypic and genetic analyses showed that disruption of the AtKAS2 by T-DNA insertion caused embryo lethality, and the development of the embryos was arrested at the globular stage. Taken together, our results suggest that AtKAS2 is required for embryo development in Arabidopsis during the transition from the globular to the heart stage.

  8. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

    Science.gov (United States)

    Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2017-02-01

    Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Clinical aspects of endothelial NO-synthase gene polymorphism in professional sportsmen (literature review

    Directory of Open Access Journals (Sweden)

    S. N. Malakhova

    2016-08-01

    Full Text Available The article deals the questions of properties and biological role of nitric oxide molecule, which is characterized with vasodilating, stress limiting and neurotransmitter properties, and participates in reactions of oxidative stress, glutamate and calcium cascade and inflammation. Researches of the relationship of endothelial NO-synthase and the risk of cardiovascular, ischemic and respiratory diseases in population of untrained persons and athletes continue. The aim of this study was to establish, according to the literature, the present state of the influence of endothelial NO-synthase gene polymorphism on the formation of the physical qualities of strength, endurance and speed in professional athletes, its changes and influence on functional state of athlete. At the present stage of development of medical genetics it is proved, that the implementation of the action of each individual isoform of the enzyme NO-synthase is caused by the presence of polymorphism. The most studied are the T-786С polymorphism of the eNOS gene promoter, G894T (Glu298Asp polymorphism of the 7th exon, 4a/b–4th intron of eNOS, but their influence is not definitively proven, and is caused by the presence of homo- or heterozygotes. Genetic researches among professional athletes prove the significant role of nitric oxide system in enhancing of physical working capacity, but do not allow evaluating the characteristics of this effect, taking into account multi-directional training process and the level of sports qualification, as well as predicting future professional success and the preservation of the proper functioning of the cardiovascular, respiratory and other body systems. Conclusions. Thus, contemporary researches allow stating that oxide synthesis has a leading role in the functioning of the organism, and in particular athletes, but do not allow assessing its effects on the body of athletes of different sports qualification. It is proved that polymorphism of the

  10. (SNP) of endothelial nitric oxide synthase gene and serum level of ...

    African Journals Online (AJOL)

    T-786C single-nucleotide polymorphism (SNP) of endothelial nitric oxide synthase gene and serum level of vascular endothelial relaxant factor (VERF) in non-diabetic patients with coronary artery disease.

  11. Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase.

    Science.gov (United States)

    Malumbres, M; Mateos, L M; Lumbreras, M A; Guerrero, C; Martín, J F

    1994-01-01

    The thrC gene of Brevibacterium lactofermentum was cloned by complementation of Escherichia coli thrC auxotrophs. The gene was located by deletion mapping and complementation analysis in a 2.9-kb Sau3AI-HindIII fragment of the genome. This fragment also complemented a B. lactofermentum UL1035 threonine auxotroph that was deficient in threonine synthase. A 1,892-bp DNA fragment of this region was sequenced; this fragment contained a 1,446-bp open reading frame that encoded a 481-amino-acid protein having a deduced M(r) of 52,807. The gene was expressed in E. coli, by using the phage T7 system, as a 53-kDa protein. The promoter region subcloned in promoter-probe plasmids was functional in E. coli. A Northern analysis revealed that the gene was expressed as a monocistronic 1,400-nucleotide transcript. The transcription start point of the thrC gene was located by S1 mapping 6 bp upstream from the translation initiation codon, which indicated that this promoter was one of the leaderless transcription-initiating sequences. The threonine synthase overexpressed in B. lactofermentum UL1035 was purified almost to homogeneity. The active form corresponded to a monomeric 52.8-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme required pyridoxal phosphate as its only cofactor to convert homoserine phosphate into threonine. Images PMID:8074505

  12. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    Leyva et al. 1995), UV treatments and blue light (Hartmann et al. 1998; Wade et al. 2001; Zhou et al. 2007), elicitor treatments such as salicylic acid and. Keywords. chalcone synthase; real-time PCR; silymarin; anthocyanin; Silybum marianum.

  13. Evolution of Homospermidine Synthase in the Convolvulaceae: A Story of Gene Duplication, Gene Loss, and Periods of Various Selection Pressures

    National Research Council Canada - National Science Library

    Elisabeth Kaltenegger; Eckart Eich; Dietrich Ober

    2013-01-01

    .... To study the processes that followed this gene duplication event and gave rise to HSS, we identified sequences encoding HSS and deoxyhypusine synthase from various species of the Convolvulaceae...

  14. 6-MSAS-like polyketide synthase genes occur in lichenized ascomycetes.

    Science.gov (United States)

    Schmitt, Imke; Kautz, Stefanie; Lumbsch, H Thorsten

    2008-02-01

    Lichenized and non-lichenized filamentous ascomycetes produce a great variety of polyketide secondary metabolites. Some polyketide synthase (PKS) genes from non-lichenized fungi have been characterized, but the function of PKS genes from lichenized species remains unknown. Phylogenetic analysis of keto synthase (KS) domains allows prediction of the presence or absence of particular domains in the PKS gene. In the current study we screened genomic DNA from lichenized fungi for the presence of non-reducing and 6-methylsalicylic acid synthase (6-MSAS)-type PKS genes. We developed new degenerate primers in the acyl transferase (AT) region to amplify a PKS fragment spanning most of the KS region, the entire linker between KS and AT, and half of the AT region. Phylogenetic analysis shows that lichenized taxa possess PKS genes of the 6-MSAS-type. The extended alignment confirms overall phylogenetic relationships between fungal non-reducing, 6-MSAS-type and bacterial type I PKS genes.

  15. Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

    Science.gov (United States)

    Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

    2016-12-01

    Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

  16. RNA sequencing on Solanum lycopersicum trichomes identifies transcription factors that activate terpene synthase promoters.

    Science.gov (United States)

    Spyropoulou, Eleni A; Haring, Michel A; Schuurink, Robert C

    2014-05-27

    Glandular trichomes are production and storage organs of specialized metabolites such as terpenes, which play a role in the plant's defense system. The present study aimed to shed light on the regulation of terpene biosynthesis in Solanum lycopersicum trichomes by identification of transcription factors (TFs) that control the expression of terpene synthases. A trichome transcriptome database was created with a total of 27,195 contigs that contained 743 annotated TFs. Furthermore a quantitative expression database was obtained of jasmonic acid-treated trichomes. Sixteen candidate TFs were selected for further analysis. One TF of the MYC bHLH class and one of the WRKY class were able to transiently transactivate S. lycopersicum terpene synthase promoters in Nicotiana benthamiana leaves. Strikingly, SlMYC1 was shown to act synergistically with a previously identified zinc finger-like TF, Expression of Terpenoids 1 (SlEOT1) in transactivating the SlTPS5 promoter. High-throughput sequencing of tomato stem trichomes led to the discovery of two transcription factors that activated several terpene synthase promoters. Our results identified new elements of the transcriptional regulation of tomato terpene biosynthesis in trichomes, a largely unexplored field.

  17. Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

    Science.gov (United States)

    Xiong, S; Chirala, S S; Wakil, S J

    2000-04-11

    To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

  18. A functional promoter polymorphism of neuronal nitric oxide synthase moderates prefrontal functioning in schizophrenia.

    Science.gov (United States)

    Reif, Andreas; Schecklmann, Martin; Eirich, Elisa; Jacob, Christian P; Jarczok, Tomasz A; Kittel-Schneider, Sarah; Lesch, Klaus-Peter; Fallgatter, Andreas J; Ehlis, Ann-Christine

    2011-08-01

    Cognitive deficits in tasks involving the prefrontal cortex such as working memory or verbal fluency are a key component of schizophrenia. This led to the hypofrontality hypothesis of schizophrenia, which is widely accepted even though molecular underpinnings are elusive. While disturbances of glutamatergic neurotransmission might play a role, other components have rarely been investigated. Recently, the promoter region of nitric oxide (NO) synthase-I (NOS-I, encoded by the gene NOS1), impacting on prefrontal glutamate transmission, has repeatedly been associated with schizophrenia. We thus tested whether an associated schizophrenia risk variant (rs41279104), leading to reduced expression of the transcript, influences prefrontal brain functioning. Forty-three patients suffering from chronic schizophrenia and 44 controls were genotyped for NOS1 rs41279104 and investigated by means of functional near-infrared spectroscopy (fNIRS), while completing a working-memory task (2-back test) and a verbal fluency test (VFT). After matching for genotype, behavioural and brain activation data of 26 patients and 28 comparable controls were correlated to rs41279104. Healthy controls showed significant activation of large parts of the lateral prefrontal cortex during both tasks, whereas task-related changes in oxygenation were significantly reduced in patients. Schizophrenia patients also performed worse in both tasks. The NOS1 schizophrenia risk genotype rs41279104 AA/AG was associated with slower reaction time in the 2-back task, as well as with reduced right-hemispheric activation of the frontal cortex for VFT in patients only. Our fNIRS data extend previous studies suggesting disturbed prefrontal functioning in schizophrenia and suggest that genetic variation of NOS1 has a role in cognitive dysfunction, probably by mediating glutamatergic tone.

  19. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    Science.gov (United States)

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  20. Endothelial nitric oxide synthase gene Glu298Asp polymorphism ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... eNOS haplotypes associated with gestational hypertension or preeclampsia. Pharmacogenomics, 9(10):. 1467-73. Serrano NC, Casas JP, Diaz LA, Paez C, Mesa CM, Cifuentes R,. Monterrosa A, Bautista A, Hawe E, Hingorani AD, Vallance P, Lopez-. Jaramillo P (2004). Endothelial NO synthase genotype ...

  1. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  2. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana

    Energy Technology Data Exchange (ETDEWEB)

    Hemleben, V.; Frey, M.; Rall, S.; Koch, M.; Kittel, M.; Kreuzaler, F.; Ragg, H.; Fautz, E.; Hahlbrock, K.

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  3. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana.

    Science.gov (United States)

    Hemleben, V; Frey, M; Rall, S; Koch, M; Kittel, M; Kreuzaler, F; Ragg, H; Fautz, E; Hahlbrock, K

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  4. Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

    Science.gov (United States)

    Ishida, Mariko; Kitao, Naoko; Mizuno, Kouichi; Tanikawa, Natsu; Kato, Misako

    2009-02-01

    Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

  5. Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.

    Science.gov (United States)

    Martin, Diane M; Fäldt, Jenny; Bohlmann, Jörg

    2004-08-01

    Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

  6. Endothelial nitric oxide synthase gene haplotypes and diabetic nephropathy among Asian Indians

    DEFF Research Database (Denmark)

    Ahluwalia, Tarun Veer Singh; Ahuja, Monica; Rai, Taranjit Singh

    2008-01-01

    Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, including diabetic nephropathy. Endothelial-derived nitric oxide synthase (eNOS) gene polymorphisms affect eNOS activity and are associated with endothelial dysfunction. We evaluated the association...... of the constitutive endothelial nitric oxide synthase gene (eNOS) polymorphisms with type 2 diabetic nephropathy. We genotyped three polymorphisms of eNOS (Two SNPs: -786T > C, 894G > T and one 27-bp repeat polymorphism in Intron 4 (27VNTR)) in type 2 diabetic nephropathy patients (cases: n = 195) and type 2 diabetic...

  7. Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.

    Science.gov (United States)

    Kato, Shota; Takaichi, Shinichi; Ishikawa, Takahiro; Asahina, Masashi; Takahashi, Senji; Shinomura, Tomoko

    2016-01-05

    Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2) s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. We identified genes

  8. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  9. Association of a Soybean Raffinose Synthase Gene with Low Raffinose and Stachyose Seed Phenotype

    Directory of Open Access Journals (Sweden)

    Emily C. Dierking

    2008-11-01

    Full Text Available Oligosaccharides are an important component of soybean [ (L. Merr.] meal in terms of metabolizable energy for monogastric animals. Sucrose, raffinose, and stachyose are the three main oligosaccharides present in soybean meal. Of the three, only sucrose is nutritionally useful. When raffinose and stachyose are fermented by microbes present in the gut, the results are flatulence and discomfort, which ultimately lead to poor weight gain. The long term objective of this research is ultimately to increase the nutritional value of soybean meal by elevating the metabolizable energy at the expense of raffinose and stachyose through the manipulation of soybean raffinose synthase, the key enzyme for raffinose and stachyose biosynthesis. The objectives of this work were to develop molecular genetic information about soybean raffinose synthases and to evaluate the candidate raffinose synthase genes in a soybean germplasm accession (PI 200508 that contains low levels of raffinose and stachyose. Our results indicate the soybean genome contains at least two expressed genes similar to other characterized raffinose synthases. A novel allele of one of these putative soybean raffinose synthase genes was discovered from the PI 200508 that completely associates with the low raffinose and stachyose phenotype. Molecular marker assays specific for the PI 200508 allele were developed to allow direct selection for the low raffinose and low stachyose phenotype.

  10. The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation.

    Science.gov (United States)

    Peng, Gang; Wang, Chunyan; Song, Song; Fu, Xiumin; Azam, Muhammad; Grierson, Don; Xu, Changjie

    2013-10-01

    Three 1-deoxy-D-xylulose-5-phosphate synthases (DXS) and three phytoene synthases (PSY) were identified in citrus, from Affymetrix GeneChip Citrus Genome Array, GenBank and public orange genome databases. Tissue-specific expression analysis of these genes was carried out on fruit peel and flesh, flower and leaf of Satsuma mandarin (Citrus unshiu Marc.) in order to determine their roles in carotenoid accumulation in different tissues. Expression of CitDXS1 and CitPSY1 was highest in all test tissues, while that of CitDXS2 and CitPSY2 was lower, and that of CitDXS3 and CitPSY3 undetectable. The transcript profiles of CitDXS1 and CitPSY1 paralleled carotenoid accumulation in flesh of Satsuma mandarin and orange (Citrus sinensis Osbeck) during fruit development, and CitPSY1 expression was also associated with carotenoid accumulation in peel, while the CitDXS1 transcript level was only weakly correlated with carotenoid accumulation in peel. Similar results were obtained following correlation analysis between expression of CitDXS1 and CitPSY1 and carotenoid accumulation in peel and flesh of 16 citrus cultivars. These findings identify CitPSY1 and CitDXS1 as the main gene members controlling carotenoid biosynthesis in citrus fruit. Furthermore, chromoplasts were extracted from flesh tissue of these citrus, and chromoplasts of different shape (spindle or globular), different size, and color depth were observed in different cultivars, indicating chromoplast abundance, number per gram tissue, size and color depth were closely correlated with carotenoid content in most cultivars. The relationship between carotenoid biosynthesis and chromoplast development was discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  11. The polyketide components of waxes and the Cer-cqu gene cluster encoding a novel polyketide synthase, the β-diketone synthase, DKS

    DEFF Research Database (Denmark)

    von Wettstein, Penny

    2017-01-01

    Cer-cqu gene cluster encoding a novel polyketide synthase-the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms...... long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols....

  12. S-Adenosylmethionine modulates inducible nitric oxide synthase gene expression in rat liver and isolated hepatocytes.

    Science.gov (United States)

    Majano, P L; García-Monzón, C; García-Trevijano, E R; Corrales, F J; Cámara, J; Ortiz, P; Mato, J M; Avila, M A; Moreno-Otero, R

    2001-12-01

    Hepatocellular availability of S-adenosylmethionine, the principal biological methyl donor, is compromised in situations of liver damage. S-Adenosylmethionine administration alleviates experimental liver injury and increases survival in cirrhotic patients. The mechanisms behind these beneficial effects of S-adenosylmethionine are not completely known. An inflammatory component is common to many of the pathological conditions in which S-adenosylmethionine grants protection to the liver. This notion led us to study the effect of S-adenosylmethionine administration on hepatic nitric oxide synthase-2 induction in response to bacterial lipopolysaccharide and proinflammatory cytokines. The effect of S-adenosylmethionine on nitric oxide synthase-2 expression was assessed in rats challenged with bacterial lipopolysaccharide and in isolated rat hepatocytes treated with proinflammatory cytokines. Interactions between S-adenosylmethionine and cytokines on nuclear factor kappa B activation and nitric oxide synthase-2 promoter transactivation were studied in isolated rat hepatocytes and HepG2 cells, respectively. S-Adenosylmethionine attenuated the induction of nitric oxide synthase-2 in the liver of lipopolysaccharide-treated rats and in cytokine-treated hepatocytes. S-Adenosylmethionine accelerated the resynthesis of inhibitor kappa B alpha, blunted the activation of nuclear factor kappa B and reduced the transactivation of nitric oxide synthase-2 promoter. Our findings indicate that the hepatoprotective actions of S-adenosylmethionine may be mediated in part through the modulation of nitric oxide production.

  13. Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716

    NARCIS (Netherlands)

    van Walraven, H. S.; Lutter, R.; Walker, J. E.

    1993-01-01

    The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha,

  14. Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host.

    Science.gov (United States)

    Yamada, Yuuki; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2015-06-01

    Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.

  15. Characterization of a sabinene synthase gene from rough lemon (Citrus jambhiri).

    Science.gov (United States)

    Kohzaki, Keisuke; Gomi, Kenji; Yamasaki-Kokudo, Yumiko; Ozawa, Rika; Takabayashi, Junji; Akimitsu, Kazuya

    2009-10-15

    We previously isolated two putative monoterpene synthase genes, RlemTPS1 and RlemTPS2, from rough lemon (Citrus jambhiri) and showed that gene expression of RlemTPS2 was induced by microbial attack. The protein product of RlemTPS2 was obtained using a prokaryotic expression system, and GC and GC-MS of monoterpene synthesis by RlemTPS2 determined that RlemTPS2 encodes a sabinene synthase. Sabinene has antifungal activity toward Alternaria alternata. Furthermore, site-directed mutagenesis identified one amino acid, Ile, located at the front of the metal ion binding motif as an important residue for the product specificity of sabinene synthase.

  16. ISOLATION AND IDENTIFICATION OF A THERMOTOLERANT PLANT GROWTH PROMOTING PSEUDOMONAS PUTIDA PRODUCING TREHALOSE SYNTHASE

    Directory of Open Access Journals (Sweden)

    Ali Sk.Z.

    2013-08-01

    Full Text Available A thermotolerant plant growth promoting Pseudomonas isolate growing at 40oC producing trehalose synthase (TreS was isolated from rhizosphere soil under semi arid conditions of India. Trehalose synthase was extracted; purified and enzymatic activity was examined at various temperatures and pH. The optimum temperature and pH was 38oC and pH 7.5 and the activity declined at above or below the optimum pH and temperature. The enzyme was active on maltose and trehalose among saccharides tested. The enzyme had a higher catalytic activity for maltose with a trehalose yield of 72% than for trehalose where 30% yield of maltose was achieved, indicating maltose as preferred substrate. The isolate showed multiple plant growth promoting traits (indole acetic acid (IAA, phosphate solubilization, siderophore and ammonia both at ambient (28oC and high temperature (40oC. Based on phenotypic and 16SrRNA analysis the isolate was identified as Pseudomonas putida (Accession No. GU396283.

  17. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

    Directory of Open Access Journals (Sweden)

    Mirian Perez Maluf

    2009-01-01

    Full Text Available In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

  18. Genome-wide identification and phylogenetic analysis of the chalcone synthase gene family in rice.

    Science.gov (United States)

    Hu, Lifang; He, Haohua; Zhu, Changlan; Peng, Xiaosong; Fu, Junru; He, Xiaopeng; Chen, Xiaorong; Ouyang, Linjuan; Bian, Jianmin; Liu, Shiqiang

    2017-01-01

    The enzymes of the chalcone synthase family are also known as type III polyketide synthases (PKS), and produce a series of secondary metabolites in bacteria, fungi and plants. In a number of plants, genes encoding PKS comprise a large multigene family. Currently, detailed reports on rice (Oryza sativa) PKS (OsPKS) family genes and tissue expression profiling are limited. Here, 27 candidate OsPKS genes were identified in the rice genome,and 23 gene structures were confirmed by EST and cDNA sequencing; phylogenetic analysis has indicated that these 23 OsPKS members could be clustered into three groups (I-III). Comparative analysis has shown OsPKS08 and OsPKS26 could be classified with the CHS genes of other species. Two members OsPKS10 and OsPKS21 were grouped into anther specific chalcone synthase-like (ASCL) clade. Intron/exon structure analysis revealed that nearly all of the OsPKS members contained one phase-1 intron at a conserved Cys. Analysis of chromosomal localization and genome distribution showed that some of the members were distributed on a chromosome as a cluster. Expression data exhibited widespread distribution of the rice OsPKS gene family within plant tissues, suggesting functional diversification of the OsPKS genes. Our results will contribute to future study of the complexity of the OsPKS gene family in rice.

  19. Distribution of genotypes and alleles of the aldosterone-synthase gene in patients with abdominal obesity

    Directory of Open Access Journals (Sweden)

    D. L. Brovin

    2015-01-01

    Full Text Available We observed 140 patients with abdominal obesity (AO (IDF, 2005, the residents of St. Petersburg (44.6 ± 0.6 years. Metabolic syndrome (MS (IDF, 2005 was diagnosed in 49.2% of patients with AO. The most frequent component of MS in patients with AO was arterial hypertension (AH. The distribution of genotypes and -alleles of the aldosterone-synthase gene in patients with AO and in the comparison group (56 subjects without AO, 41.0 ± 1.1 years didn't differ (p> 0.05. Levels of both systolic and diastolic blood pressure (BP were higher in carriers of -344T allele of aldosterone-synthase gene. Plasma renin activity, plasma aldosterone and glucose levels, anthropometric parameters, serum blood lipids and carbohydrate metabolism indices in obese patients with different genotypes of aldosterone-synthase gene didn't differ. -344T allele of aldosterone-synthase gene in patients with AO is associated with the increased risk of AH.

  20. Cloning and characterization of ATP synthase CF1 α gene from ...

    African Journals Online (AJOL)

    ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

  1. Gene expression and characterization of isoprene synthase from Populus alba.

    Science.gov (United States)

    Sasaki, Kanako; Ohara, Kazuaki; Yazaki, Kazufumi

    2005-04-25

    Isoprene synthase cDNA from Populus alba (PaIspS) was isolated by RT-PCR. This PaIspS mRNA, which was predominantly observed in the leaves, was strongly induced by heat stress and continuous light irradiation, and was substantially decreased in the dark, suggesting that isoprene emission was regulated at the transcriptional level. The subcellular localization of PaIspS protein with green fluorescent protein fusion was shown to be in plastids. PaIspS expressed in Escherichia coli was characterized enzymatically: it had an optimum pH of approximately 8.0, and an optimum temperature 40 degrees C. Its preference for divalent cations for its activity was also studied.

  2. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

    Directory of Open Access Journals (Sweden)

    Marquez Rodolfo

    2004-09-01

    Full Text Available Abstract Background Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase and insulin-like growth factor binding protein-1 (IGFBP-1, is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE. The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase. However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3 is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase, whose products are required for gluconeogenesis. Results In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. Conclusions These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

  3. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

    Directory of Open Access Journals (Sweden)

    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  4. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    comparative analysis of grass genomes and as a source of beneficial genes for agriculture. Recent studies have shown that there is conservation of gene order at the chromosome level in rice, sorghum and maize. Therefore, a high-resolution alignment between these genomes will be needed to utilize them constructively ...

  5. Identification of the trehalose-6-phosphate synthase gene family in ...

    Indian Academy of Sciences (India)

    2015-03-04

    Mar 4, 2015 ... 3Jiamusi Branch of Heilongjiang Academy of Agricultural Sciences, Jiamusi 154005, Heilongjiang Province,. People's Republic of China ..... Clustering analysis of differential gene expression patterns. Genes with similar ... cluster software PermutMatrixEN, and the Pearson corre- lation method with the ...

  6. Identification of the trehalose-6-phosphate synthase gene family in ...

    Indian Academy of Sciences (India)

    The estimated divergence time of wheat TPS gene pairs and wheat–rice orthologues suggested that wheat and rice have a common ancestor. The number of TPS genes in the wheat genome was estimated to be at least 12, which is close to the number found in rice, Arabidopsis and soybean. Moreover, it has been reported ...

  7. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshigai, Emi [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Machida, Toru [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okuyama, Tetsuya [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okumura, Tadayoshi [Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga (Japan); Department of Surgery, Kansai Medical University, Hirakata, Osaka (Japan); Ikeya, Yukinobu [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga (Japan); Nishino, Hoyoku [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto (Japan); Nishizawa, Mikio, E-mail: nishizaw@sk.ritsumei.ac.jp [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan)

    2013-09-13

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

  8. Role of hyaluronan synthase 2 to promote CD44-dependent oral cavity squamous cell carcinoma progression.

    Science.gov (United States)

    Wang, Steven J; Earle, Christine; Wong, Gabriel; Bourguignon, Lilly Y W

    2013-04-01

    CD44 is a transmembrane receptor found on many different benign and malignant cells. Hyaluronan (HA), a major component of the extracellular matrix, is the primary ligand for CD44 receptors. In cancer cells, HA interaction with CD44 promotes multiple signaling pathways that influence tumor cell progression behaviors in a variety of solid tumors. Increasing evidence indicates that HA and CD44 signaling play an important role in oral cavity squamous cell carcinoma progression. HA is primarily synthesized by hyaluronan synthases, and the current study investigated the role of hyaluronan synthase 2 (HAS 2) in oral cavity carcinoma progression behaviors. Analysis of HAS 2 mRNA and protein expression, HA production, and HAS 2-mediated tumor cell proliferation and migration behaviors with and without HAS 2 suppression were carried out on 2 established oral cavity cancer cell lines. Immunohistochemical analysis of HAS 2 and CD44 expression in oral cavity carcinoma tumor specimens was performed. HAS 2 was expressed in the 2 oral cancer cell lines, HSC-3 and SCC-4. Suppression of HAS 2 expression resulted in CD44-dependent decreased tumor cell migration, decreased tumor cell growth, and increased cisplatin sensitivity, suggesting the importance of tumor cell HA production to promote in vitro tumor progression behaviors in oral cancer cells. Increased HAS 2 expression in oral cavity carcinoma clinical specimens was associated with poor clinicopathologic characteristics and worse disease-free survival. HAS 2 may be a potential therapeutic target for the treatment of oral cavity cancer. Copyright © 2012 Wiley Periodicals, Inc.

  9. Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes.

    Science.gov (United States)

    Last, R L; Bissinger, P H; Mahoney, D J; Radwanski, E R; Fink, G R

    1991-01-01

    The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis. PMID:1840915

  10. Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters.

    Science.gov (United States)

    Marchegiani, Elisabetta; Sidhu, Yaadwinder; Haynes, Ken; Lebrun, Marc-Henri

    2015-06-01

    Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms With Acute Rejection in Liver Transplant Recipients.

    Science.gov (United States)

    Azarpira, Negar; Namazi, Soha; Malahi, Sayan; Kazemi, Kourosh

    2016-06-01

    Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients. We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism. Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02). Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

  12. Nucleosomal promoter variation generates gene expression noise.

    Science.gov (United States)

    Brown, Christopher R; Boeger, Hinrich

    2014-12-16

    Gene product molecule numbers fluctuate over time and between cells, confounding deterministic expectations. The molecular origins of this noise of gene expression remain unknown. Recent EM analysis of single PHO5 gene molecules of yeast indicated that promoter molecules stochastically assume alternative nucleosome configurations at steady state, including the fully nucleosomal and nucleosome-free configuration. Given that distinct configurations are unequally conducive to transcription, the nucleosomal variation of promoter molecules may constitute a source of gene expression noise. This notion, however, implies an untested conjecture, namely that the nucleosomal variation arises de novo or intrinsically (i.e., that it cannot be explained as the result of the promoter's deterministic response to variation in its molecular surroundings). Here, we show--by microscopically analyzing the nucleosome configurations of two juxtaposed physically linked PHO5 promoter copies--that the configurational variation, indeed, is intrinsically stochastic and thus, a cause of gene expression noise rather than its effect.

  13. Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP.

    Science.gov (United States)

    Cirovic, Natasa; Kecmanovic, Miljana; Keckarevic, Dusan; Keckarevic Markovic, Milica

    2017-10-01

    Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana (C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3% in dried plants, usually punishable by the law. Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism (RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy. An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of producing THC in concentrations higher than 0.3%. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  14. Cloning and characterization of a monoterpene synthase gene from flowers of Camelina sativa.

    Science.gov (United States)

    Borghi, Monica; Xie, De-Yu

    2017-10-26

    CsTPS1 encodes for a monoterpene synthase that contributes to the emission of a blend of volatile compounds emitted from flowers of Camelina sativa. The work describes the in vitro characterization of a monoterpene synthase and its regulatory region that we cloned from Camelina sativa (Camelina). Here, we named this gene as C. sativa terpene synthase 1 (CsTPS1). In vitro experiments performed with the CsTPS1 protein after expression and purification from Escherichia coli (E. coli) showed production of a blend of monoterpene volatile organic compounds, of which the emission was also detected in the floral bouquet of wild-type Camelina plants. Quantitative-PCR measurements revealed a high abundance of CsTPS1 transcripts in flowers and experiments performed with the GUS reporter showed high CsTPS1 expression in the pistil, in the cells of the wall of the ovary and in the stigma. Subcellular localization of the CsTPS1 protein was investigated with a GFP reporter construct that showed expression in plastids. The CsTPS1 gene identified in this study belongs to a mid-size family of 60 genes putatively codifying for TPS enzymes. This enlarged family of TPS genes suggests that Camelina has the structural framework for the production of terpenes and other secondary metabolites of relevance for the consumers.

  15. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    Energy Technology Data Exchange (ETDEWEB)

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  16. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of gene family, 1 and 2. Third member, full-length cDNA (3) was isolated by rapid amplification of cDNA ends (RACE), whose open reading frame contained 1239 bp ...

  17. Functional analysis of a gene encoding threonine synthase from rice ...

    African Journals Online (AJOL)

    The predicted amino acid sequence of OsTS is highly homologous to that of Arabidopsis TS and many bacterial TS encoded by thrC gene. The OsTS protein harbors a signature binding motif for pyridoxal- 5' -phosphate at the amino terminus. A thrC mutant strain of Escherichia coli was complemented by OsTS expression.

  18. Expression divergence of cellulose synthase (CesA) genes after a recent whole genome duplication event in Populus.

    Science.gov (United States)

    Takata, Naoki; Taniguchi, Toru

    2015-01-01

    Secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules. Gene duplication is an important mechanism for functional divergence of genes. Secondary cell wall-associated cellulose synthase genes (CesA4, CesA7 and CesA8) are duplicated in Populus plants due to a recent whole genome duplication event. Here, we demonstrate that duplicate CesA genes show tissue-dependent expression divergence in Populus plants. Real-time PCR analysis of Populus CesA genes suggested that Pt × tCesA8-B was more highly expressed than Pt × tCesA8-A in phloem and secondary xylem tissue of mature stem. Histochemical and histological analyses of transformants expressing a GFP-GUS fusion gene driven by Populus CesA promoters revealed that the duplicate CesA genes showed different expression patterns in phloem fibers, secondary xylem, root cap and leaf trichomes. We predicted putative cis-regulatory motifs that regulate expression of secondary cell wall-associated CesA genes, and identified 19 motifs that are highly conserved in the CesA gene family of eudicotyledonous plants. Furthermore, a transient transactivation assay identified candidate transcription factors that affect levels and patterns of expression of Populus CesA genes. The present study reveals that secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules.

  19. [Cellulose synthase genes that control the fiber formation of flax (Linum usitatissimum L.)].

    Science.gov (United States)

    Galinovskiĭ, D V; Anisimova, N V; Raĭskiĭ, A P; Leont'ev, V N; Titok, V V; Hotyleva, L V

    2014-01-01

    Four cellulose synthase genes were identified by analysis of their class-specific regions (CSRII) in plants of fiber flax during the "rapid growth" stage. These genes were designated as LusCesA1, LusCesA4, LusCesA7 and LusCesA9. LusCesA4, LusCesA7, and LusCesA9 genes were expressed in the stem; LusCesA1 and LusCesA4 genes were expressed in the apex part of plants, and the LusCesA4 gene was expressed in the leaves of fiber flax. The expression of the LusCesA7 and LusCesA9 genes was specific to the stems of fiber flax. These genes may influence the quality of the flax fiber.

  20. Horizontal Gene Transfer of Phytochelatin Synthases from Bacteria to Extremophilic Green Algae.

    Science.gov (United States)

    Olsson, Sanna; Penacho, Vanessa; Puente-Sánchez, Fernando; Díaz, Silvia; Gonzalez-Pastor, José Eduardo; Aguilera, Angeles

    2017-01-01

    Transcriptomic sequencing together with bioinformatic analyses and an automated annotation process led us to identify novel phytochelatin synthase (PCS) genes from two extremophilic green algae (Chlamydomonas acidophila and Dunaliella acidophila). These genes are of intermediate length compared to known PCS genes from eukaryotes and PCS-like genes from prokaryotes. A detailed phylogenetic analysis gives new insight into the complicated evolutionary history of PCS genes and provides evidence for multiple horizontal gene transfer events from bacteria to eukaryotes within the gene family. A separate subgroup containing PCS-like genes within the PCS gene family is not supported since the PCS genes are monophyletic only when the PCS-like genes are included. The presence and functionality of the novel genes in the organisms were verified by genomic sequencing and qRT-PCR. Furthermore, the novel PCS gene in Chlamydomonas acidophila showed very strong induction by cadmium. Cloning and expression of the gene in Escherichia coli clearly improves its cadmium resistance. The gene in Dunaliella was not induced, most likely due to gene duplication.

  1. Mutations in the dihydropteroate synthase gene of Pneumocystis jiroveci isolates from Portuguese patients with Pneumocystis pneumonia

    DEFF Research Database (Denmark)

    Costa, M C; Helweg-Larsen, J; Lundgren, Bettina

    2003-01-01

    mutations were not significantly more frequent in patients exposed to sulpha drugs compared with patients not exposed (P=0.390). The results of this study suggest that DHPS gene mutations are frequent in the Portuguese immunocompromised population but do not seem associated with previous sulpha exposure......The aim of this study was to evaluate the frequency of mutations of the P. jiroveci dihydropteroate synthase (DHPS) gene in an immunocompromised Portuguese population and to investigate the possible association between DHPS mutations and sulpha exposure. In the studied population, DHPS gene...

  2. Mutations in the dihydropteroate synthase gene of Pneumocystis jiroveci isolates from Portuguese patients with Pneumocystis pneumonia

    DEFF Research Database (Denmark)

    Costa, M C; Helweg-Larsen, J; Lundgren, Bettina

    2003-01-01

    The aim of this study was to evaluate the frequency of mutations of the P. jiroveci dihydropteroate synthase (DHPS) gene in an immunocompromised Portuguese population and to investigate the possible association between DHPS mutations and sulpha exposure. In the studied population, DHPS gene...... mutations were not significantly more frequent in patients exposed to sulpha drugs compared with patients not exposed (P=0.390). The results of this study suggest that DHPS gene mutations are frequent in the Portuguese immunocompromised population but do not seem associated with previous sulpha exposure...

  3. IDENTIFICATION AND CHARACTERIZATION OF THE SUCROSE SYNTHASE 2 GENE (Sus2 IN DURUM WHEAT

    Directory of Open Access Journals (Sweden)

    Mariateresa eVolpicella

    2016-03-01

    Full Text Available Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for sucrose synthase in durum wheat (cultivars Ciccio and Svevo is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur and 5-BIL42. The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modelling approaches. The combined results of SUS2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

  4. Polymorphisms in the endothelial nitric oxide synthase gene in thalidomide embryopathy.

    Science.gov (United States)

    Vianna, Fernanda Sales Luiz; Fraga, Lucas Rosa; Tovo-Rodrigues, Luciana; Tagliani-Ribeiro, Alice; Biondi, Flavia; Maximino, Claudia Marques; Sanseverino, Maria Teresa Vieira; Hutz, Mara Helena; Schuler-Faccini, Lavínia

    2013-11-30

    Thalidomide is one of the most potent teratogens known to humans. It is currently used for many clinical situations such as treatment of leprosy reactions and multiple myeloma. However, the teratogenic mechanisms by which it produces morphological defects still remain unclear. One of the hypotheses is the blockage of angiogenesis by reduction of nitric oxide (NO). In this study, we evaluated two functional polymorphisms of the endothelial nitric oxide synthase (eNOS) gene which is a constitutively expressed enzyme responsible for production of NO. The promoter -786T>C exon 7 (896G>T) polymorphisms were genotyped using real-time PCR for 28 individuals with thalidomide embryopathy (TE), 27 first-degree relatives of these individuals, and 68 individuals from the general population. Their allele, genotypic, and haplotypic frequencies were compared. A significant difference was observed in the -786T>C polymorphism genotypes (p=0.03) between the groups affected by TE and those unaffected (non-relatives). The TT genotype of the 896G>T polymorphism was observed in 10.7% of those affected and 2.9% of those unaffected, but the difference was not statistically significant (p=0.09). The haplotypic analysis indicated that the wild haplotype -786T/896G was distributed differently in the affected and unaffected groups (p=0.004). These results indicate that the individuals with TE have a higher frequency of alleles associated with lower expression of eNOS, indicating that this may be a genotype susceptible to TE. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Expression analysis of cellulose synthase and main cytoskeletal protein genes in flax (Linum usitatissimum L.).

    Science.gov (United States)

    Galinousky, Dmitry; Padvitski, Tsimafei; Bayer, Galina; Pirko, Yaroslav; Pydiura, Nikolay; Anisimova, Natallia; Nikitinskaya, Tatyana; Khotyleva, Liubov; Yemets, Alla; Kilchevsky, Aleksandr; Blume, Yaroslav

    2017-08-09

    Fiber flax is an important source of natural fiber and a comprehensive model for the plant fiber biogenesis studies. Cellulose-synthase (CesA) and cytoskeletal genes are known to be important for the cell wall biogenesis in general and for the biogenesis of flax fibers in particular. Currently, knowledge about activity of these genes during the plant growth is limited. In this study, we have investigated flax fiber biogenesis by measuring expression of CesA and cytoskeletal genes at two stages of the flax development (seedlings and stems at the rapid growth stage) in several flax subspecies (elongatum, mediterraneum, crepitans). RT-qPCR has been used to quantify the expression of LusСesA1, LusСesA4, LusСesA7, LusСesA6, Actin, and α-Tubulin genes in plant samples. We report that CesA genes responsible for the secondary cell wall synthesis (LusCesA4, LusCesA7) have different expression pattern compared with CesA genes responsible for the primary cell wall synthesis (LusCesA1, LusCesA6): an average expression of LusCesA4 and LusCesA7 genes is relatively high in seedlings and further increases in stems at the rapid growth stage, whereas an average expression of LusCesA1 and LusCesA6 genes decreases. Interestingly, LusCesA1 is the only studied gene with different expression dynamics between the flax subspecies: its expression decreases by 5.2-10.7 folds in elongatum and mediterraneum but does not change in crepitans subspecies when the rapid growth stage and seedlings are compared. The expression of cytoskeleton genes (coding actin and α-tubulin) is relatively stable and significantly higher than the expression of cellulose-synthase genes in all the studied samples. © 2017 International Federation for Cell Biology.

  6. Expression of thromboxane A2 receptor gene and thromboxane A2 synthase in bovine corpora lutea.

    Science.gov (United States)

    Lei, Z M; Rao, C V; Chakraborty, C

    1992-08-01

    Studies were undertaken to investigate the expression of thromboxane (TXA2) receptor gene, from mRNA to functional receptor protein in terms of ligand binding, along with the cellular and subcellular distribution of the enzyme that catalyzes the formation of the ligand for the receptors. Bovine corpora lutea contained a single TXA2 receptor mRNA transcript of 2.8 kb. All the cell types in bovine corpora lutea contained immunoreactive TXA2 synthase, TXB2, TXA2 receptor transcripts, and receptor protein that bound the TXA2 antagonist 9,11-dimethylmethano-11,12-methano-16 (3-iodo-4-hydroxyphenyl)-13-14-dihydro-13-aza-15 alpha beta-omega-tetranor TXA2. The large luteal cells (20-35 microns) contained more receptor transcripts, receptor protein, and immunoreactive TXA2 synthase than did the small luteal cells (12-19 microns), luteal blood vessels, and nonluteal cells (7-12 microns). After correction for the cellular area differences, small luteal cells were seen to contain more receptor protein than did large luteal cells and nonluteal cells. All the cells showed an increase of TXA2 receptors and catalytically active TXA2 synthase from mid-luteal phase to early pregnancy, suggesting the possibility that TXA2 could be a luteotropic eicosanoid. Bovine lung homogenates (a positive control), bovine luteal plasma membranes-mitochondria-lysosomes fraction, rough-smooth endoplasmic reticulum-Golgi fraction, and highly purified nuclei contained 65-kDa immunoreactive protein, presumably representing TXA2 synthase. In addition, the luteal fractions, but not bovine lung, contained other small and large molecular-size immunoreactive proteins. Immunogold electron microscopy showed that immunoreactive TXA2 synthase was present primarily in plasma membranes, rough endoplasmic reticulum, nuclear membranes, and chromatin; and immunoreactive TXB2 was present primarily in different-size vesicles and nuclear chromatin. In summary, the present studies demonstrate for the first time that

  7. Differential expression of biphenyl synthase gene family members in fire-blight-infected apple 'Holsteiner Cox'.

    Science.gov (United States)

    Chizzali, Cornelia; Gaid, Mariam M; Belkheir, Asma K; Hänsch, Robert; Richter, Klaus; Flachowsky, Henryk; Peil, Andreas; Hanke, Magda-Viola; Liu, Benye; Beerhues, Ludger

    2012-02-01

    Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

  8. Identification and molecular characterization of nitric oxide synthase (NOS) gene in the intertidal copepod Tigriopus japonicus.

    Science.gov (United States)

    Jeong, Chang-Bum; Kang, Hye-Min; Seo, Jung Soo; Park, Heum Gi; Rhee, Jae-Sung; Lee, Jae-Seong

    2016-02-10

    In copepods, no information has been reported on the structure or molecular characterization of the nitric oxide synthase (NOS) gene. In the intertidal copepod Tigriopus japonicus, we identified a NOS gene that is involved in immune responses of vertebrates and invertebrates. In silico analyses revealed that nitric oxide (NO) synthase domains, such as the oxygenase and reductase domains, are highly conserved in the T. japonicus NOS gene. The T. japonicus NOS gene was highly transcribed in the nauplii stages, implying that it plays a role in protecting the host during the early developmental stages. To examine the involvement of the T. japonicus NOS gene in the innate immune response, the copepods were exposed to lipopolysaccharide (LPS) and two Vibrio sp. After exposure to different concentrations of LPS and Vibrio sp., T. japonicus NOS transcription was significantly increased over time in a dose-dependent manner, and the NO/nitrite concentration increased as well. Taken together, our findings suggest that T. japonicus NOS transcription is induced in response to an immune challenge as part of the conserved innate immunity. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. [Oxidative stress promotes hepatocyte apoptosis mediated by glycogen synthase kinase 3β].

    Science.gov (United States)

    Zhang, Xiangying; Guo, Yuanyuan; Zhang, Li; Wen, Tao; Piao, Zhengfu; Shi, Hongbo; Chen, Dexi; Duan, Zhongping; Ren, Feng

    2015-01-01

    To analyze the role of glycogen synthase kinase 3β (GSK3β) in hepatocyte apoptosis induced by oxidative stress. Human HL-7702 hepatoma cells were induced by H₂O₂/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H₂O₂/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide (PI) double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase (LDH) assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved caspase-3, c-Jun N-terminal kinase (JNK) and cytochrome C (CytC) proteins were examined using Western blotting. Oxidative stress triggered by H₂O₂/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased. GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.

  10. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    Science.gov (United States)

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  11. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes

    Science.gov (United States)

    Kroken, Scott; Glass, N. Louise; Taylor, John W.; Yoder, O. C.; Turgeon, B. Gillian

    2003-01-01

    Fungal type I polyketides (PKs) are synthesized by PK synthases (PKSs) and include well known secondary metabolites such as the anticholesterol drug lovastatin and the potent natural carcinogen aflatoxin. Other type I PKs are known to be virulence factors for some plant pathogens and pigments such as melanin. In this study, a phylogenomic approach was used to investigate the origin and diversity of fungal genes encoding putative PKSs that are predicted to synthesize type I PKs. The resulting genealogy, constructed by using the highly conserved PKS ketosynthase (KS) domain, indicated that: (i) Species within subphylum Pezizomycotina (phylum Ascomycota) but not early diverging ascomycetes, like Saccharomyces cerevisiae (Saccharomycotina) or Schizosaccharomyces pombe (Taphrinomycotina), had large numbers (7–25) of PKS genes. (ii) Bacteria and fungi had separate groups of PKS genes; the few exceptions are the likely result of horizontal gene transfer from bacteria to various sublineages of fungi. (iii) The bulk of genes encoding fungal PKSs fell into eight groups. Four groups were predicted to synthesize variously reduced PKs, and four groups were predicted to make unreduced PKs. (iv) Species within different classes of Pezizomycotina shared the same groups of PKS genes. (v) Different fungal genomes shared few putative orthologous PKS genes, even between closely related genomes in the same class or genus. (vi) The discontinuous distributions of orthologous PKSs among fungal species can be explained by gene duplication, divergence, and gene loss; horizontal gene transfer among fungi does not need to be invoked. PMID:14676319

  12. The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes

    DEFF Research Database (Denmark)

    Schneider, Lizette Marais; Adamski, Nikolai M.; Christensen, Caspar Elo

    2016-01-01

    five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase...

  13. Molecular Evolution of Trehalose-6-Phosphate Synthase (TPS) Gene Family in Populus, Arabidopsis and Rice

    Science.gov (United States)

    Yang, Hai-Ling; Liu, Yan-Jing; Wang, Cai-Ling; Zeng, Qing-Yin

    2012-01-01

    Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling. Plant TPS proteins contain both a TPS and a trehalose-6-phosphate phosphatase (TPP) domain, which are coded by a multi-gene family. The plant TPS gene family has been divided into class I and class II. A previous study showed that the Populus, Arabidopsis, and rice genomes have seven class I and 27 class II TPS genes. In this study, we found that all class I TPS genes had 16 introns within the protein-coding region, whereas class II TPS genes had two introns. A significant sequence difference between the two classes of TPS proteins was observed by pairwise sequence comparisons of the 34 TPS proteins. A phylogenetic analysis revealed that at least seven TPS genes were present in the monocot–dicot common ancestor. Segmental duplications contributed significantly to the expansion of this gene family. At least five and three TPS genes were created by segmental duplication events in the Populus and rice genomes, respectively. Both the TPS and TPP domains of 34 TPS genes have evolved under purifying selection, but the selective constraint on the TPP domain was more relaxed than that on the TPS domain. Among 34 TPS genes from Populus, Arabidopsis, and rice, four class I TPS genes (AtTPS1, OsTPS1, PtTPS1, and PtTPS2) were under stronger purifying selection, whereas three Arabidopsis class I TPS genes (AtTPS2, 3, and 4) apparently evolved under relaxed selective constraint. Additionally, a reverse transcription polymerase chain reaction analysis showed the expression divergence of the TPS gene family in Populus, Arabidopsis, and rice under normal growth conditions and in response to stressors. Our findings provide new insights into the mechanisms of gene family expansion and functional evolution. PMID:22905132

  14. Rhizobacteria activates (+)-δ-cadinene synthase genes and induces systemic resistance in cotton against beet armyworm (Spodoptera exigua).

    Science.gov (United States)

    Zebelo, Simon; Song, Yuanyuan; Kloepper, Joseph W; Fadamiro, Henry

    2016-04-01

    Gossypol is an important allelochemical produced by the subepidermal glands of some cotton varieties and important for their ability to respond to changing biotic stress by exhibiting antibiosis against some cotton pests. Plant growth-promoting rhizobacteria (PGPR) are root-colonizing bacteria that increase plant growth and often elicit defence against plant pathogens and insect pests. Little is known about the effect of PGPR on cotton plant-insect interactions and the potential biochemical and molecular mechanisms by which PGPR enhance cotton plant defence. Here, we report that PGPR (Bacillus spp.) treated cotton plants showed significantly higher levels of gossypol compared with untreated plants. Similarly, the transcript levels of the genes (i.e. (+)-δ-cadinene synthase gene family) involved in the biosynthesis of gossypol were higher in PGPR-treated plants than in untreated plants. Furthermore, the levels of jasmonic acid, an octadecanoid-derived defence-related phytohormone and the transcript level of jasmonic acid responsive genes were higher in PGPR-treated plants than in untreated plants. Most intriguingly, Spodoptera exigua showed reduced larval feeding and development on PGPR-treated plants. These findings demonstrate that treatment of plants with rhizobacteria may induce significant biochemical and molecular changes with potential ramifications for plant-insect interactions. © 2015 John Wiley & Sons Ltd.

  15. Functional analysis of the Brassica napus L. phytoene synthase (PSY) gene family.

    Science.gov (United States)

    López-Emparán, Ada; Quezada-Martinez, Daniela; Zúñiga-Bustos, Matías; Cifuentes, Víctor; Iñiguez-Luy, Federico; Federico, María Laura

    2014-01-01

    Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds

  16. Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, B.; Witton, C.J.

    2008-01-01

    BACKGROUND: Most human cancer cells have structural aberrations of chromosomal regions leading to loss or gain of gene specific alleles. This study aimed to assess the range of gene copies per nucleus of thymidylate synthase (TYMS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR...

  17. Deletion of a Chitin Synthase Gene in a Citric Acid Producing Strain of Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Rinker, Torri E.; Baker, Scott E.

    2007-01-29

    Citric acid production by the filamentous fungus Aspergillus niger is carried out in a process that causes the organism to drastically alter its morphology. This altered morphology includes hyphal swelling and highly limited polar growth resulting in clumps of swollen cells that eventually aggregate into pellets of approximately 100 microns in diameter. In this pelleted form, A. niger has increased citric acid production as compared to growth in filamentous form. Chitin is a crucial component of the cell wall of filamentous fungi. Alterations in the deposition or production of chitin may have profound effects on the morphology of the organism. In order to study the role of chitin synthesis in pellet formation we have deleted a chitin synthase gene (csmA) in Aspergillus niger strain ATCC 11414 using a PCR based deletion construct. This class of chitin synthases is only found in filamentous fungi and is not present in yeasts. The csmA genes contain a myosin motor domain at the N-terminus and a chitin synthesis domain at the C-terminus. They are believed to contribute to the specialized polar growth observed in filamentous fungi that is lacking in yeasts. The csmA deletion strain (csmAΔ) was subjected to minimal media with and without osmotic stabilizers as well as tested in citric acid production media. Without osmotic stabilizers, the mutant germlings were abnormally swollen, primarily in the subapical regions, and contained large vacuoles. However, this swelling is ultimately not inhibitory to growth as the germlings are able to recover and undergo polar growth. Colony formation was largely unaffected in the absence of osmotic stabilizers. In citric acid production media csmAΔ was observed to have a 2.5 fold increase in citric acid production. The controlled expression of this class of chitin synthases may be useful for improving production of organic acids in filamentous fungi.

  18. Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance.

    Science.gov (United States)

    Li, Bin; Xu, Wen Wen; Guan, Xin Yuan; Qin, Yan Ru; Law, Simon; Lee, Nikki Pui Yue; Chan, Kin Tak; Tam, Pui Ying; Li, Yuk Yin; Chan, Kwok Wah; Yuen, Hiu Fung; Tsao, Sai Wah; He, Qing Yu; Cheung, Annie L M

    2016-03-01

    Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. In vitro and in vivo functional assays were performed to study the effects of Id1-E2F1-IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1-E2F1-IGF2 regulatory axis has important implications for cancer prognosis and treatment. ©2015 American Association for Cancer Research.

  19. Gene structure, phylogeny and expression profile of the sucrose synthase gene family in cacao (Theobroma cacao L.).

    Science.gov (United States)

    Li, Fupeng; Hao, Chaoyun; Yan, Lin; Wu, Baoduo; Qin, Xiaowei; Lai, Jianxiong; Song, Yinghui

    2015-09-01

    In higher plants, sucrose synthase (Sus, EC 2.4.1.13) is widely considered as a key enzyme involved in sucrose metabolism. Although, several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, to date detailed information about the Sus genes is lacking for cacao. This study reports the identification of six novel Sus genes from economically important cacao tree. Analyses of the gene structure and phylogeny of the Sus genes demonstrated evolutionary conservation in the Sus family across cacao and other plant species. The expression of cacao Sus genes was investigated via real-time PCR in various tissues, different developmental phases of leaf, flower bud and pod. The Sus genes exhibited distinct but partially redundant expression profiles in cacao, with TcSus1, TcSus5 and TcSus6, being the predominant genes in the bark with phloem, TcSus2 predominantly expressing in the seed during the stereotype stage. TcSus3 and TcSus4 were significantly detected more in the pod husk and seed coat along the pod development, and showed development dependent expression profiles in the cacao pod. These results provide new insights into the evolution, and basic information that will assist in elucidating the functions of cacao Sus gene family.

  20. Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species.

    Science.gov (United States)

    Kita, Tomoko; Komatsu, Katsuko; Zhu, Shu; Iida, Osamu; Sugimura, Koji; Kawahara, Nobuo; Taguchi, Hiromu; Masamura, Noriya; Cai, Shao-Qing

    2016-03-01

    Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. (E)-β-farnesene synthase genes affect aphid (Myzus persicae) infestation in tobacco (Nicotiana tabacum).

    Science.gov (United States)

    Yu, Xiudao; Jones, Huw D; Ma, Youzhi; Wang, Genping; Xu, Zhaoshi; Zhang, Baoming; Zhang, Yongjun; Ren, Guangwei; Pickett, John A; Xia, Lanqin

    2012-03-01

    Aphids are major agricultural pests which cause significant yield losses of the crop plants each year. (E)-β-farnesene (EβF) is the alarm pheromone involved in the chemical communication between aphids and particularly in the avoidance of predation. In the present study, two EβF synthase genes were isolated from sweet wormwood and designated as AaβFS1 and AaβFS2, respectively. Overexpression of AaβFS1 or AaβFS2 in tobacco plants resulted in the emission of EβF ranging from 1.55 to 4.65 ng/day/g fresh tissues. Tritrophic interactions involving the peach aphids (Myzus persicae), predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaβFS1 and AaβFS2 could repel peach aphids, but not as strongly as expected. However, AaβFS1 and AaβFS2 lines exhibited strong and statistically significant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagon arrangement showed transgenic tobacco plants could repel aphids and attract lacewing larvae, thus minimizing aphid infestation. Therefore, we demonstrated a potentially valuable strategy of using EβF synthase genes from sweet wormwood for aphid control in tobacco or other economic important crops in an environmentally benign way.

  2. Tracking sesamin synthase gene expression through seed maturity in wild and cultivated sesame species--a domestication footprint.

    Science.gov (United States)

    Pathak, N; Bhaduri, A; Bhat, K V; Rai, A K

    2015-09-01

    Sesamin and sesamolin are the major oil-soluble lignans present in sesame seed, having a wide range of biological functions beneficial to human health. Understanding sesame domestication history using sesamin synthase gene expression could enable delineation of the sesame putative progenitor. This report examined the functional expression of sesamin synthase (CYP81Q1) during capsule maturation (0-40 days after flowering) in three wild Sesamum species and four sesame cultivars. Among the cultivated accessions, only S. indicum (CO-1) exhibited transcript abundance of sesamin synthase along with high sesamin content similar to S. malabaricum, while the other cultivated sesame showed low expression. The sesamin synthase expression analysis, coupled with quantification of sesamin level, indicates that sesamin synthase was not positively favoured during domestication. The sesamin synthase expression pattern and lignan content, along with phylogenetic analysis suggested a close relationship of cultivated sesame and the wild species S. malabaricum. The high genetic identity between the two species S. indicum and S. malabaricum points towards the role of the putative progenitor S. malabaricum in sesame breeding programmes to broaden the genetic base of sesame cultivars. This study emphasises the need to investigate intraspecific and interspecific variation in the primary, secondary and tertiary gene pools to develop superior sesame genotypes. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  3. RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate Karenia mikimotoi.

    Directory of Open Access Journals (Sweden)

    Kei Kimura

    Full Text Available The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1-A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates.

  4. Elevated expression of thymidylate synthase cycle genes in cisplatin-resistant human ovarian carcinoma A2780 cells

    Energy Technology Data Exchange (ETDEWEB)

    Scanlon, K.J.; Kashani-Sabet, M.

    1988-02-01

    Activity of the thymidylate synthase cycle was compared in the human ovarian carcinoma cell line A2780 and a subline that is resistant to cisplatin by a factor of 3. Resistant cells exhibited a 3-fold increase in mRNA for both dihydrofolate reductase and thymidylate synthase when compared with the parent line. Resistance to cisplatin also resulted in a 2.5-fold increase in enzyme activity for dihydrofolate reductase and thymidylate synthase; however, this increase did not result from amplification of the genes for these two enzymes. These data suggest that the initial step of cisplatin resistance in A2780 cells is a consequence of enhanced expression of the thymidylate synthase cycle.

  5. Absence of dihydropteroate synthase gene mutations in Pneumocystis jirovecii isolated from Swedish patients.

    Science.gov (United States)

    Beser, Jessica; Dini, Leigh; Botero-Kleiven, Silvia; Krabbe, Margareta; Lindh, Johan; Hagblom, Per

    2012-04-01

    Pneumocystis jirovecii remains an important cause of pneumonia in the immunocompromised host, with the largest group of patients at risk for P. jirovecii pneumonia (PCP) in Sweden being those with haematological diseases. Widespread prophylaxis and treatment for P. jirovecii with sulfa-containing drugs have effectively decreased the incidence of PCP, but concerns have been raised about the possible emergence of P. jirovecii isolates that are resistant to these drugs. Two point mutations in the gene coding for the dihydropteroate synthase enzyme (DHPS) in P. jirovecii have been shown to be associated with prior exposure to sulfa drugs. We retrospectively studied the occurrence of P. jirovecii DHPS mutations in isolates recovered from 103 Swedish patients. The DHPS gene, including the polymorphic positions 165 and 171, were amplified and sequenced by pyrosequencing technology. All the clinical specimens showed a wild-type pattern indicating that the occurrence of P. jirovecii DHPS mutations in Sweden is very low or absent.

  6. Apoptosis of bladder transitional cell carcinoma T24 cells induced by adenovirus-mediated inducible nitric oxide synthase gene transfection.

    Science.gov (United States)

    Tan, Jing; Zeng, Qing; Jiang, Xian-Zheng; He, Le-Ye; Wang, Jin-Rong; Yao, Kun; Wang, Chang-Hui

    2013-10-01

    To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells, and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma. Firstly, construct recombinant adenovirus vector pAd-iNOS of iNOS, followed by transfection of pAd-iNOS into HECK293 packaging cells. Thirdly, harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures. Finally, transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism. As shown by this study, the recombinant adenovirus rAd-iNOS was constructed successfully. The virus titer was 5.8×10(8) PFU/mL and recombinant was verified by PCR analysis. Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration, P53 protein expression up-regulation, as well as promotion of T24 cell apoptosis. The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAd-iNOS was confirmed to induce intracellular iNOS over-expression, high production of NO, up-regulation of intracellular P53 expression and promotion of cell apoptosis.

  7. Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269.

    Science.gov (United States)

    Huang, Sha; Romanchuk, Gail; Pattridge, Katherine; Lesley, Scott A; Wilson, Ian A; Matthews, Rowena G; Ludwig, Martha

    2007-08-01

    The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome-wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin-dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E. coli MetH. The fourth module of E. coli MetH is required for reductive remethylation of the cob(II)alamin form of the cofactor and binds the methyl donor for this reactivation, S-adenosylmethionine (AdoMet). Measurements of the rates of methionine formation in the presence and absence of TM0269 and AdoMet demonstrate that both TM0269 and AdoMet are required for reactivation of the inactive cob(II)alamin form of TM0268. These activity measurements confirm the structure-based assignment of the function of the TM0269 gene product. In the presence of TM0269, AdoMet, and reductants, the measured activity of T. maritima MetH is maximal near 80 degrees C, where the specific activity of the purified protein is approximately 15% of that of E. coli methionine synthase (MetH) at 37 degrees C. Comparisons of the structures and sequences of TM0269 and the reactivation domain of E. coli MetH suggest that AdoMet may be bound somewhat differently by the homologous proteins. However, the conformation of a hairpin that is critical for cobalamin binding in E. coli MetH, which constitutes an essential structural element, is retained in the T. maritima reactivation protein despite striking divergence of the sequences.

  8. Chalcone synthase family genes have redundant roles in anthocyanin biosynthesis and in response to blue/UV-A light in turnip (Brassica rapa; Brassicaceae).

    Science.gov (United States)

    Zhou, Bo; Wang, Yu; Zhan, Yaguang; Li, Yuhua; Kawabata, Saneyuki

    2013-12-01

    The epidermis of Brassica rapa (turnip) cv. Tsuda contains light-induced anthocyanins, visible signs of activity of chalcone synthase (CHS), a key anthocyanin biosynthetic enzyme, which is encoded by the CHS gene family. To elucidate the regulation of this light-induced pigmentation, we isolated Brassica rapa CHS1-CHS6 (BrCHS1-CHS6) and characterized their cis-elements and expression patterns. Epidermises of light-exposed swollen hypocotyls (ESHS) were harvested to analyze transcription levels of BrCHS genes by real-time PCR. Different promoters for the genes were inserted into tobacco to examine pCHS-GUS activity by histochemistry. Yeast-one-hybridization was used to detect binding activity of BrCHS motifs to transcription factors. Transcript levels of BrCHS1, -4, and -5 and anthocyanin-biosynthesis-related genes F3H, DFR, and ANS were high, while those of BrCHS2, -3, and -6 were almost undetectable in pigmented ESHS. However, in leaves, CHS5, F3H, and ANS expression was higher than in nonpigmented ESHS, but transcription of DFR was not detected. In the analysis of BrCHS1 and BrCHS3 promoter activity, GUS activity was strong in pigmented flowers of BrPCHS1-GUS-transformed tobacco plants, but nearly absent in BrPCHS3-GUS-transformed plants. Transcript levels of regulators, BrMYB75 and BrTT8, were strongly associated with the anthocyanin content and were light-induced. Coregulated cis-elements were found in promoters of BrCHS1,-4, and -5, and BrMYB75 and BrTT8 had high binding activities to the BrCHS Unit 1 motif. The chalcone synthase gene family encodes a redundant set of light-responsive, tissue-specific genes that are expressed at different levels and are involved in flavonoid biosynthesis in Tsuda turnip.

  9. Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily1[w

    Science.gov (United States)

    Martin, Diane M.; Fäldt, Jenny; Bohlmann, Jörg

    2004-01-01

    Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (−)-limonene synthase, (−)-α/β-pinene synthase, and (−)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-α-farnesene synthase, and E-α-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed. PMID:15310829

  10. Fatty acid synthase 2 contributes to diapause preparation in a beetle by regulating lipid accumulation and stress tolerance genes expression

    Science.gov (United States)

    Tan, Qian-Qian; Liu, Wen; Zhu, Fen; Lei, Chao-Liang; Wang, Xiao-Ping

    2017-01-01

    Diapause, also known as dormancy, is a state of arrested development that allows insects to survive unfavorable environmental conditions. Diapause-destined insects store large amounts of fat when preparing for diapause. However, the extent to which these accumulated fat reserves influence diapause remains unclear. To address this question, we investigated the function of fatty acid synthase (FAS), which plays a central role in lipid synthesis, in stress tolerance, the duration of diapause preparation, and whether insects enter diapause or not. In diapause-destined adult female cabbage beetles, Colaphellus bowringi, FAS2 was more highly expressed than FAS1 at the peak stage of diapause preparation. FAS2 knockdown suppressed lipid accumulation and subsequently affected stress tolerance genes expression and water content. However, silencing FAS2 had no significant effects on the duration of diapause preparation or the incidence of diapause. FAS2 transcription was suppressed by juvenile hormone (JH) and the JH receptor methoprene-tolerant (Met). These results suggest that the absence of JH-Met induces FAS2 expression, thereby promoting lipid storage in diapause-destined female beetles. These results demonstrate that fat reserves regulate stress tolerance genes expression and water content, but have no significant effect on the duration of diapause preparation or the incidence of diapause. PMID:28071706

  11. Monogalactosyldiacylglycerol: An abundant galactosyllipid of Cirsium brevicaule A. GRAY leaves inhibits the expression of gene encoding fatty acid synthase.

    Science.gov (United States)

    Inafuku, Masashi; Takara, Kensaku; Taira, Naoyuki; Nugara, Ruwani N; Kamiyama, Yasuo; Oku, Hirosuke

    2016-05-15

    The leaves of Cirsium brevicaule A. GRAY (CL) significantly decreased hepatic lipid accumulation and the expression of fatty acid synthase gene (FASN) in mice. We aimed to purify and identify the active compound(s) from CL and determine the inhibitory mechanism of expression of FASN. We purified monogalactosyldiacylglycerol (MGDG) from extracts of CL (CL-MGDG) and showed that it was the active CL component through analyses of its effects on the expression of genes of human breast cancer cell line, SKBR-3. The content and fatty acid composition of CL-MGDG are distinctly different from those of other vegetable-derived MGDGs. Treatment of SKBR-3 cells with MGDG decreased the level of FASN mRNA as well as the levels of mRNA encoding other protein involved in lipogenesis. Further, MGDG treatments significantly inhibited luciferase activities of constructs containing liver X receptor response element in FASN promoter region without altering the levels of mRNA encoding transcription factors. MGDG and the FASN inhibitor C75 decreased the viabilities of SKBR-3 cells in a concentration-dependent manner. CL-MGDG more potently inhibited cell viability than a commercial MGDG preparation. CL represents a good source of glycoglycerolipids with potential as functional ingredients of food. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. Over-expression of the nitric oxide synthase associated gene1 (NOA1 in cucumber improves chilling stress tolerance

    Directory of Open Access Journals (Sweden)

    Xingwang Liu

    2016-11-01

    Full Text Available Nitric oxide (NO is a gaseous signaling molecule in plants, transducing information as a result of exposure to low temperatures. However, the underlying molecular mechanism linking NO with chilling stress is not well understood. Here, we functionally characterized the cucumber (Cucumis sativus nitric oxide synthase-associated gene, NITRIC OXIDE ASSOCIATED 1(CsNOA1. Expression analysis of CsNOA1, using quantitative real-time PCR, in situ hybridization and a promoter::β-glucuronidase (GUS reporter assay, revealed that it is expressed mainly in the root and shoot apical meristem (SAM, and that expression is up-regulated by low temperatures. A CsNOA1-GFP fusion protein was found to be localized to the mitochondria, and ectopic expression of CsNOA1 in the A. thaliana noa1 mutant partially rescued the normal phenotype. Transgenic cucumber plants revealed that the gene is required by seedlings to tolerate chilling stress: constitutive over-expression of CsNOA1 led to a greater accumulation of soluble sugars, starch, and an up-regulation of Cold-regulatory C-repeat binding factor3 (CBF3 expression. Conversely, suppression of CsNOA1 expression resulted in the opposite phenotype and a reduced NO content compared to wild type plants. Additionally, analyses using inhibitors suggested that NO can be synthesized via a CsNOA1 dependent pathway that is independent of the nitrate reductase (NR pathway.

  13. Association of endothelial nitric oxide synthase gene polymorphisms with endometrial carcinoma: a preliminary study.

    Science.gov (United States)

    Oztürk, Ebru; Dikensoy, Ebru; Balat, Ozcan; Uğur, Mete Gürol; Balcı, Sibel Oğuzkan; Aydın, Abdullah; Kazancı, Ulkü; Pehlivan, Sacide

    2011-01-01

    To investigate the relationship between specific endothelial nitric oxide synthase (eNOS) gene polymorphisms and endometrial cancer (ECa). The study group consisted of 89 patients histologically diagnosed with the endometrioid type of endometrial carcinoma. The control group consisted of 60 randomly selected individuals who had undergone total hysterectomy. Genomic DNA was isolated from paraffin-embedded endometrial tissues. We investigated the G894T polymorphisms (G894T) and variable number tandem repeats polymorphisms in intron 4 (VNTR intron 4) in the eNOS gene by using polymerase chain reaction (PCR) and/or restriction fragment length polymorphism (RFLP). The genotype distributions and allele frequencies of the two groups were compared. Analysis of the VNTR intron 4 polymorphisms in eNOS gene revealed that the frequency of the AA genotype was significantly higher in the control group, whereas the frequency of the BB genotype was significantly higher in the ECa group. Analysis of the G894T polymorphisms in eNOS gene revealed a significantly higher frequency of the GG genotype in the control group but a significantly higher frequency of the TT genotype in the endometrial cancer group. The G894T and VNTR intron 4 polymorphisms in eNOS gene could be an intriguing susceptibility factor that modulates an individual's risk of ECa in the Turkish population.

  14. Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites*

    Science.gov (United States)

    Zhao, Ya-e; Wang, Zheng-hang; Xu, Yang; Xu, Ji-ru; Liu, Wen-yan; Wei, Meng; Wang, Chu-ying

    2012-01-01

    To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi’an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites. PMID:23024043

  15. Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites.

    Science.gov (United States)

    Zhao, Ya-e; Wang, Zheng-hang; Xu, Yang; Xu, Ji-ru; Liu, Wen-yan; Wei, Meng; Wang, Chu-ying

    2012-10-01

    To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi'an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi'an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%-99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites.

  16. Positive selection and functional divergence of farnesyl pyrophosphate synthase genes in plants.

    Science.gov (United States)

    Qian, Jieying; Liu, Yong; Chao, Naixia; Ma, Chengtong; Chen, Qicong; Sun, Jian; Wu, Yaosheng

    2017-02-04

    Farnesyl pyrophosphate synthase (FPS) belongs to the short-chain prenyltransferase family, and it performs a conserved and essential role in the terpenoid biosynthesis pathway. However, its classification, evolutionary history, and the forces driving the evolution of FPS genes in plants remain poorly understood. Phylogeny and positive selection analysis was used to identify the evolutionary forces that led to the functional divergence of FPS in plants, and recombinant detection was undertaken using the Genetic Algorithm for Recombination Detection (GARD) method. The dataset included 68 FPS variation pattern sequences (2 gymnosperms, 10 monocotyledons, 54 dicotyledons, and 2 outgroups). This study revealed that the FPS gene was under positive selection in plants. No recombinant within the FPS gene was found. Therefore, it was inferred that the positive selection of FPS had not been influenced by a recombinant episode. The positively selected sites were mainly located in the catalytic center and functional areas, which indicated that the 98S and 234D were important positively selected sites for plant FPS in the terpenoid biosynthesis pathway. They were located in the FPS conserved domain of the catalytic site. We inferred that the diversification of FPS genes was associated with functional divergence and could be driven by positive selection. It was clear that protein sequence evolution via positive selection was able to drive adaptive diversification in plant FPS proteins. This study provides information on the classification and positive selection of plant FPS genes, and the results could be useful for further research on the regulation of triterpenoid biosynthesis.

  17. [Diversity of polyketide synthase genes (PKS) in metagenomic community of the freshwater sponge].

    Science.gov (United States)

    Kaliuzhnaia, O V; Kulakova, N V; Itskovich, B V

    2012-01-01

    Screening of metagenomic DNA of microbial community, associated with Baikalian sponge Lubomirskia baicalensis, was made to show the presence of polyketide synthase genes (PKS). PKS enzymatic systems take part in synthesis of a great number of biologically-active substances. Cloning and sequencing of amplified products of the ketosynthase domain section of PKS gene cluster has revealed 15 fragments of PKS genes differing from each other's on 35-65% by aminoacid sequences. BLASTX analysis has shown that all these sequences belong to the KS-domains identified in various groups of microorganisms: alpha-, beta-, delta-Proteobacteria, Verrucomicrobia, Cyanobacteria, Chlorophyta. Some sequences were related to the genes which are taking part in biosynthesis of curacin A (CurI, CurJ), stigmatellin (StiC, StiG), nostophycin (NpnB), and cryptophycins (CrpB). The homology of the found sequences with those of the EMBL database varies within 50-82% confirming the presence in fresh-water sponge community the genes for synthesis of the new, yet not studied polyketide substances, possessing the biotechnological potential.

  18. Nicotianamine synthase overexpression positively modulates iron homeostasis-related genes in high iron rice

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2013-05-01

    Full Text Available Nearly one-third of the world population, mostly women and children, suffer from iron malnutrition and its consequences, such as anemia or impaired mental development. Biofortification of rice, which is a staple crop for nearly half of the world’s population, can significantly contribute in alleviating iron deficiency. NFP rice (transgenic rice expressing nicotianamine synthase, ferritin and phytase genes has a more than six-fold increase in iron content in polished rice grains, resulting from the synergistic action of nicotianamine synthase (NAS and ferritin transgenes. We investigated iron homeostasis in NFP plants by analyzing the expression of 28 endogenous rice genes known to be involved in the homeostasis of iron and other metals, in iron-deficient and iron-sufficient conditions. RNA was collected from different tissues (roots, flag leaves, grains and at three developmental stages during grain filling. NFP plants showed increased sensitivity to iron-deficiency conditions and changes in the expression of endogenous genes involved in nicotianamine (NA metabolism, in comparison to their non-transgenic siblings. Elevated transcript levels were detected in NFP plants for several iron transporters. In contrast, expression of OsYSL2, which encodes a member of Yellow Stripe-like protein family, and a transporter of the NA-Fe(II complex was reduced in NFP plants under low iron conditions, indicating that expression of OsYSL2 is regulated by the endogenous iron status. Expression of the transgenes did not significantly affect overall iron homeostasis in NFP plants, which establishes the engineered push-pull mechanism as a suitable strategy to increase rice endosperm iron content.

  19. Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice.

    Directory of Open Access Journals (Sweden)

    Shigang Zheng

    Full Text Available Resveratrol (Res is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS, existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 μg/g FW in seedlings and 3.053 μg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering.

  20. Multi-level gene expression profiles affected by thymidylate synthase and 5-fluorouracil in colon cancer

    Directory of Open Access Journals (Sweden)

    Chu Edward

    2006-04-01

    Full Text Available Abstract Background Thymidylate synthase (TS is a critical target for cancer chemotherapy and is one of the most extensively studied biomarkers for fluoropyrimidine-based chemotherapy. In addition to its critical role in enzyme catalysis, TS functions as an RNA binding protein to regulate the expression of its own mRNA translation and other cellular mRNAs, such as p53, at the translational level. In this study, a comprehensive gene expression analysis at the levels of both transcriptional and post-transcriptional regulation was conducted to identify response markers using human genome array with TS-depleted human colon cancer HCT-C18 (TS- cells and HCT-C18 (TS+ cells stably transfected with the human TS cDNA expression plasmid. Results A total of 38 genes were found to be significantly affected by TS based on the expression profiles of steady state mRNA transcripts. However, based on the expression profiles of polysome associated mRNA transcripts, over 149 genes were affected by TS overexpression. This indicates that additional post-transcriptionally controlled genes can be captured with profiling polysome associated mRNA population. This unique approach provides a comprehensive overview of genes affected by TS. Additional novel post-transcriptionally regulated genes affected by 5-fluorouracil (5-FU treatment were also discovered via similar approach. Conclusion To our knowledge, this is the first time that a comprehensive gene expression profile regulated by TS and 5-FU was analyzed at the multiple steps of gene regulation. This study will provide candidate markers that can be potentially used for predicting therapeutic outcomes for fluoropyrimidine-based cancer chemotherapy.

  1. Hypersensitive Response of Plasmid-Encoded AHL Synthase Gene to Lifestyle and Nutrient by Ensifer adhaerens X097

    Directory of Open Access Journals (Sweden)

    Yanhua Zeng

    2017-06-01

    Full Text Available It is known that some bacteria, especially members of the family Rhizobiaceae, have multiple N-acyl homoserine lactones (AHL synthase genes and produce multiple AHL signals. However, how bacteria selectively utilize these multiple genes and signals to cope with changing environments is poorly understood. Ensifer adhaerens is an important microorganism in terms of biotechnology, ecology and evolutionary. In this study, we investigated the AHL-based QS system of E. adhaerens X097 and its response to different lifestyles or nutrients. Draft genome sequence data indicated that X097 harbored three distinct AHL synthase genes (ensI1, 2, 3 and seven luxR homologs, which was different from other E. adhaerens strains. In vitro expression indicated that plasmid-encoded ensI1 and ensI2 directed production of multiple AHLs, while chromosome-encoded ensI3 only directed production of C14-HSL. Predicted three dimensional structure of EnsI3 was quite different from that of EnsI1 and EnsI2. X097 produced different AHL profiles in Luria-Bertani (LB and NFB medium, under biofilm and planktonic lifestyle, respectively. Notably, expression of ensI1 and ensI2 but not ensI3 is hypersensitive to different lifestyles and nutrients. The hypersensitive response of plasmid-encoded AHL synthase genes to different culture conditions may shed a light on the phylogenetic development of AHL synthase genes in Rhizobiaceae family.

  2. Disruption of Bcchs4, Bcchs6 or Bcchs7 chitin synthase genes in Botrytis cinerea and the essential role of class VI chitin synthase (Bcchs6).

    Science.gov (United States)

    Morcx, Serena; Kunz, Caroline; Choquer, Mathias; Assie, Sébastien; Blondet, Eddy; Simond-Côte, Elisabeth; Gajek, Karina; Chapeland-Leclerc, Florence; Expert, Dominique; Soulie, Marie-Christine

    2013-03-01

    Chitin synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulié et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI chitin synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. The Phytoene synthase gene family of apple (Malus x domestica) and its role in controlling fruit carotenoid content.

    Science.gov (United States)

    Ampomah-Dwamena, Charles; Driedonks, Nicky; Lewis, David; Shumskaya, Maria; Chen, Xiuyin; Wurtzel, Eleanore T; Espley, Richard V; Allan, Andrew C

    2015-07-28

    Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation. The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in 'Granny Smith' and 'Royal Gala' apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms. The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars.

  4. Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

    Science.gov (United States)

    Yao, Lin; Tan, Chong; Song, Jinzhu; Yang, Qian; Yu, Lijie; Li, Xinling

    2016-01-01

    Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  5. Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei

    Directory of Open Access Journals (Sweden)

    Yue Ming

    2009-06-01

    Full Text Available Abstract Background Trehalose synthase (TreS which converts maltose to trehalose is considered to be a potential biocatalyst for trehalose production. This enzymatic process has the advantage of simple reaction and employs an inexpensive substrate. Therefore, new TreS producing bacteria with suitable enzyme properties are expected to be isolated from extreme environment. Results Six TreS producing strains were isolated from a specimen obtained from soil of the Tibetan Plateau using degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified using thermal asymmetric interlaced PCR. The gene contained a 1626 bp open reading frame encoding 541 amino acids. The gene was expressed in Escherichia coli, and the recombinant TreS was purified and characterized. The purified TreS had a molecular mass of 65 kDa and an activity of 18.5 U/mg. The optimum temperature and pH for the converting reaction were 37°C and 6, respectively. Hg2+, Zn2+, Cu2+and SDS inhibited the enzyme activity at different levels whereas Mn2+ showed an enhancing effect by 10%. Conclusion In this study, several TreS producing strains were screened from a source of soil bacteria. The characterization of the recombinant TreS of Enterobacter hormaechei suggested its potential application. Consequently, a strategy for isolation of TreS producing strains and cloning of novel treS genes from natural sources was demonstrated.

  6. Adaptive changes in geranylgeranyl pyrophosphate synthase gene expression level under ethanol stress conditions in Oenococcus oeni.

    Science.gov (United States)

    Cafaro, C; Bonomo, M G; Salzano, G

    2014-01-01

    The aim of this study was to investigate the effect of ethanol exposure on the expression level of geranylgeranyl pyrophosphate synthase gene involved in the metabolism of Oenococcus oeni to probe the mechanisms of ethanol tolerance correlated with adaptive changes. The evaluation of ten potential internal control genes and the comparative study of their stability were performed to select the most stable internal controls for the normalization of expression data. The expression level analysis by qPCR and changes after exposure to ethanol stresses highlighted a significant increase in the presence of higher ethanol concentrations. The analysis of results suggest that O. oeni adjusts the expression of genes to adapt to stress conditions and the high expression level of ggpps would allow a flow of isoprenoid precursors towards the carotenoids and related pathways to stabilize bacterial cell membranes, improving the cell membrane disturbances and preventing cell death induced by ethanol. The involvement of ggpps gene in physiological changes of bacterial behaviour confirmed the exposure to stress requires the activation of defence mechanism to be more tolerant to adverse conditions. Improving the knowledge of stress tolerance and adaptation mechanisms of O. oeni is essential to enhance the efficiency of the malolactic starter in wine and to obtain the development of starters able to survive to direct inoculation with a large benefit for wine technology. © 2013 The Society for Applied Microbiology.

  7. Silybin content and overexpression of chalcone synthase genes in Silybum marianum L. plants under abiotic elicitation.

    Science.gov (United States)

    El-Garhy, Hoda A S; Khattab, Salah; Moustafa, Mahmoud M A; Abou Ali, Rania; Abdel Azeiz, Ahmed Z; Elhalwagi, Abeer; El Sherif, Fadia

    2016-11-01

    Silymarin, a Silybum marianum seed extract containing a mixture of flavonolignans including silybin, is being used as an antihepatotoxic therapy for liver diseases. In this study, the enhancing effect of gamma irradiation on plant growth parameters of S. marianum under salt stress was investigated. The effect of gamma irradiation, either as a single elicitor or coupled with salinity, on chalcone synthase (CHS) gene expression and silybin A + B yield was also evaluated. The silybin A + B content in S. marianum fruits was estimated by liquid chromatography-mass spectrometry (LC-MS/MS). An increase in silybin content was accompanied by up-regulation of the CHS1, CHS2 and CHS3 genes, which are involved in the silybin biosynthetic pathway. The highest silybin A + B production (0.77 g/100 g plant DW) and transcript levels of the three studied genes (100.2-, 91.9-, and 24.3-fold increase, respectively) were obtained with 100GY gamma irradiation and 4000 ppm salty water. The CHS2 and CHS3 genes were partially sequenced and submitted to the NCBI database under the accession numbers KT252908.1 and KT252909.1, respectively. Developing new approaches to stimulate silybin biosynthetic pathways could be a useful tool to potentiate the use of plants as renewable resources of medicinal compounds. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. Overexpression of a synthetic insect-plant geranyl pyrophosphate synthase gene in Camelina sativa alters plant growth and terpene biosynthesis.

    Science.gov (United States)

    Xi, Jing; Rossi, Lorenzo; Lin, Xiuli; Xie, De-Yu

    2016-07-01

    A novel plastidial homodimeric insect-plant geranyl pyrophosphate synthase gene is synthesized from three different cDNA origins. Its overexpression in Camelina sativa effectively alters plant development and terpenoid metabolism. Geranyl pyrophosphate synthase (GPPS) converts one isopentenyl pyrophosphate and dimethylallyl pyrophosphate to GPP. Here, we report a synthetic insect-plant GPPS gene and effects of its overexpression on plant growth and terpenoid metabolism of Camelina sativa. We synthesized a 1353-bp cDNA, namely PTP-MpGPPS. This synthetic cDNA was composed of a 1086-bp cDNA fragment encoding a small GPPS isomer of the aphid Myzus persicae (Mp), 240-bp Arabidopsis thaliana cDNA fragment encoding a plastidial transit peptide (PTP), and a 27-bp short cDNA fragment encoding a human influenza hemagglutinin tag peptide. Structural modeling showed that the deduced protein was a homodimeric prenyltransferase. Confocal microscopy analysis demonstrated that the PTP-MpGPPS fused with green florescent protein was localized in the plastids. The synthetic PTP-MpGPPS cDNA driven by 2 × 35S promoters was introduced into Camelina (Camelina sativa) by Agrobacterium-mediated transformation and its overexpression in transgenic plants were demonstrated by western blot. T2 and T3 progeny of transgenic plants developed larger leaves, grew more and longer internodes, and flowered earlier than wild-type plants. Metabolic analysis showed that the levels of beta-amyrin and campesterol were higher in tissues of transgenic plants than in those of wild-type plants. Fast isoprene sensor analysis demonstrated that transgenic Camelina plants emitted significantly less isoprene than wild-type plants. In addition, transcriptional analyses revealed that the expression levels of gibberellic acid and brassinosteroids-responsive genes were higher in transgenic plants than in wild-type plants. Taken together, these data demonstrated that this novel synthetic insect-plant GPPS cDNA was

  9. Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants

    Directory of Open Access Journals (Sweden)

    Soltis Pamela S

    2006-02-01

    Full Text Available Abstract Background The glycogen synthase kinase 3 (GSK3/SHAGGY-like kinases (GSKs are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences. Results We found that the structure of GSK genes is generally conserved in Arabidopsis and rice, although we documented examples of exon expansion and intron loss. Our phylogenetic analyses of 139 sequences revealed four major clades of GSK genes that correspond to the four subgroups initially recognized in Arabidopsis. ESTs from basal angiosperms were represented in all four major clades; GSK homologs from the basal angiosperm Persea americana (avocado appeared in all four clades. Gymnosperm sequences occurred in clades I, III, and IV, and a sequence of the red alga Porphyra was sister to all green plant sequences. Conclusion Our results indicate that (1 the plant-specific GSK gene lineage was established early in the history of green plants, (2 plant GSKs began to diversify prior to the origin of extant seed plants, (3 three of the four major clades of GSKs present in Arabidopsis and rice were established early in the evolutionary history of extant seed plants, and (4 diversification into four major clades (as initially reported in Arabidopsis occurred either just prior to the origin of the angiosperms or very early in angiosperm history.

  10. A reporter gene system for the precise measurement of promoter activity in Thermus thermophilus HB27.

    Science.gov (United States)

    Fujita, Atsushi; Sato, Takaaki; Koyama, Yoshinori; Misumi, Yoshio

    2015-11-01

    We developed a reporter gene system that enables precise analysis of promoter activity in Thermus thermophilus HB27. The reporter vector employs a promoterless β-galactosidase gene of Thermus spp. strain T2. However, T. thermophilus HB27 strain has three genes (TTP0042, TTP0220 and TTP0222) whose products have β-galactosidase activity, which would interfere with correct measurements of promoter activities. Thus, to eliminate this background activity, we disrupted all three of these genes to generate a host strain for measuring promoter expression as β-galactosidase activity. In addition, T. thermophilus strains also produce carotenoids called thermoxanthins that are yellow pigments. To avoid the influence of these carotenoids on the β-galactosidase assay, we also disrupted the phytoene synthase gene (crtB). The reporter gene system developed here is a powerful tool for studying transcriptional activity and the mechanisms that regulate gene expression in T. thermophilus HB27. We also showed that the crtB gene cassette could be used in repeated gene-disruption experiments to screen transformants by colony colour, thus eliminating the need for antibiotic resistance markers.

  11. Different chitin synthase genes are required for various developmental and plant infection processes in the rice blast fungus Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ling-An Kong

    2012-02-01

    Full Text Available Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs. Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.

  12. Differential expression of cellulose synthase (CesA) gene transcripts in potato as revealed by QRT-PCR

    NARCIS (Netherlands)

    Olawole, O.; Jacobsen, E.; Vincken, J.P.; Visser, R.G.F.

    2009-01-01

    Two transgenic potato lines, csr2–1 and csr4–8 that contained two different antisense cellulose synthase (CesA) genes, csr2 and csr4, respectively were crossed. The aim, amongst others, was to investigate the possibility of generating double transformants to validate a hypothetical presence of the

  13. Gene variations of nitric oxide synthase regulate the effects of a saturated fat rich meal on endothelial function

    Science.gov (United States)

    Objective: Endothelial nitric oxide synthase gene variations have been linked to a higher risk for cardiovascular diseases by unknown mechanisms. Our aim was to determine if two SNPs located in NOS3 (E298D and i19342) interfere with microvascular endothelial function (MEF) and/or oxidative stress du...

  14. Violent suicidal behaviour in bipolar disorder is associated with nitric oxide synthase 3 gene polymorphism.

    Science.gov (United States)

    Oliveira, J; Debnath, M; Etain, B; Bennabi, M; Hamdani, N; Lajnef, M; Bengoufa, D; Fortier, C; Boukouaci, W; Bellivier, F; Kahn, J-P; Henry, C; Charron, D; Krishnamoorthy, R; Leboyer, M; Tamouza, R

    2015-09-01

    Given the importance of nitric oxide system in oxidative stress, inflammation, neurotransmission and cerebrovascular tone regulation, we postulated its potential dysfunction in bipolar disorder (BD) and suicide. By simultaneously analysing variants of three isoforms of nitric oxide synthase (NOS) genes, we explored interindividual genetic liability to suicidal behaviour in BD. A total of 536 patients with BD (DSM-IV) and 160 healthy controls were genotyped for functionally relevant NOS1, NOS2 and NOS3 polymorphisms. History of suicidal behaviour and violent suicide attempt was documented for 511 patients with BD. Chi-squared test was used to perform genetic association analyses and logistic regression to test for gene-gene interactions. NOS3 rs1799983 T homozygous state was associated with violent suicide attempts (26.4% vs. 10.8%, in patients and controls, P = 0.002, corrected P (Pc) = 0.004, OR: 2.96, 95% CI = 1.33-6.34), and this association was restricted to the early-onset BD subgroup (37.9% vs. 10.8%, in early-onset BD and controls, P = 0.0003, Pc = 0.0006 OR: 5.05, 95% CI: 1.95-12.45), while we found no association with BD per se and no gene-gene interactions. Our results bring further evidence for the potential involvement of endothelial NOS gene variants in susceptibility to suicidal behaviour. Future exploration of this pathway on larger cohort of suicidal behaviour is warranted. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Invertebrate Trehalose-6-Phosphate Synthase Gene: Genetic Architecture, Biochemistry, Physiological Function, and Potential Applications

    Directory of Open Access Journals (Sweden)

    Bin Tang

    2018-01-01

    Full Text Available The non-reducing disaccharide trehalose is widely distributed among various organisms. It plays a crucial role as an instant source of energy, being the major blood sugar in insects. In addition, it helps countering abiotic stresses. Trehalose synthesis in insects and other invertebrates is thought to occur via the trehalose-6-phosphate synthase (TPS and trehalose-6-phosphate phosphatase (TPP pathways. In many insects, the TPP gene has not been identified, whereas multiple TPS genes that encode proteins harboring TPS/OtsA and TPP/OtsB conserved domains have been found and cloned in the same species. The function of the TPS gene in insects and other invertebrates has not been reviewed in depth, and the available information is quite fragmented. The present review discusses the current understanding of the trehalose synthesis pathway, TPS genetic architecture, biochemistry, physiological function, and potential sensitivity to insecticides. We note the variability in the number of TPS genes in different invertebrate species, consider whether trehalose synthesis may rely only on the TPS gene, and discuss the results of in vitro TPS overexpression experiment. Tissue expression profile and developmental characteristics of the TPS gene indicate that it is important in energy production, growth and development, metamorphosis, stress recovery, chitin synthesis, insect flight, and other biological processes. We highlight the molecular and biochemical properties of insect TPS that make it a suitable target of potential pest control inhibitors. The application of trehalose synthesis inhibitors is a promising direction in insect pest control because vertebrates do not synthesize trehalose; therefore, TPS inhibitors would be relatively safe for humans and higher animals, making them ideal insecticidal agents without off-target effects.

  16. Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

    Science.gov (United States)

    Liu, Weixin; Xu, Biyu; Jin, Zhiqiang

    2014-01-01

    Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage. PMID:24505384

  17. Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis.

    Directory of Open Access Journals (Sweden)

    Holger Jenke-Kodama

    2006-10-01

    Full Text Available Modular polyketide synthases (PKSs of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT, ketoreductase (KR, and dehydratase (DH-KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.

  18. Molecular Characterization of Dihydrofolate Reductase-Thymidylate Synthase Gene Concerning Antifulate Resistance of Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    N Hosseinzadeh

    2009-12-01

    Full Text Available "nBackground: The recently reported resistance to antimalarials contributes to making the control of ma­laria more difficult. There is a need to evaluate the current antimalaria regimens to prevent this emerging problem. The aim of this study was to determine dihydrofolate reductase-thymidylate synthase gene mu­tation (pvdhfr regarding antifulate resistance in Plasmodium vivax. "nMethods: From 2007 to 2009, 117 P. vivax infected blood samples collected from two regions of Hor­mozgan Province, south of Iran were analyzed using PCR, semi-nested-PCR and RFLP methods. "nResults: Eighty four isolates (71.8 % showed no mutation in pvdhfr gene of P. vivax known as wild type and 33 (28.2% of the samples revealed nine single (7.7%, twenty two double (18.8% and two (1.7% triple mutations. "nConclusion: Genetic diversity was observed by molecular methods in pvdhfr gene of p. vivax in Hor­mozgan Province suggests that the antifolate falciparum malaria drug (fansidar is proportionally affecting P. vivax dhfr mutation. Therefore, more studies to evaluate antimalarial drugs that should preferably be effective against both P. vivax and P. falciparum are recommended.

  19. Nonsense mutation inside anthocyanidin synthase gene controls pigmentation in yellow raspberry (Rubus idaeus L..

    Directory of Open Access Journals (Sweden)

    Muhammad Zubair Rafique

    2016-12-01

    Full Text Available Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry (Anne. A 5-bp insertion in the coding region was identified and designated as ans+5. The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect i.e. nonsense-mRNA mediated decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans+5 and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

  20. Identification of genes encoding granule-bound starch synthase involved in amylose metabolism in banana fruit.

    Directory of Open Access Journals (Sweden)

    Hongxia Miao

    Full Text Available Granule-bound starch synthase (GBSS is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.

  1. Nonsense Mutation Inside Anthocyanidin Synthase Gene Controls Pigmentation in Yellow Raspberry (Rubus idaeus L.).

    Science.gov (United States)

    Rafique, Muhammad Z; Carvalho, Elisabete; Stracke, Ralf; Palmieri, Luisa; Herrera, Lorena; Feller, Antje; Malnoy, Mickael; Martens, Stefan

    2016-01-01

    Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry ("Anne"). A 5 bp insertion in the coding region was identified and designated as ans+5. The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild-type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect, i.e., nonsense-mediated mRNA decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans+5 and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

  2. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  3. Expression of terpene synthase genes associated with the formation of volatiles in different organs of Vitis vinifera.

    Science.gov (United States)

    Matarese, Fabiola; Cuzzola, Angela; Scalabrelli, Giancarlo; D'Onofrio, Claudio

    2014-09-01

    Plants produce a plethora of volatile organic compounds (VOCs) which are important in determining the quality and nutraceutical properties of horticultural food products, including the taste and aroma of wine. Given that some of the most prevalent grape aroma constituents are terpenoids, we investigated the possible variations in the relative expression of terpene synthase (TPS) genes that depend on the organ. We thus analysed mature leaves, young leaves, stems, young stems, roots, rachis, tendrils, peduncles, bud flowers, flowers and berries of cv Moscato bianco in terms of their VOC content and the expression of 23 TPS genes. In terms of the volatile characterization of the organs by SPME/GC-MS analysis, flower buds and open flowers appeared to be clearly distinct from all the other organs analysed in terms of their high VOC concentration. Qualitatively detected VOCs clearly separated all the vegetative organs from flowers and berries, then the roots and rachis from other vegetative organs and flowers from berries, which confirms the specialization in volatile production among different organs. Our real-time RT-PCR results revealed that the majority of TPS genes analysed exhibited detectable transcripts in all the organs investigated, while only some were found to be expressed specifically in one or just a few organs. In most cases, we found that the known products of the in vitro assay of VvTPS enzymes corresponded well to the terpenes found in the organs in which the encoding gene was expressed, as in the case of (E)-β-caryophyllene synthases, α-terpineol synthase and α-farnesene synthase. In addition, we found groups of homologous TPS genes, such as (E)-β-caryophyllene and β-ocimene synthases, expressed distinctively in the various tissues. This thus confirmed the subfunctionalization events and a specialization on the basis of the organs in which they are mostly expressed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. The human tyrosine hydroxylase gene promoter.

    Science.gov (United States)

    Kessler, Mark A; Yang, Ming; Gollomp, Kandace L; Jin, Hao; Iacovitti, Lorraine

    2003-04-10

    13.329 kilobases of the single copy human tyrosine hydroxylase (hTH) gene were isolated from a genomic library. The 5' flanking 11 kilobases fused to the reporter green fluorescent protein (GFP) drove high level expression in TH+ cells of the substantia nigra of embryonic and adult transgenic mice as determined by double label fluorescence microscopy. To provide a basis for future analysis of polymorphisms and structure-function studies, the previously unreported distal 10.5 kilobases of the hTH promoter were sequenced with an average coverage of 20-fold, the remainder with 4-fold coverage. Sequence features identified included four perfect matches to the bicoid binding element (BBE, consensus: BBTAATCYV) all of which exhibited specific binding by electrophoretic mobility shift assay (EMSA). Comparison to published sequences of mouse and rat TH promoters revealed five areas of exceptional homology shared by these species in the upstream TH promoter region -2 kb to -9 kb relative to the transcription start site. Within these conserved regions (CRs I-V), potential recognition sites for NR4A2 (Nurr1), HNF-3beta, HOXA4, and HOXA5 were shared across human, mouse, and rat TH promoters.

  5. The Polyketide Components of Waxes and the Cer-cqu Gene Cluster Encoding a Novel Polyketide Synthase, the β-Diketone Synthase, DKS

    Directory of Open Access Journals (Sweden)

    Penny von Wettstein-Knowles

    2017-07-01

    Full Text Available The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue–grey color. Identification of the barley Cer-c, -q and -u genes forming the 101 kb Cer-cqu gene cluster encoding a novel polyketide synthase—the β-diketone synthase (DKS, a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane aliphatic that forms long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols.

  6. Genetic analysis of polyketide synthase and peptide synthase genes of ‎cyanobacteria as a mining tool for new pharmaceutical compounds

    Directory of Open Access Journals (Sweden)

    Bahareh Nowruzi

    2017-06-01

    Full Text Available Cyanobacteria are considered a promising source for new ‎pharmaceutical lead compounds and a large number of chemically diverse and ‎bioactive metabolites have been obtained from cyanobacteria. Despite of ‎several worldwide studies on prevalence of NRPSs and PKSs among the ‎cyanobacteria, none of them included Iranian cyanobacteria of Kermanshah ‎province. Therefore, the aim of this study was to amplify the NRPS and PKS ‎genes. These stains were the most frequent species of that zone. The DNA ‎sequences of the 16s rRNA region were determined and the strains were named ‎as Scytonema sp. N11, Calothrix sp. N42 and terrestrial Nostoc SP. N66. ‎Lastly, to show that the presence of these genes correlates with natural product ‎synthesis, we conducted biochemical assays to detect the presence of ‎antimicrobial effects in organic extracts. The results of these experiments ‎suggest that both NRPS and PKS genes are presence in all three strains. ‎Finally the sequences presented in this study have been deposited in GenBank ‎for getting the accession numbers (NRPS A domains and (PKS KS domains. ‎According to these results, it is concluded that the antibiogram bioassay and ‎molecular detection of peptide synthetase and polyketide synthase genes in ‎cyanobacterial strains may be useful techniques for the assessment of natural ‎product -producing species and possible role of peptide synthetase and ‎polyketide synthase enzyme complexes in the biosynthesis of biologically ‎active compounds.‎

  7. Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes.

    Science.gov (United States)

    Mlalazi, Bulukani; Welsch, Ralf; Namanya, Priver; Khanna, Harjeet; Geijskes, R Jason; Harrison, Mark D; Harding, Rob; Dale, James L; Bateson, Marion

    2012-11-01

    Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

  8. Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.

    Directory of Open Access Journals (Sweden)

    Dullat Harpreet K

    2011-03-01

    Full Text Available Abstract Background In conifers, terpene synthases (TPSs of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress. Results The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs and full-length cDNAs in several spruce (Picea species allowed us to estimate that a conifer genome contains at least 69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in any of the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruce TPSs: 12 from Sitka spruce (P. sitchensis, 5 from white spruce (P. glauca, and 4 from hybrid white spruce (P. glauca × P. engelmannii, which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpene synthases. Conclusions The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in the oleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity at the molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway spruce diterpene synthases revealed the natural occurrence of TPS sequence variants between closely related spruce species, confirming a previous prediction from site-directed mutagenesis and modelling.

  9. Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.)

    Science.gov (United States)

    2011-01-01

    Background In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress. Results The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs) and full-length cDNAs in several spruce (Picea) species allowed us to estimate that a conifer genome contains at least 69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in any of the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruce TPSs: 12 from Sitka spruce (P. sitchensis), 5 from white spruce (P. glauca), and 4 from hybrid white spruce (P. glauca × P. engelmannii), which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpene synthases. Conclusions The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in the oleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity at the molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway spruce diterpene synthases revealed the natural occurrence of TPS sequence variants between closely related spruce species, confirming a previous prediction from site-directed mutagenesis and modelling. PMID:21385377

  10. Thymidylate Synthase Gene Polymorphism and Survival of Colorectal Cancer Patients Receiving Adjuvant 5-Fluorouracil

    Science.gov (United States)

    Sulzyc-Bielicka, Violetta; Bielicki, Dariusz; Binczak-Kuleta, Agnieszka; Kaczmarczyk, Mariusz; Pioch, Wiesława; Machoy-Mokrzynska, Anna; Ciechanowicz, Andrzej; Gołębiewska, Magdalena

    2013-01-01

    Limited studies indicate a possible association of 5′-UTR thymidylate synthase enhancer region polymorphism and treatment outcome in patients medicated with 5-fluorouracil (5-FU). The study was designed to verify the relationship in patients with colorectal cancer (CRC), a Polish population that received 5-FU-based adjuvant chemotherapy. The study analyzed 145 Astler-Coller B2 and C CRC patients. Genotyping for a variable number of tandem repeats and G to C single-nucleotide polymorphism in the 5′-UTR of the thymidylate synthase (TS) gene was carried out. TS genotypes were classified into high expression (high TS) and low expression types (low TS). High TS was found in 22.8% of patients. The right-side tumors were more frequently associated with high TS than the left-side tumors (p=0.024). High TS was only found in 9.3% of rectal tumors, but in 29.7% of colon cancers (p=0.0042). Disease-free survival after 20 months (DFS 20) was longer in subjects with low TS than in high TS (p=0.043). Patients who underwent chemotherapy had longer DFS 20 in the low TS than in the high TS subgroup (p=0.051). The low TS was found to be an independent good prognostic factor for DFS 20 in the whole group as well as in the subgroup treated with chemotherapy (p=0.024 and p=0.034, respectively). Patients with low TS did not show any differences in DFS 20 whether they were treated with adjuvant chemotherapy or not. Proximal CRC tumors are characterized by higher TS expression genotypes than distal tumors, and are at significantly greater risk of early recurrence during the first 20 months after surgery. PMID:23968134

  11. Organophosphate Pesticide Exposures, Nitric Oxide Synthase Gene Variants, and Gene?Pesticide Interactions in a Case?Control Study of Parkinson?s Disease, California (USA)

    OpenAIRE

    Paul, KC; Sinsheimer, JS; Rhodes, SL; Cockburn, M; Bronstein, J; Ritz, B

    2015-01-01

    Background: Nitric oxide synthase (NOS) genes are candidates for Parkinson?s disease (PD) because NOS enzymes produce nitric oxide (NO), a pro-oxidant that can damage neurons. Widely used organophosphate (OP) pesticides can induce oxidative stress and are reported to increase PD risk. Additionally, two single nucleotide polymorphisms (SNPs) from the PON1 (paraoxonase 1) gene influence the ability to metabolize OPs. Objective: Here, we investigated contributions of NOS genes and OP pesticides ...

  12. Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

    Science.gov (United States)

    Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

    2016-09-01

    The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems ( Pcloned from a halophyte, and it might contain a diff erent metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  13. Ectopic expression of the rice lumazine synthase gene contributes to defense responses in transgenic tobacco.

    Science.gov (United States)

    Wu, Tingquan; Guo, An; Zhao, Yanying; Wang, Xiaomeng; Wang, Ying; Zhao, Dan; Li, Xiaojie; Ren, Haiying; Dong, Hansong

    2010-06-01

    Lumazine synthase (LS) catalyzes the penultimate reaction in the multistep riboflavin biosynthesis pathway, which is involved in plant defenses. Plant defenses are often subject to synergistic effects of jasmonic acid and ethylene whereas LS is a regulator of jasmonic acid signal transduction. However, little is known about whether the enzyme contributes to defense responses. To study the role of LS in plant pathogen defenses, we generated transgenic tobacco expressing the rice (Oryza sativa) LS gene, OsLS. OsLS was cloned and found to have strong identity with its homologues in higher plants and less homology to microbial orthologues. The OsLS protein localized to chloroplasts in three OsLS-expressing transgenic tobacco (LSETT) lines characterized as enhanced in growth and defense. Compared with control plants, LSETT had higher content of both riboflavin and the cofactors flavin mononucleotide and flavin adenine dinucleotide. In LSETT, jasmonic acid and ethylene were elevated, the expression of defense-related genes was induced, levels of resistance to pathogens were enhanced, and resistance was effective to viral, bacterial, and oomycete pathogens. Extents of OsLS expression correlated with increases in flavin, jasmonic acid, and ethylene content, and correlated with increases in resistance levels, suggesting a role for OsLS in defense responses.

  14. Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

    Science.gov (United States)

    Valera, Maria José; Torija, Maria Jesús; Mas, Albert; Mateo, Estibaliz

    2015-02-01

    The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

  15. An evolutionary analysis of lateral gene transfer in thymidylate synthase enzymes.

    Science.gov (United States)

    Stern, Adi; Mayrose, Itay; Penn, Osnat; Shaul, Shaul; Gophna, Uri; Pupko, Tal

    2010-03-01

    Thymidylate synthases (Thy) are key enzymes in the synthesis of deoxythymidylate, 1 of the 4 building blocks of DNA. As such, they are essential for all DNA-based forms of life and therefore implicated in the hypothesized transition from RNA genomes to DNA genomes. Two evolutionally unrelated Thy enzymes, ThyA and ThyX, are known to catalyze the same biochemical reaction. Both enzymes are sporadically distributed within each of the 3 domains of life in a pattern that suggests multiple nonhomologous lateral gene transfer (LGT) events. We present a phylogenetic analysis of the evolution of the 2 enzymes, aimed at unraveling their entangled evolutionary history and tracing their origin back to early life. A novel probabilistic evolutionary model was developed, which allowed us to compute the posterior probabilities and the posterior expectation of the number of LGT events. Simulation studies were performed to validate the model's ability to accurately detect LGT events, which have occurred throughout a large phylogeny. Applying the model to the Thy data revealed widespread nonhomologous LGT between and within all 3 domains of life. By reconstructing the ThyA and ThyX gene trees, the most likely donor of each LGT event was inferred. The role of viruses in LGT of Thy is finally discussed.

  16. Diversifying selection on flavanone 3-hydroxylase and isoflavone synthase genes in cultivated soybean and its wild progenitors.

    Directory of Open Access Journals (Sweden)

    Hao Cheng

    Full Text Available Soybean isoflavone synthase (IFS and flavanone 3-hydroxylase (F3H are two key enzymes catalyzing the biosynthesis of isoflavonoids and flavonoids, both of which play diverse roles in stress responses. However, little is known about the evolutionary pattern of these genes in cultivated soybean and its wild progenitors. Herein, we investigated the nucleotide polymorphisms in Isoflavone synthase (IFS1, IFS2 and Flavanone 3-hydroxylase (F3H2 genes from 33 soybean accessions, including 17 cultivars (Glycine max and 16 their wild progenitors (Glycine soja. Our data showed that the target genes shared the levels of nucleotide polymorphism with three reference genes involved in plant-microbe interactions, but possessed a much higher nucleotide polymorphism than other reference genes. Moreover, no significant genetic differentiation was found between cultivated soybean and its wild relatives in three target genes, despite of considering bottleneck and founder effect during domestication. These results indicate that IFS and F3H genes could have experienced gene introgressions or diversifying selection events during domestication process. Especially, F3H2 gene appears to evolve under positive selection and enjoy a faster evolutionary rate than IFS1 and IFS2 genes.

  17. Structural, Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine1[W

    Science.gov (United States)

    Parage, Claire; Tavares, Raquel; Réty, Stéphane; Baltenweck-Guyot, Raymonde; Poutaraud, Anne; Renault, Lauriane; Heintz, Dimitri; Lugan, Raphaël; Marais, Gabriel A.B.; Aubourg, Sébastien; Hugueney, Philippe

    2012-01-01

    Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed. PMID:22961129

  18. Haplotype analysis of sucrose synthase gene family in three Saccharum species

    Science.gov (United States)

    2013-01-01

    Background Sugarcane is an economically important crop contributing about 80% and 40% to the world sugar and ethanol production, respectively. The complicated genetics consequential to its complex polyploid genome, however, have impeded efforts to improve sugar yield and related important agronomic traits. Modern sugarcane cultivars are complex hybrids derived mainly from crosses among its progenitor species, S. officinarum and S. spontanuem, and to a lesser degree, S. robustom. Atypical of higher plants, sugarcane stores its photoassimilates as sucrose rather than as starch in its parenchymous stalk cells. In the sugar biosynthesis pathway, sucrose synthase (SuSy, UDP-glucose: D-fructose 2-a-D-glucosyltransferase, EC 2.4.1.13) is a key enzyme in the regulation of sucrose accumulation and partitioning by catalyzing the reversible conversion of sucrose and UDP into UDP-glucose and fructose. However, little is known about the sugarcane SuSy gene family members and hence no definitive studies have been reported regarding allelic diversity of SuSy gene families in Saccharum species. Results We identified and characterized a total of five sucrose synthase genes in the three sugarcane progenitor species through gene annotation and PCR haplotype analysis by analyzing 70 to 119 PCR fragments amplified from intron-containing target regions. We detected all but one (i.e. ScSuSy5) of ScSuSy transcripts in five tissue types of three Saccharum species. The average SNP frequency was one SNP per 108 bp, 81 bp, and 72 bp in S. officinarum, S. robustom, and S. spontanuem respectively. The average shared SNP is 15 between S. officinarum and S. robustom, 7 between S. officinarum and S. spontanuem , and 11 between S. robustom and S. spontanuem. We identified 27, 35, and 32 haplotypes from the five ScSuSy genes in S. officinarum, S. robustom, and S. spontanuem respectively. Also, 12, 11, and 9 protein sequences were translated from the haplotypes in S. officinarum, S. robustom, S

  19. Acoustic stimulation promotes the expression of inducible nitric oxide synthase in the vestibule of guinea pigs.

    Science.gov (United States)

    Watanabe, Ken-ichi; Inai, Shunta; Hess, Alexander; Michel, Olaf; Yagi, Toshiaki

    2004-08-01

    Loud acoustic stimulation is known to cause inner ear disturbance. We examined immunohistochemically the vestibule of 12 guinea pigs after acoustic stimulation. The animals were divided into two equal groups: a control group and an acoustic stimulation group. The temporal bones were fixed by means of a cardiac infusion of fixative and immunohistochemically stained for inducible nitric oxide synthase (iNOS). The temporal bones in the control group did not show any iNOS. In the acoustic stimulation group, immunoreactivity for iNOS was detected in the supporting cells and sensory cells of the sensory epithelium, in the dark cell areas and in the vestibular ganglion cells. These findings suggest that free radicals are involved in the pathogenesis of noise-induced inner ear damage. Furthermore, free radicals may cause vestibular damage, as is seen in noise-induced inner ear damage.

  20. Dihydropteroate synthase (DHPS) gene mutation study in HIV-Infected Indian patients with Pneumocystis jirovecii pneumonia.

    Science.gov (United States)

    Tyagi, Anuj Kumar; Mirdha, Bijay Ranjan; Luthra, Kalpana; Guleria, Randeep; Mohan, Anant; Singh, Urvashi Balbir; Samantaray, Jyotish Chandra; Dar, Lalit; Iyer, Venkateswaran K; Chaudhry, Rama

    2010-11-24

    Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations' (55th and 57th codon) association with prior sulfa prophylaxis failure has been reported from both developed and developing countries. We conducted a prospective study to determine the prevalence of P. jirovecii DHPS mutations from 2006 to 2009 on P. jirovecii isolates obtained from HIV-infected patients with a clinical diagnosis of Pneumocystis carinii pneumonia (PCP) admitted to our tertiary care reference health center in New Delhi, India. Detection of P. jirovecii cysts was performed by direct fluorescent antibody (DFA) staining and by Grocott's-Gomori methenamine silver staining (GMS). DNA detection was performed by polymerase chain reaction (PCR) using primers for the major surface glycoprotein (MSG) gene. P. jirovecii DHPS gene was amplified by nested PCR protocol and sequenced for detecting mutations at the 55th and 57th codons. Out of 147 HIV-positive patients with suspected Pneumocystis pneumonia (PCP), 16 (10.8%) PCP positive cases were detected. Of 16 cases, nine (56.2%) were positive by DFA staining, four (25%) were positive by Grocott's-Gomori methenamine silver staining, and all 16 were positive by MSG PCR. DHPS mutations at the 55th and 57th codons were observed in 6.2% of HIV patients studied, which was relatively low compared to reports from developed nations.   Prevalence of Pneumocystis jirovecii DHPS mutations associated with cotrimoxazole treatment failure may be low in the Indian subpopulation of HIV-positive patients and warrants larger studies to elucidate the true picture of Pneumocystis jirovecii sulfa drug resistance in India.

  1. Thymidylate synthase gene (TYMS polymorphisms in sporadic and hereditary breast cancer

    Directory of Open Access Journals (Sweden)

    Junior José da Silva Nogueira

    2012-12-01

    Full Text Available Abstract Background Breast cancer (BC is a genetic disorder characterized by growth and proliferation of breast cells in a disorderly. In Brazil, there are approximately 49.240 new cases of BC, every year. The BC etiology is still poorly understood. The BC can be sporadic (SBC or hereditary (HBC. Recent studies have correlated gene polymorphisms with the BC, such as alterations in thymidylate synthase gene (TYMS, which are used to improve diagnosis and prevention of the disease. Polymorphisms in the TYMS gene 5’-UTR region, usually present reps double (2R and/or triple (3R. Studies have shown that homozygous 3R/3R is overexpressed compared with 2R/2R genotype, and these polymorphic variations may contribute to individual susceptibility to the development of BC. In this context, the objective of this study was to evaluate the frequency of the TYMS 2R and 3R polymorphisms, comparing genotypic and allelic distribution with SBC and HBC patients. Methods In this study we included a total of 204 subjects, 70 with BC (33 with SBC, and 37 with HBC and 134 healthy subjects (controls. The Polymerase Chain Reaction was the method used. Results Results demonstrated a high frequency of the 3R allele at BC, SBC, and HBC groups. The frequency of genotype 2R/3R was significantly higher in BC group. This work showed association between the 2R/3R variants (OR = 4.14, CI95% = 1.77-9.71 in the development of SBC, and 2R/2R (OR = 0.233, CI95% = 1.63-7.65 and 2R/3R (OR = 3.53, CI95% = 0.06-0.81 for developing HBC. To BC, there was association with the genotype 2R/3R (OR: 3.79, CI95% = 2.03-7.08. Conclusion Our results show relation to the development of BC in association with the analyzed polymorphisms.

  2. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  3. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

    Science.gov (United States)

    Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

    2013-12-05

    The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

  4. Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

    Science.gov (United States)

    Singh, Anup Kumar; Dwivedi, Varun; Rai, Avanish; Pal, Shaifali; Reddy, Sajjalavarahalli Gangireddy Eswara; Rao, Dodaghatta Krishnarao Venkata; Shasany, Ajit Kumar; Nagegowda, Dinesh A

    2015-12-01

    Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides

  5. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    Directory of Open Access Journals (Sweden)

    Smrati Mishra

    Full Text Available Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  6. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    Science.gov (United States)

    Mishra, Smrati; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  7. Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon.

    Science.gov (United States)

    Toyomasu, Tomonobu; Miyamoto, Koji; Shenton, Matthew R; Sakai, Arisa; Sugawara, Chizu; Horie, Kiyotaka; Kawaide, Hiroshi; Hasegawa, Morifumi; Chuba, Masaru; Mitsuhashi, Wataru; Yamane, Hisakazu; Kurata, Nori; Okada, Kazunori

    2016-11-18

    Cultivated rice (Oryza sativa) possesses various labdane-related diterpene synthase genes, homologs of ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    Science.gov (United States)

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  9. Epidemiology and clinical relevanceof Pneumocystis jirovecii Frenkel, 1976 dihydropteroate synthase gene mutations*

    Directory of Open Access Journals (Sweden)

    Matos O.

    2010-09-01

    Full Text Available A review was conducted to examine the published works that studied the prevalence of Pneumocystis jirovecii dihydropteroate synthase (DHPS mutations in patients with P. jirovecii pneumonia (PcP, in develop and developing countries, and that focused the problem of the possible association of these mutations with exposure to sulpha or sulphone drugs and their influence in the PcP outcome. Studies conducted in United States of America presented higher P. jirovecii mutations rates, in comparison with European countries, and in developing countries, lower rates of DHPS mutations were reported, due to limited use of sulpha drugs. A significant association was reported between the use of sulpha or sulphone agents for PcP prophylaxis in HIV-infected patients and the presence of DHPS mutations. However these mutations were also detected in PcP patients who were not currently receiving sulpha or sulphone agents. The outcome and mortality of HIV-infected patients with PcP harbouring DHPS gene mutations were related primarily to the underlying severity of illness and the initial severity of PcP, more than to the presence of mutations.

  10. Carotenoid content and expression of phytoene synthase and phytoene desaturase genes in bitter melon (Momordica charantia).

    Science.gov (United States)

    Tuan, Pham Anh; Kim, Jae Kwang; Park, Nam Il; Lee, Sook Young; Park, Sang Un

    2011-06-15

    Momordica charantia, a tropical plant, produces a fruit that has a β-carotene concentration five times higher than that of carrot. To elucidate the molecular basis of β-carotene accumulation in M. charantia, the gene expression levels of phytoene synthase (McPSY) and phytoene desaturase (McPDS) were determined. These levels were particularly high in the flowers of M. charantia. During fruit maturation, the expression levels of McPSY and McPDS decreased during the mid-stages but increased in the fully mature fruit. In addition, carotenoids accumulated as the peel changed from green to orange. Thus, McPSY and McPDS expression correlated with carotenoid accumulation during fruit maturation. Principal component analysis (PCA) also was used to evaluate the differences among the profiles of seven carotenoids identified in the fruit at several maturation stages. Riper fruits had higher carotenoid concentrations than less ripe fruits. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  11. Endothelial nitric oxide synthase: From biochemistry and gene structure to clinical implications of NOS3 polymorphisms.

    Science.gov (United States)

    Oliveira-Paula, Gustavo H; Lacchini, Riccardo; Tanus-Santos, Jose E

    2016-01-10

    Nitric oxide (NO) is an important vasodilator with a well-established role in cardiovascular homeostasis. While mediator is synthesized from L-arginine by neuronal, endothelial, and inducible nitric oxide synthases (NOS1,NOS3 and NOS2 respectively), NOS3 is the most important isoform for NO formation in the cardiovascular system. NOS3 is a dimeric enzyme whose expression and activity are regulated at transcriptional, posttranscriptional,and posttranslational levels. The NOS3 gene, which encodes NOS3, exhibits a number of polymorphic sites including single nucleotide polymorphisms (SNPs), variable number of tandem repeats (VNTRs), microsatellites, and insertions/deletions. Some NOS3 polymorphisms show functional effects on NOS3 expression or activity, thereby affecting NO formation. Interestingly, many studies have evaluated the effects of functional NOS3 polymorphisms on disease susceptibility and drug responses. Moreover, some studies have investigated how NOS3 haplotypes may impact endogenous NO formation and disease susceptibility. In this article,we carried out a comprehensive review to provide a basic understanding of biochemical mechanisms involved in NOS3 regulation and how genetic variations in NOS3 may translate into relevant clinical and pharmacogenetic implications.

  12. Cloning and expression analysis of chalcone synthase gene from Coleus forskohlii.

    Science.gov (United States)

    Awasthi, Praveen; Mahajan, Vidushi; Jamwal, Vijay Lakshmi; Kapoor, Nitika; Rasool, Shafaq; Bedi, Yashbir S; Gandhi, Sumit G

    2016-09-01

    Flavonoids are an important class of secondary metabolites that play various roles in plants such as mediating defense, floral pigmentation and plant-microbe interaction. Flavonoids are also known to possess antioxidant and antimicrobial activities. Coleus forskohlii (Willd.) Briq. (Lamiaceae) is an important medicinal herb with a diverse metabolic profile, including production of a flavonoid, genkwanin. However, components of the flavonoid pathway have not yet been studied in this plant. Chalcone synthase (CHS) catalyses the first committed step of flavonoid biosynthetic pathway. Full-length cDNA, showing homology with plant CHS gene was isolated from leaves of C. forskohlii and named CfCHS (GenBank accession no. KF643243). Theoretical translation of CfCHS nucleotide sequence shows that it encodes a protein of 391 amino acids with a molecular weight of 42.75 kDa and pI 6.57. Expression analysis of CfCHS in different tissues and elicitor treatments showed that methyl jasmonate (MeJA) strongly induced its expression. Total flavonoids content and antioxidant activity of C. forskohlii also got enhanced in response to MeJA, which correlated with increased CfCHS expression. Induction of CfCHS by MeJA suggest its involvement in production of flavonoids, providing protection from microbes during herbivory or mechanical wounding. Further, our in silico predictions and experimental data suggested that CfCHS may be posttranscriptionally regulated by miR34.

  13. Identification and characterization of a novel trehalose synthase gene derived from saline-alkali soil metagenomes.

    Directory of Open Access Journals (Sweden)

    Ling Jiang

    Full Text Available A novel trehalose synthase (TreS gene was identified from a metagenomic library of saline-alkali soil by a simple activity-based screening system. Sequence analysis revealed that TreS encodes a protein of 552 amino acids, with a deduced molecular weight of 63.3 kDa. After being overexpressed in Escherichia coli and purified, the enzymatic properties of TreS were investigated. The recombinant TreS displayed its optimal activity at pH 9.0 and 45 °C, and the addition of most common metal ions (1 or 30 mM had no inhibition effect on the enzymatic activity evidently, except for the divalent metal ions Zn(2+ and Hg(2+. Kinetic analysis showed that the recombinant TreS had a 4.1-fold higher catalytic efficiency (Kcat/K m for maltose than for trehalose. The maximum conversion rate of maltose into trehalose by the TreS was reached more than 78% at a relatively high maltose concentration (30%, making it a good candidate in the large-scale production of trehalsoe after further study. In addition, five amino acid residues, His172, Asp201, Glu251, His318 and Asp319, were shown to be conserved in the TreS, which were also important for glycosyl hydrolase family 13 enzyme catalysis.

  14. Metagenomic Survey of Potential Symbiotic Bacteria and Polyketide Synthase Genes in an Indonesian Marine Sponge

    Directory of Open Access Journals (Sweden)

    Nia M. Kurnia

    2017-01-01

    Full Text Available There has been emerging evidence that the bacteria associated with marine sponges are the key producers of many complex bioactive compounds. The as-yet uncultured candidate bacterial genus “Candidatus Entotheonella” of the marine sponge Theonella swinhoei from Japan have recently been recognized as the source of numerous pharmacologically relevant polyketides and modified peptides, as previously reported by the Piel group (Wilson et al. 2014. This work reported the presence of “Candidatus Entotheonella sp.” in the highly complex microbiome of an Indonesian marine sponge from Kapoposang Island, South Sulawesi. We further identified the Kapoposang sponge specimen used in this work as Rhabdastrella sp. based on the integrated morphological, histological, and cytochrome oxidase subunit I (COI gene analyses. To detect the polyketide biosynthetic machinery called type I polyketide synthase (PKS in this Indonesian Rhabdastrella sp., we amplified and cloned the ketosynthase-encoding DNA regions of approximately 700 bp from the uncultured sponge's microbiome. Further sequencing and analysis of several randomly chosen clones indicated that all of them are mostly likely involved in the biosynthesis of methyl-branched fatty acids. However, employing a PKS-targeting primer designed in this work led to the isolation of four positive clones. BlastX search and subsequent phylogenetic analysis showed that one of the positive clones, designed as RGK32, displayed high homology with ketosynthase domains of many type I PKS systems and may belong to the subclass cis-AT PKS group.

  15. Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2016-02-01

    Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

  16. Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments

    OpenAIRE

    Lanfranconi, Mariana P.; Alvarez, Adri?n F; Alvarez, H?ctor M.

    2015-01-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained...

  17. Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P. carinii pneumonia

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Benfield, Thomas; Eugen-Olsen, J

    1999-01-01

    Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential for folate...... biosynthesis. We assessed whether mutations in the DHPS gene of P. carinii were associated with exposure to sulpha drugs and influenced outcome from PCP....

  18. A Malus crabapple chalcone synthase gene, McCHS, regulates red petal color and flavonoid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Deqiang Tai

    Full Text Available Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, 'Royalty' and 'Flame', have dark red and white petals respectively, while the intermediate cultivar 'Radiant' has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

  19. Identification and characterization of genetic variation in the folylpolyglutamate synthase gene.

    Science.gov (United States)

    Leil, Tarek A; Endo, Chiaki; Adjei, Araba A; Dy, Grace K; Salavaggione, Oreste E; Reid, Joel R; Ames, Matthew M; Adjei, Alex A

    2007-09-15

    Folylpolyglutamate synthase (FPGS) catalyzes the polyglutamation of folic acid, methotrexate, and pemetrexed to produce highly active metabolites. To characterize genetic variation in the FPGS gene, FPGS, have resequenced the gene in four different ethnic populations. Thirty-four single nucleotide polymorphisms were identified including five nonsynonymous coding single nucleotide polymorphisms that altered the FPGS protein sequence: F13L and V22I polymorphisms in the mitochondrial isoform of FPGS, and R466/424C, A489/447V, and S499/457F polymorphisms, which exist in both the mitochondrial and cytosolic isoforms. When expressed in AuxB1 cells, the A447V cytosolic variant was functionally similar to the wild-type cytosolic (WT Cyt) allozyme, whereas the R424C and S457F cytosolic variants were reduced by approximately 2-fold in protein expression compared with WT Cyt (P glutamate was reduced by 12.3-fold (R424C, P < 0.01) and 6.2-fold (S457F, P < 0.01), whereas the intrinsic clearance of methotrexate was reduced by 4.2-fold (R424C, P < 0.05) and 5.4-fold (S457F, P < 0.05) in these two cytosolic variants when compared with the WT Cyt isoform. Additionally, the in vitro enzyme velocity at saturating pemetrexed concentrations was reduced by 1.6-fold (R424C, P < 0.05) and 2.6-fold (S457F, P < 0.01) compared with WT Cyt. AuxB1 cells harboring these same cytosolic variant allozymes displayed significant increases in the EC(50) for folic acid and in the IC(50) values for both methotrexate and pemetrexed relative to the WT Cyt form of FPGS. These observations suggest that genetic variations in FPGS may alter the efficacy of antifolate therapy in cancer patients.

  20. Late-onset cutaneous porphyria in a patient heterozygous for a uroporphyrinogen III synthase gene mutation.

    Science.gov (United States)

    Aguilera, P; Badenas, C; Whatley, S D; To-Figueras, J

    2016-12-01

    Deficiency of uroporphyrinogen III synthase (UROS) causes congenital erythropoietic porphyria (CEP). The disease, originating from the inheritance of mutations within the UROS gene, presents a recessive form of transmission. In a few patients, a late-onset CEP-like phenotype without UROS mutations appears to be associated with a myelodysplastic syndrome. We report a 60-year-old man with late-onset signs of cutaneous porphyria and accumulation in urine, plasma and faeces of type I porphyrin isomers characteristic of CEP. Analysis of DNA from peripheral leucocytes, skin and bone marrow aspirate showed that he was a heterozygous carrier of a Cys73Arg (c.217 T>C) mutation within UROS. Sequencing of cDNA from peripheral blood confirmed heterozygosity and expression of the normal allele. Measurement of UROS enzymatic activity in erythrocytes showed values ~70% of normal, indirectly indicating expression of the normal allele. Differently from other cases of late-onset uroporphyria, the patient did not present thrombocytopenia or any evidence of a myelodysplastic syndrome. Five years of clinical follow-up showed persistence of skin signs and increased excretion of porphyrins, independently of lifestyle factors or changes in medication regimes. We hypothesize acquired mosaicism (in the bone marrow) affecting the UROS gene. Thus, unstable cellular clones initiated overproduction of isomer I porphyrins leading to a CEP phenotype. This could be explained either by a clonal expansion of the porphyric (Cys73Arg) allele or by loss of function of the normal allele. Cellular turnover would facilitate release of uroporphyrins into circulation and subsequent skin lesions. This is the first case of a CEP heterozygous carrier presenting clinical manifestations. © 2016 British Association of Dermatologists.

  1. Endothelial Nitric Oxide Synthase Gene Variation Associated With Chronic Kidney Disease After Liver Transplant

    Science.gov (United States)

    Bambha, Kiran; Kim, W. Ray; Rosen, Charles B.; Pedersen, Rachel A.; Rys, Cynthia; Kolbert, Christopher P.; Cunningham, Julie M.; Therneau, Terry M.

    2010-01-01

    OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with risk of developing chronic kidney disease (CKD), a prevalent comorbidity, after liver transplant (LT). PATIENTS AND METHODS: This study consists of a cohort of adult (≥18 years) primary-LT recipients who had normal renal function before LT and who survived 1 year or more after LT at a high-volume US LT program between January 1, 1990, and December 31, 2000. Patients with adequate renal function (estimated glomerular filtration rate, ≥40 mL/min per 1.73 m2 during follow-up; n=308) and patients with incident CKD (estimated glomerular filtration rate, <40 mL/min per 1.73 m2 after LT; n=92) were identified. To investigate the association of 6 candidate genes with post-LT CKD, we selected SNPs that have been associated with renal function in the literature. Hazard ratios were estimated using Cox regression, adjusted for potential confounding variables. RESULTS: The variant allele (298Asp) of the Glu298Asp SNP in the endothelial nitric oxide synthase gene (NOS3) was significantly associated with CKD after LT (P=.05; adjusted for multiple comparisons). The 5-year incidence of CKD was 70% among patients homozygous for the NOS3 variant allele (298Asp) compared with 42% among those not homozygous for the NOS3 variant allele. Specifically, homozygosity for the NOS3 variant allele conferred a 2.5-fold increased risk of developing CKD after LT (P=.005, adjusted for confounding variables). CONCLUSION: Homozygosity for the variant allele of NOS3 (298Asp) is associated with CKD after LT and may be useful for identifying recipients at higher risk of post-LT CKD. PMID:20810793

  2. Expression of an (E-β-farnesene synthase gene from Asian peppermint in tobacco affected aphid infestation

    Directory of Open Access Journals (Sweden)

    Xiudao Yu

    2013-10-01

    Full Text Available Aphids are major agricultural pests that cause significant yield losses in crop plants each year. (E-β-farnesene (EβF is the main or only component of an alarm pheromone involved in chemical communication within aphid species and particularly in the avoidance of predation. EβF also occurs in the essential oil of some plant species, and is catalyzed by EβF synthase. By using oligonucleotide primers designed from the known sequence of an EβF synthase gene from black peppermint (Mentha × piperita, two cDNA sequences, MaβFS1 and MaβFS2, were isolated from Asian peppermint (Mentha asiatica. Expression pattern analysis showed that the MaβFS1 gene exhibited higher expression in flowers than in roots, stems and leaves at the transcriptional level. Overexpression of MaβFS1 in tobacco plants resulted in emission of pure EβF ranging from 2.62 to 4.85 ng d− 1 g− 1 of fresh tissue. Tritrophic interactions involving peach aphids (Myzus persicae, and predatory lacewing (Chrysopa septempunctata larvae demonstrated that transgenic tobacco expressing MaβFS1 had lower aphid infestation. This result suggested that the EβF synthase gene from Asian peppermint could be a good candidate for genetic engineering of agriculturally important crop plants.

  3. Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

    Directory of Open Access Journals (Sweden)

    Catalina Sanz

    Full Text Available Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

  4. Functional analysis of Gossypium hirsutum cellulose synthase catalytic subunit 4 promoter in transgenic Arabidopsis and cotton tissues.

    Science.gov (United States)

    Kim, Hee Jin; Murai, Norimoto; Fang, David D; Triplett, Barbara A

    2011-02-01

    Gossypium hirsutum cellulose synthase catalytic subunit 4 (GhCesA4) plays an important role in cellulose biosynthesis during cotton fiber development. The transcript levels of GhCesA4 are significantly up-regulated as secondary cell wall cellulose is produced in developing cotton fibers. To understand the molecular mechanisms involved in transcriptional regulation of GhCesA4, β-glucuronidase (GUS) activity regulated by a GhCesA4 promoter (-2574/+56) or progressively deleted promoters were determined in both cotton tissues and transgenic Arabidopsis. The spatial regulation of GhCesA4 expression was similar between cotton tissues and transgenic Arabidopsis. GUS activity regulated by the GhCesA4 promoter (-2574/+56) was found in trichomes and root vascular tissues in both cotton and transgenic Arabidopsis. The -2574/-1824 region was responsible for up-regulation of GhCesA4 expression in trichomes and root vascular tissues in transgenic Arabidopsis. The -1824/-1355 region negatively regulated GhCesA4 expression in most Arabidopsis vascular tissues. For vascular expression in stems and leaves, the -898/-693 region was required. The -693/-320 region of the GhCesA4 promoter was necessary for basal expression of GhCesA4 in cotton roots as well as Arabidopsis roots. Exogenous phytohormonal treatments on transgenic Arabidopsis revealed that phytohormones may be involved in the differential regulation of GhCesA4 during cotton fiber development. Published by Elsevier Ireland Ltd.

  5. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively). Copyright © 2011 Elsevier GmbH. All rights reserved.

  6. Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633.

    Science.gov (United States)

    Chen, Ming; Yi, Bing; Zhu, Ni; Wei, Xin; Zhang, Guan-Xin; Huang, Shengdong; Sun, Jianxin

    2016-01-01

    Posttranslational modification, such as phosphorylation, plays an essential role in regulating activation of endothelial NO synthase (eNOS). In the present study, we aim to determine whether eNOS could be phosphorylated and regulated by a novel serine/threonine-protein kinase Pim1 in vascular endothelial cells (ECs). Using immunoprecipitation and protein kinase assays, we demonstrated that Pim1 specifically interacts with eNOS, which leads to a marked phosphorylation of eNOS at Ser-633 and increased production of nitric oxide (NO). Intriguingly, in response to VEGF stimulation, eNOS phosphorylation at Ser-633 exhibits two distinct phases: transient phosphorylation occurring between 0 and 60 min and sustained phosphorylation occurring between 2 and 24 h, which are mediated by the protein kinase A (PKA) and Pim1, respectively. Inhibiting Pim1 by either pharmacological inhibitor SMI-4a or the dominant-negative form of Pim1 markedly attenuates VEGF-induced tube formation, while Pim1 overexpression significantly increases EC tube formation and migration in an NO-dependent manner. Importantly, Pim1 expression and eNOS phosphorylation at Ser-633 were substantially decreased in high glucose-treated ECs and in the aorta of db/db diabetic mice. Increased Pim1 expression ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay. Our findings demonstrate Pim1 as a novel kinase that is responsible for the phosphorylation of eNOS at Ser-633 and enhances EC sprouting of aortic rings from diabetic mice, suggesting that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  7. Nitric Oxide Synthase Promotes Distension-Induced Tracheal Venular Leukocyte Adherence

    Science.gov (United States)

    Moldobaeva, Aigul; Rentsendorj, Otgonchimeg; Jenkins, John; Wagner, Elizabeth M.

    2014-01-01

    The process of leukocyte recruitment to the airways in real time has not been extensively studied, yet airway inflammation persists as a major contributor to lung pathology. We showed previously in vivo, that neutrophils are recruited acutely to the large airways after periods of airway distension imposed by the application of positive end-expiratory pressure (PEEP). Given extensive literature implicating products of nitric oxide synthase (NOS) in lung injury after ventilatory over-distension, we questioned whether similar mechanisms exist in airway post-capillary venules. Yet, endothelial nitric oxide has been shown to be largely anti-inflammatory in other systemic venules. Using intravital microscopy to visualize post-capillary tracheal venules in anesthetized, ventilated mice, the number of adherent leukocytes was significantly decreased in eNOS-/- mice under baseline conditions (2±1 cell/60 min observation) vs wild type (WT) C57BL/6 mice (7±2 cells). After exposure to PEEP (8 cmH2O for 1 min; 5 times), adherent cells increased significantly (29±5 cells) in WT mice while eNOS-/- mice demonstrated a significantly decreased number of adherent cells (11±4 cells) after PEEP. A similar response was seen when thrombin was used as the pro-inflammatory stimulus. In addition, mouse tracheal venular endothelial cells studied in vitro after exposure to cyclic stretch (18% elongation) or thrombin both demonstrated increased p-selectin expression that was significantly attenuated by NG-nitro-L-arginine methyl ester, N-acetylcysteine amide (NACA) and excess BH4. In vivo treatment with the ROS inhibitor NACA or co-factor BH4 abolished completely the PEEP-induced leukocyte adherence. These results suggest that pro-inflammatory stimuli cause leukocyte recruitment to tracheal endothelium in part due to eNOS uncoupling. PMID:25181540

  8. Sequence of the bchG gene from Chloroflexus aurantiacus: relationship between chlorophyll synthase and other polyprenyltransferases

    Science.gov (United States)

    Lopez, J. C.; Ryan, S.; Blankenship, R. E.

    1996-01-01

    The sequence of the Chloroflexus aurantiacus open reading frame thought to be the C. aurantiacus homolog of the Rhodobacter capsulatus bchG gene is reported. The BchG gene product catalyzes esterification of bacteriochlorophyllide a by geranylgeraniol-PPi during bacteriochlorophyll a biosynthesis. Homologs from Arabidopsis thaliana, Synechocystis sp. strain PCC6803, and C. aurantiacus were identified in database searches. Profile analysis identified three related polyprenyltransferase enzymes which attach an aliphatic alcohol PPi to an aromatic substrate. This suggests a broader relationship between chlorophyll synthases and other polyprenyltransferases.

  9. Vascular gene transfer of the human inducible nitric oxide synthase: characterization of activity and effects on myointimal hyperplasia.

    OpenAIRE

    E.; Tzeng; Shears, L. L.; Robbins, P. D.; Pitt, B.R.; Geller, D. A.; Watkins, S C; Simmons, R.L.; Billiar, T R

    1996-01-01

    BACKGROUND: Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible No synthase (iNOS) gene transfer even at low efficiency will provide adequate local no production to achieve this goal. MATERIALS AND METHODS: A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle ce...

  10. Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

    Directory of Open Access Journals (Sweden)

    Emilia Wilmowicz

    2016-03-01

    Full Text Available Allene oxide synthase (AOS encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

  11. Analysis of THCA synthase gene expression in cannabis: a preliminary study by real-time quantitative PCR.

    Science.gov (United States)

    Cascini, Fidelia; Passerotti, Stella; Boschi, Ilaria

    2013-09-10

    In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression. Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability. As results we found minor gene expression in samples from leaves of young plants. Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Correlation between RAGE gene promoter methylation and diabetic retinal inflammation.

    Science.gov (United States)

    Kan, Shifeng; Wu, Jing; Sun, Chengxi; Hao, Jing; Wu, Zhen

    2018-01-01

    The methylation status of the receptor for advanced glycation end products (RAGE) gene promoter in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic retinopathy (DR) patients was evaluated to investigate the correlation between RAGE gene promoter methylation and diabetic retinal inflammation. Eighty patients admitted and diagnosed as type 2 DR in Qilu Hospital, Shandong University during the period from October, 2013 to October, 2015 were enrolled in this study. They were the observation group and 40 healthy subjects were enrolled in the control group. PBMCs were collected from patients using density gradient centrifugation, and the methylation status of RAGE gene promoters was detected using methylation-specific PCP (MSP). Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) levels of in the serum were measured using enzyme-linked immunosorbent assay (ELISA). PBMCs in patients with positive RAGE gene promoter methylation were isolated and cultured and RAGE gene promoter methylation was inhibited using the demethylating agent, 5'-aza-2'-deoxycytidine (5-aza-dC). The methylation status of RAGE gene promoters in PBMCs was detected via MSP. IL-1β, IL-6 and TNF-α levels in the supernatant of PBMC culture solution were evaluated using ELISA. MSP results showed that there were 26 cases (32.50%) of RAGE gene promoter methylation in PBMCs in DR patients. RAGE gene promoters were methylated in all normal healthy subjects. IL-1β, IL-6 and TNF-α levels in serum for positive RAGE gene promoter methylation group were significantly lower than those in negative RAGE gene promoter methylation group (pRAGE gene promoter methylation of PBMCs in patients with positive RAGE gene promoter methylation. The inhibition of methylation in RAGE gene promoter increased the levels of IL-1β, IL-6 and TNF-α in supernatant of culture solution. In conclusion, RAGE gene promoter hypomethylation was detected in DR patients, indicating that RAGE gene promoter

  13. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Pneumocystis jiroveci dihydropteroate synthase gene mutations among colonized individuals and Pneumocystis pneumonia patients from Spain.

    Science.gov (United States)

    Friaza, Vicente; Morilla, Rubén; Respaldiza, Nieves; de la Horra, Carmen; Calderón, Enrique J

    2010-11-01

    Cotrimoxazole, an association of trimethoprim and sulfamethoxazole, and dapsone, are mainstays for the prophylaxis and treatment of Pneumocystis pneumonia (PcP). The inability to culture Pneumocystis prevents routine susceptibility testing and detection of drug resistance. Instead, molecular techniques have been used to detect Pneumocystis jiroveci dihydropteroate synthase (DHPS) mutations that cause sulfa resistance in other microorganisms. The most frequent DHPS mutations occur at nucleotide positions 165 and 171, which lead to an amino acid change at positions 55 and 57. Several studies suggest that these mutations are associated with the failure of chemoprophylaxis for PcP. The aim was to establish the frequency and characteristics of P jiroveci DHPS mutations among colonized individuals and PcP patients from Spain. A total of 50 colonized individuals and 25 PcP patients were studied. DHPS polymorphisms were identified by restriction fragment length polymorphism assay. The analysis provided a rate of 28% of DHPS gene mutations in our population, with the presence of all possible polymorphisms described. The presence of mutations was higher in PcP patients than in colonized subjects (40% vs 22%), probably because of the chemoprophylaxis used in PcP patients. The comparison between patients with and without DHPS mutations did not show statistical differences due to age, sex, steroid use, sulfa drug exposure, or smoking. A high rate of DHPS mutations in our area of Spain, not only confined to patients previously exposed to sulfa drugs, is shown in this study. As well as PcP patients, colonized individuals who harbor P jiroveci strains with DHPS mutations could play a major role in the transmission cycle of these mutations, representing a reservoir and source of infection for susceptible individuals. Further research is thus warranted to assess the true scope of the problem and to design rational preventive strategies.

  15. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    Science.gov (United States)

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  16. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...

  17. Isolating Barley ( Hordeum vulgare L.) B1 Hordein Gene Promoter ...

    African Journals Online (AJOL)

    Gene expression is a complex multi-step process. For the efficient expression of foreign genes in plants, it is essential to optimize every step of the process for the plant machinery, which includes choosing suitable promoters. Promoters play the most important role in determining the temporal and spatial expression pattern ...

  18. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    Science.gov (United States)

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

    2008-01-01

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  19. Precise regulation of gene expression dynamics favors complex promoter architectures.

    Directory of Open Access Journals (Sweden)

    Dirk Müller

    2009-01-01

    Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

  20. Identifying promoters for gene expression in Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Daniel G. Olson

    2015-12-01

    Full Text Available A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase (lacZ and NADPH-alcohol dehydrogenase (adhB in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ, and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB.

  1. A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome*

    Science.gov (United States)

    Suchanowska, Anna; Kaczmarek, Radoslaw; Duk, Maria; Lukasiewicz, Jolanta; Smolarek, Dorota; Majorczyk, Edyta; Jaskiewicz, Ewa; Laskowska, Anna; Wasniowska, Kazimiera; Grodecka, Magdalena; Lisowska, Elwira; Czerwinski, Marcin

    2012-01-01

    Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NORint, and NOR2, in which Gal(α1–4), GalNAc(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)Gal(α1–4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1–4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1–4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1–4)Gal and Gal(α1–4)GalNAc moieties. PMID:22965229

  2. A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome.

    Science.gov (United States)

    Suchanowska, Anna; Kaczmarek, Radoslaw; Duk, Maria; Lukasiewicz, Jolanta; Smolarek, Dorota; Majorczyk, Edyta; Jaskiewicz, Ewa; Laskowska, Anna; Wasniowska, Kazimiera; Grodecka, Magdalena; Lisowska, Elwira; Czerwinski, Marcin

    2012-11-02

    Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(β1-3)Gal(α1-4), and Gal(α1-4)GalNAc(β1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

  3. [Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii].

    Science.gov (United States)

    Cortés, Liliana Jazmín; Duque, Sofía; López, Miryam Consuelo; Moncada, Diego; Molina, Diego; Gómez-Marín, Jorge Enrique; Gunturiz, María Luz

    2014-01-01

    There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

  4. Thymidylate synthase (TS tandem repeat promoter polymorphism and susceptibility to colorectal cancer of romanian subjects

    Directory of Open Access Journals (Sweden)

    Mihai TOMA

    2010-05-01

    Full Text Available The risk of colorectal cancer (CRC is influence by polymorphisms located in the genes encoding enzymes of the folate pathway. The aim of this study was to evaluate if 2R/3R TS (rs34743033 polymorphism is involved in predisposition for colorectal in Romanian subjects. In the present case-control study, 75 sporadic CRC subjects and 60 healthy controls were genotyped by PCR method. The frequency of 3R/3R genotype was 40% in control group and 42.7% in cancer group. We found that there was no statistically significant association between the risk for CRC and 2R/3R TS polymorphism in Romanian subjects.

  5. Functional analysis of the α-1,3-glucan synthase genes agsA and agsB in Aspergillus nidulans: agsB is the major α-1,3-glucan synthase in this fungus.

    Directory of Open Access Journals (Sweden)

    Akira Yoshimi

    Full Text Available Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and (13C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.

  6. Understanding the molecular mechanism of transcriptional regulation of banana Sucrose phosphate synthase (SPS) gene during fruit ripening: an insight into the functions of various cis-acting regulatory elements.

    Science.gov (United States)

    Choudhury, Swarup Roy; Roy, Sujit; Singh, Sanjay Kumar; Sengupta, Dibyendu N

    2010-05-01

    Recently, we have reported the characterization of promoter region of Sucrose phosphate synthase (SPS) gene in banana and investigated the role of some cis-elements/motifs, present in the promoter of SPS, in the transcriptional regulation of the gene. DNA-protein interaction studies have demonstrated the presence of specific trans-acting factors which showed specific interactions with ethylene, auxin, low temperature and light responsive elements in regulating SPS transcription. Transient expression analyses have demonstrated the functional significance of the various cis-acting regulatory elements present in banana SPS promoter in regulating SPS expression during ripening. (1) Here, we have further discussed the possible role of these regulatory sequences in the regulation of transcriptional network and comment on their function in relation to sucrose metabolism during banana fruit ripening.

  7. Cloning of the sdsA gene encoding solanesyl diphosphate synthase from Rhodobacter capsulatus and its functional expression in Escherichia coli and Saccharomyces cerevisiae.

    Science.gov (United States)

    Okada, K; Kamiya, Y; Zhu, X; Suzuki, K; Tanaka, K; Nakagawa, T; Matsuda, H; Kawamukai, M

    1997-10-01

    Different organisms produce different species of isoprenoid quinones, each with its own distinctive length. These differences in length are commonly exploited in microbial classification. The side chain length of quinone is determined by the nature of the polyprenyl diphosphate synthase that catalyzes the reaction. To determine if the side chain length of ubiquinone (UQ) has any distinct role to play in the metabolism of the cells in which it is found, we cloned the solanesyl diphosphate synthase gene (sdsA) from Rhodobacter capsulatus SB1003 and expressed it in Escherichia coli and Saccharomyces cerevisiae. Sequence analysis revealed that the sdsA gene encodes a 325-amino-acid protein which has similarity (27 to 40%) with other prenyl diphosphate synthases. Expression of the sdsA gene complemented a defect in the octaprenyl diphosphate synthase gene of E. coli and the nonrespiratory phenotype resulting from a defect in the hexaprenyl diphosphate synthase gene of S. cerevisiae. Both E. coli and S. cerevisiae expressing the sdsA gene mainly produced solanesyl diphosphate, which resulted in the synthesis of UQ-9 without any noticeable effect on the growth of the cells. Thus, it appears that UQ-9 can replace the function of UQ-8 in E. coli and UQ-6 in S. cerevisiae. Taken together with previous results, the results described here imply that the side chain length of UQ is not a critical factor for the survival of microorganisms.

  8. Cloning and Functional Characterization of a Gene for Capsanthin-Capsorubin Synthase from Tiger Lily (Lilium lancifolium Thunb. ‘Splendens’)

    OpenAIRE

    Jeknić, Zoran; Morré, Jeffrey T.; Jeknić, Stevan; Jevremović, Slađana; Subotić, Angelina; Chen, Tony H.H.

    2012-01-01

    The orange color of tiger lily (Lolium lancifolium ‘Splendens’) flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-de...

  9. Gene polymorphism of endothelial NO-synthase, angiotensin-converting enzyme and receptors of angiotensin II type 1 in patients with coronary artery disease and type II diabetes

    Directory of Open Access Journals (Sweden)

    Yakovleva L.M.

    2013-12-01

    Full Text Available Purpose: to study the clinical manifestations of coronary artery disease (CAD in patients with diabetes mellitus (DM II type and the possible association of mutations in the genes of endothelial NO-synthase (eNOS, angiotensin-converting enzyme (ACE and receptor of angiotensin II type 1 (AT2R1 with the development of type II diabetes in these patients. Mate¬rials and methods. The study involved 318 patients with CAD, of whom 54 (17,0% patients had type II DM and 264 (83,0% patients -without diabetes. Study of allelic polymorphism of T-786S of eNOS gene promoter, insertion-deletion (I/D polymorphism of ACE gene and polymorphism A1166S of AT2R1 gene was performed by polymerase chain reaction. Results. In patients with CAD and DM type II such risk factors as hypertension, obesity, family history with early onset of cardiovascular disease were significantly more common; CAD manifestation occurs at a younger age, myocardial ischemia more often has silent nature. In the presence of type II DM, coronary artery lesions were of diffuse and multivessel character, other vascular pools are often involved in the atherosclerotic process. An independent as¬sociation of type II diabetes with hypertension, obesity, and CC genotype of eNOS gene T-786S polymorphism was identified in patients with CAD. Negative independent influence of ACE gene I/D polymorphism of D allele and AT2R1 gene A1166S polymorphism C allele on renal function in patients with CAD and type II DM was proved.

  10. Cloning and characterization of a flavonol synthase gene from Scutellaria baicalensis

    National Research Council Canada - National Science Library

    Kim, Yeon Bok; Kim, KwangSoo; Kim, Yeji; Tuan, Pham Anh; Kim, Haeng Hoon; Cho, Jin Woong; Park, Sang Un

    2014-01-01

    .... We isolated a cDNA clone encoding flavonol synthase from Scutellaria baicalensis (SbFLS). The SbFLS cDNA is 1011 bp long, encodes 336 amino acid residues, and belongs to a family of 2-oxoglutarate-dependent dioxygenases...

  11. Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

    OpenAIRE

    Grogan, D W; Cronan, J E

    1984-01-01

    Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and C...

  12. Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

    Science.gov (United States)

    Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

    2015-08-01

    Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Discovery of bacterial polyhydroxyalkanoate synthase (PhaC)-encoding genes from seasonal Baltic Sea ice and cold estuarine waters.

    Science.gov (United States)

    Pärnänen, Katariina; Karkman, Antti; Virta, Marko; Eronen-Rasimus, Eeva; Kaartokallio, Hermanni

    2015-01-01

    Polyhydroxyalkanoates (PHAs) are macromolecules produced by bacteria as means for storing carbon and energy in intracellular granules. PHAs have physical properties similar to those of plastics and have become of interest to industry as materials for environmentally friendly bioplastic production. There is an ongoing search for new PHA-producing bacterial strains and PHA-synthesizing enzymes tolerating extreme conditions to find ways of producing PHAs at cold temperatures and high solute concentrations. Moreover, the study of PHA producers in the sea-ice biome can aid in understanding the microbial ecology of carbon cycling in ice-associated ecosystems. In this study, PHA producers and PHA synthase genes were examined under the extreme environmental conditions of sea ice and cold seawater to find evidence of PHA production in an environment requiring adaptation to high salinity and cold temperatures. Sea ice and cold estuarine water samples were collected from the northern Baltic Sea and evidence of PHA production was gathered, using microscopy with Nile Blue A staining of PHA-granules and PCR assays detecting PHA-synthesis genes. The PHA granules and PHA synthases were found at all sampling locations, in both sea ice and water, and throughout the sampling period spanning over 10 years. Our study shows, for the first time, that PHA synthesis occurs in Baltic Sea cold-adapted bacteria in their natural environment, which makes the Baltic Sea and its cold environments an interesting choice in the quest for PHA-synthesizing bacteria and synthesis genes.

  14. [Cloning and expression of a class II chitin synthase gene PstChs II from the rust fungus Puccinia striiformis].

    Science.gov (United States)

    Liang, Xiaofei; Liu, Bo; Zhu, Lin; Zhang, Xiaoyu; Wang, Xiaojie; Huang, Lili; Kang, Zhensheng

    2009-12-01

    To clone the chitin synthase gene PstChs II from Puccinia striiformis and to analyze its expression pattern. We isolated the cDNA and genomic DNA of PstChs II via RT-PCR and PCR,analyzed the sequences with different bioinformatic tools, characterized the gene expression pattern via real-time PCR. The coding region of PstChs II (Genbank accession no: GQ329851), interrupted by 15 introns, corresponded to a 2727 bp open reading frame encoding 908 amino acids. PstChs II showed a highest 94% similarity to PgtChs II from Puccinia graminis. PstChs II had 7 transmembrance regions and several conserved chitin synthase domains and motifs such as "QXRRW", "GXGPL" and "DXD". PstChs II belonged to the class II sub-family and had a closer phylogenetic relationship to its homologs from basidiomycetes than those from ascomycetes. PstChs II expression level increased by 10-fold at the urediospore germination stage. PstChs II might be involved in the cell wall synthesis during the germ tube elongation process. The cloning and expression analysis of PstChs II served as a good foundation for further analyzing the role of this gene in the pathogenesis process.

  15. Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species

    Directory of Open Access Journals (Sweden)

    Agnieszka Waśkiewicz

    2013-03-01

    Full Text Available Beauvericin (BEA and enniatins (ENNs are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.

  16. An (E,E)-a-farnesene synthase gene of soybean has a role in defense against nematodes and is involved in synthesizing insect-induced volatiles

    Science.gov (United States)

    Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here we report the functional characterization of one member of the soybean TP S gene family, which was designated GmAFS. Recombinant GmAFS produced in E.coli catalyzed the formation of a sesquiterpene (E,E)-a-farnesene....

  17. Genetic Diversity of Polyketide Synthase/Nonribosomal Peptide Synthetase Genes in Isolates of the Barley Net Blotch Fungus Pyrenophora teres f. teres

    Science.gov (United States)

    Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes responsible for biosynthesis of diverse small molecules (e.g., mycotoxins and phytotoxins) in filamentous ascomycetes. Both PKS and NRPS genes are present in fungal genomes as large gene families but...

  18. Chitin synthase gene FgCHS8 affects virulence and fungal cell wall sensitivity to environmental stress in Fusarium graminearum.

    Science.gov (United States)

    Zhang, Ya-Zhou; Chen, Qing; Liu, Cai-Hong; Liu, Yu-Bin; Yi, Pan; Niu, Ke-Xin; Wang, Yan-Qing; Wang, An-Qi; Yu, Hai-Yue; Pu, Zhi-En; Jiang, Qian-Tao; Wei, Yu-Ming; Qi, Peng-Fei; Zheng, You-Liang

    2016-05-01

    Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) of wheat and barley and is considered to be one of the most devastating plant diseases worldwide. Chitin is a critical component of the fungal cell wall and is polymerized from UDP-N-acetyl-alpha-D-glucosamine by chitin synthase. We characterized FgCHS8, a new class of the chitin synthase gene in F. graminearum. Disruption of FgCHS8 resulted in reduced accumulation of chitin, decreased chitin synthase activity, and had no effect on conidia growth when compared with the wild-type isolate. ΔFgCHS8 had a growth rate comparable to that of the wild-type isolate in vitro. However, ΔFgCHS8 had reduced growth when grown on agar supplemented with either 0.025% SDS or 0.9 mM salicylic acid. ΔFgCHS8 produced significantly less deoxynivalenol and exhibited reduced pathogenicity in wheat spikes. Re-introduction of a functional FgCHS8 gene into the ΔFgCHS8 mutant strain restored the wild-type phenotypes. Fluorescence microscopy revealed that FgCHS8 protein was initially expressed in the septa zone, and then gradually distributed over the entire cellular membrane, indicating that FgCHS8 was required for cell wall development. Our results demonstrated that FgCHS8 is important for cell wall sensitivity to environmental stress factors and deoxynivalenol production in F. graminearum. Copyright © 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  19. Genome-Wide Analysis of the Sucrose Synthase Gene Family in Grape (Vitis vinifera): Structure, Evolution, and Expression Profiles

    Science.gov (United States)

    Zhu, Xudong; Wang, Mengqi; Li, Xiaopeng; Jiu, Songtao; Wang, Chen; Fang, Jinggui

    2017-01-01

    Sucrose synthase (SS) is widely considered as the key enzyme involved in the plant sugar metabolism that is critical to plant growth and development, especially quality of the fruit. The members of SS gene family have been identified and characterized in multiple plant genomes. However, detailed information about this gene family is lacking in grapevine (Vitis vinifera L.). In this study, we performed a systematic analysis of the grape (V. vinifera) genome and reported that there are five SS genes (VvSS1–5) in the grape genome. Comparison of the structures of grape SS genes showed high structural conservation of grape SS genes, resulting from the selection pressures during the evolutionary process. The segmental duplication of grape SS genes contributed to this gene family expansion. The syntenic analyses between grape and soybean (Glycine max) demonstrated that these genes located in corresponding syntenic blocks arose before the divergence of grape and soybean. Phylogenetic analysis revealed distinct evolutionary paths for the grape SS genes. VvSS1/VvSS5, VvSS2/VvSS3 and VvSS4 originated from three ancient SS genes, which were generated by duplication events before the split of monocots and eudicots. Bioinformatics analysis of publicly available microarray data, which was validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct temporal and spatial expression patterns of VvSS genes in various tissues, organs and developmental stages, as well as in response to biotic and abiotic stresses. Taken together, our results will be beneficial for further investigations into the functions of SS gene in the processes of grape resistance to environmental stresses. PMID:28350372

  20. Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Franchi, Nicola [Department of Biology, University of Padova, Padova (Italy); Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Piccinni, Ester [Department of Biology, University of Padova, Padova (Italy); Ferro, Diana [Department of Biology, University of Padova, Padova (Italy); Institute for Evolution and Biodiversity, Westfälische Wilhelms-Universität, Münster (Germany); Basso, Giuseppe [Department of Woman and Child Health, University of Padova, Padova (Italy); Spolaore, Barbara [CRIBI Biotechnology Centre, University of Padova, Padova (Italy); Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova (Italy); Santovito, Gianfranco, E-mail: gianfranco.santovito@unipd.it [Department of Biology, University of Padova, Padova (Italy); Ballarin, Loriano [Department of Biology, University of Padova, Padova (Italy)

    2014-07-01

    Highlights: • Ciona intestinalis have a functional phytochelatin synthase (PCS) gene (cipcs). • CiPCS amino acid sequence is phylogentically related to other metazoan PCSs. • CiPCS catalyze the synthesis of PC2. • cipcs are mostly transcribed in circulating hemocytes, in both tunic and blood lacunae. • Cadmium exposure results in a significant increase of cipcs and cipcna transcription. - Abstract: The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96 h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

  1. Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple.

    Science.gov (United States)

    Nieuwenhuizen, Niels J; Green, Sol A; Chen, Xiuyin; Bailleul, Estelle J D; Matich, Adam J; Wang, Mindy Y; Atkinson, Ross G

    2013-02-01

    Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple 'Royal Gala' expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies.

  2. Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple1[W

    Science.gov (United States)

    Nieuwenhuizen, Niels J.; Green, Sol A.; Chen, Xiuyin; Bailleul, Estelle J.D.; Matich, Adam J.; Wang, Mindy Y.; Atkinson, Ross G.

    2013-01-01

    Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple ‘Royal Gala’ expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies. PMID:23256150

  3. Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Prevents Dihydrofolate Reductase Degradation via Promoting S-Nitrosylation.

    Science.gov (United States)

    Cai, Zhejun; Lu, Qiulun; Ding, Ye; Wang, Qilong; Xiao, Lei; Song, Ping; Zou, Ming-Hui

    2015-11-01

    Dihydrofolate reductase (DHFR) is a key protein involved in tetrahydrobiopterin (BH4) regeneration from 7,8-dihydrobiopterin (BH2). Dysfunctional DHFR may induce endothelial nitric oxide (NO) synthase (eNOS) uncoupling resulting in enzyme production of superoxide anions instead of NO. The mechanism by which DHFR is regulated is unknown. Here, we investigate whether eNOS-derived NO maintains DHFR stability. DHFR activity, BH4 content, eNOS activity, and S-nitrosylation were assessed in human umbilical vein endothelial cells and in aortas isolated from wild-type and eNOS knockout mice. In human umbilical vein endothelial cells, depletion of intracellular NO by transfection with eNOS-specific siRNA or by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-both of which had no effect on DHFR mRNA levels-markedly reduced DHFR protein levels in parallel with increased DHFR polyubiquitination. Supplementation of S-nitroso-l-glutathione (GSNO), a NO donor, or MG132, a potent inhibitor of the 26S proteasome, prevented eNOS silencing and PTIO-induced DHFR reduction in human umbilical vein endothelial cells. PTIO suppressed S-nitrosylation of DHFR, whereas GSNO promoted DHFR S-nitrosylation. Mutational analysis confirmed that cysteine 7 of DHFR was S-nitrosylated. Cysteine 7 S-nitrosylation stabilized DHFR from ubiquitination and degradation. Experiments performed in aortas confirmed that PTIO or eNOS deficiency reduces endothelial DHFR, which can be abolished by MG132 supplementation. We conclude that S-nitrosylation of DHFR at cysteine 7 by eNOS-derived NO is crucial for DHFR stability. We also conclude that NO-induced stabilization of DHFR prevents eNOS uncoupling via regeneration of BH4, an essential eNOS cofactor. © 2015 American Heart Association, Inc.

  4. Interleukin-10 gene promoter polymorphism as a potential host ...

    African Journals Online (AJOL)

    Interleukin-10 gene promoter polymorphism as a potential host susceptibility factor in Pakistani patients with pulmonary tuberculosis. MS Afzal, S Anjum, A Salman, S Ashraf, ZUR Farooqi, T Ahmed, Y Waheed, I Qadri ...

  5. A WDR Gene Is a Conserved Member of a Chitin Synthase Gene Cluster and Influences the Cell Wall in Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Gea Guerriero

    2016-06-01

    Full Text Available WD40 repeat (WDR proteins are pleiotropic molecular hubs. We identify a WDR gene that is a conserved genomic neighbor of a chitin synthase gene in Ascomycetes. The WDR gene is unique to fungi and plants, and was called Fungal Plant WD (FPWD. FPWD is within a cell wall metabolism gene cluster in the Ascomycetes (Pezizomycotina comprising chsD, a Chs activator and a GH17 glucanase. The FPWD, AN1556.2 locus was deleted in Aspergillus nidulans strain SAA.111 by gene replacement and only heterokaryon transformants were obtained. The re-annotation of Aspergilli genomes shows that AN1556.2 consists of two tightly linked separate genes, i.e., the WDR gene and a putative beta-flanking gene of unknown function. The WDR and the beta-flanking genes are conserved genomic neighbors localized within a recently identified metabolic cell wall gene cluster in genomes of Aspergilli. The heterokaryons displayed increased susceptibility to drugs affecting the cell wall, and their phenotypes, observed by optical, confocal, scanning electron and atomic force microscopy, suggest cell wall alterations. Quantitative real-time PCR shows altered expression of some cell wall-related genes. The possible implications on cell wall biosynthesis are discussed.

  6. Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv.

    Science.gov (United States)

    Waddell, S J; Chung, G A; Gibson, K J C; Everett, M J; Minnikin, D E; Besra, G S; Butcher, P D

    2005-01-01

    Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.

  7. Cloning of an anthocyanidin synthase gene homolog from blackcurrant (Ribes nigrum L.) and its expression at different fruit stages.

    Science.gov (United States)

    Li, X-G; Wang, J; Yu, Z-Y

    2015-03-31

    Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, catalyzes the penultimate step in anthocyanin biosynthesis, from leucoanthocyanidins to anthocyanidins, the first colored compound in the anthocyanin pathway. In this study, a full-length, 1427-bp long cDNA named RnANS1, which is homologous to the anthocyanidin synthase gene, was cloned from blackcurrant using a homologous cloning strategy. RnANS1 is highly homologous to other plant ANS genes at both the nucleotide and amino acid sequence levels. The deduced protein contains domains conserved in the 2OG and Fe(II)-dependent oxygenase, and is phylogenetically closely related to Paeonia suffruticosa and Paeonia lactiflora. The expression of RnANS1 was upregulated during fruit maturation, and correlated with the accumulation of anthocyanins and soluble carbohydrates in the fruit. Further characterization of the structure and expression patterns of RnANS1 will clarify our understanding of anthocyanin biosynthesis in blackcurrant, and support the development of molecular approaches to manipulate anthocyanin production in this plant.

  8. Arabidopsis thaliana tryptophan synthase alpha: gene cloning, expression, and subunit interaction.

    Science.gov (United States)

    Radwanski, E R; Zhao, J; Last, R L

    1995-10-25

    The tryptophan synthase alpha subunit catalyzes the conversion of indole-3-glycerolphosphate to indole, the penultimate reaction in the biosynthesis of the essential amino acid tryptophan. A cDNA encoding Arabidopsis thaliana tryptophan synthase alpha(TSA1) was isolated by complementation of an Escherichia coli delta trpA mutation and by polymerase chain reaction amplification from a cDNA library using degenerate primers. A TSA1 genomic clone was also isolated and 5 kb of the DNA sequence determined. A single sequence in the Arabidopsis genome with homology to the TSA1 cDNA was detected by high-stringency genomic Southern blot hybridization. In contrast under hybridization conditions of reduced stringency, one or two additional homologous sequences were observed. A 1.4 kb transcript was detected in wild-type RNA with the TSA1 cDNA as a probe. Several lines of evidence, including immunoaffinity chromatography, suggest that the active A. thaliana tryptophan synthase enzyme consists of a heterosubunit complex, presumably analogous to the prokaryotic alpha 2 beta 2 complex. Immunoblot analysis indicated that the plant alpha and beta subunits are present throughout development.

  9. Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus.

    Science.gov (United States)

    Nakano, Chiaki; Motegi, Akihiro; Sato, Tsutomu; Onodera, Masayuki; Hoshino, Tsutomu

    2007-10-01

    Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.

  10. Core promoter functions in the regulation of gene expression of Drosophila dorsal target genes.

    Science.gov (United States)

    Zehavi, Yonathan; Kuznetsov, Olga; Ovadia-Shochat, Avital; Juven-Gershon, Tamar

    2014-04-25

    Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes.

  11. Congenital erythropoietic porphyria with two mutations of the uroporphyrinogen III synthase gene (Cys73Arg, Thr228Met)

    Science.gov (United States)

    Gucev, Zoran; Slavevska, Nevenka; Tasic, Velibor; Laban, Nevenka; Pop-Jordanova, Nada; Danilovski, Dragan; Woolf, Jacqueline; Cole, Duncan

    2011-01-01

    Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of uroporphyrinogen III synthase (UROS). We describe a 14-year-old girl with red urine since infancy, progressive blistering and scarring of the skin, and moderate hemolytic anemia. After years of skin damage, her face is mutilated; she has a bald patch on the scalp, hypertrichosis of the neck, areas of skin darkening, and limited joint movements of the hands. Total urine excretion and fecal total porphyrin were both markedly raised above normal levels. Sequencing of the UROS gene identified two mutations causing CEP (Cys73Arg, Thr228Met). The patient lesions are progressing. Bone marrow transplantation and/or gene therapy are proposed as the next steps in her treatment. In brief, we describe a CEP with confirmed two pathogenic mutations, severe phenotype and discuss the various treatment options available. PMID:22090724

  12. Environmental Stability of Seed Carbohydrate Profiles in Soybeans Containing Different Alleles of the Raffinose Synthase 2 (RS2) Gene.

    Science.gov (United States)

    Bilyeu, Kristin D; Wiebold, William J

    2016-02-10

    Soybean [Glycine max (L.) Merr.] is important for the high protein meal used for livestock feed formulations. Carbohydrates contribute positively or negatively to the potential metabolizable energy in soybean meal. The positive carbohydrate present in soybean meal consists primarily of sucrose, whereas the negative carbohydrate components are the raffinose family of oligosaccharides (RFOs), raffinose and stachyose. Increasing sucrose and decreasing raffinose and stachyose are critical targets to improve soybean. In three recently characterized lines, variant alleles of the soybean raffinose synthase 2 (RS2) gene were associated with increased sucrose and decreased RFOs. The objective of this research was to compare the environmental stability of seed carbohydrates in soybean lines containing wild-type or variant alleles of RS2 utilizing a field location study and a date of planting study. The results define the carbohydrate variation in distinct regional and temporal environments using soybean lines with different alleles of the RS2 gene.

  13. Diversification of the monoterpene synthase gene family (TPSb) in Protium, a highly diverse genus of tropical trees.

    Science.gov (United States)

    Zapata, Felipe; Fine, Paul V A

    2013-09-01

    Plant monoterpenes are a diverse class of secondary metabolites mediating biotic and abiotic interactions with direct effects on plant fitness. To evaluate the hypothesis that monoterpene diversity is related to functional diversification after gene duplication, we reconstructed the evolutionary history of monoterpene synthases (TPSb)--the genes underlying monoterpene synthesis--in Protium, a taxonomically and chemically diverse genus of tropical trees. We isolated multiple copies of TPSb genes from chemically divergent Protium species, reconstructed the phylogeny of this gene family, used maximum-likelihood estimation of selection coefficients, and inferred residues evolving under positive selection. We found evidence for one ancient and multiple more recent duplication events giving rise to three, and potentially five, copies of TPSb genes currently present in Protium. There was evidence for adaptive evolution in one copy with a positively selected residue likely involved in protein folding and product specificity. All other copies were inferred to be evolving under a combination of stabilizing and/or relaxed selection. Although gene copy number is consistent with the extensive phenotypic diversity in monoterpenes shown in Protium, selection analyses suggest that not all copies are undergoing divergent selection consistent with a coevolutionary arms race with enemies, but instead may be under stabilizing and relaxed selection consistent with signaling or physiological stress functionality. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

    Science.gov (United States)

    So, Kum-Kang; Chung, Yun-Jo; Kim, Jung-Mi; Kim, Beom-Tae; Park, Seung-Moon; Kim, Dae-Hyuk

    2015-12-01

    Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

  15. Genome-Wide Identification, Evolutionary and Expression Analyses of the GALACTINOL SYNTHASE Gene Family in Rapeseed and Tobacco

    Directory of Open Access Journals (Sweden)

    Yonghai Fan

    2017-12-01

    Full Text Available Galactinol synthase (GolS is a key enzyme in raffinose family oligosaccharide (RFO biosynthesis. The finding that GolS accumulates in plants exposed to abiotic stresses indicates RFOs function in environmental adaptation. However, the evolutionary relationships and biological functions of GolS family in rapeseed (Brassica napus and tobacco (Nicotiana tabacum remain unclear. In this study, we identified 20 BnGolS and 9 NtGolS genes. Subcellular localization predictions showed that most of the proteins are localized to the cytoplasm. Phylogenetic analysis identified a lost event of an ancient GolS copy in the Solanaceae and an ancient duplication event leading to evolution of GolS4/7 in the Brassicaceae. The three-dimensional structures of two GolS proteins were conserved, with an important DxD motif for binding to UDP-galactose (uridine diphosphate-galactose and inositol. Expression profile analysis indicated that BnGolS and NtGolS genes were expressed in most tissues and highly expressed in one or two specific tissues. Hormone treatments strongly induced the expression of most BnGolS genes and homologous genes in the same subfamilies exhibited divergent-induced expression. Our study provides a comprehensive evolutionary analysis of GolS genes among the Brassicaceae and Solanaceae as well as an insight into the biological function of GolS genes in hormone response in plants.

  16. Thymidylate synthase (TS) gene expression in primary lung cancer patients: a large-scale study in Japanese population.

    Science.gov (United States)

    Tanaka, F; Wada, H; Fukui, Y; Fukushima, M

    2011-08-01

    Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TS-inhibiting agents such as pemetrexed. To quantitatively measure TS gene expression in a large-scale Japanese population (n = 2621) with primary lung cancer, laser-captured microdissected sections were cut from primary tumors, surrounding normal lung tissues and involved nodes. TS gene expression level in primary tumor was significantly higher than that in normal lung tissue (mean TS/β-actin, 3.4 and 1.0, respectively; P TS gene expression level was further higher in involved node (mean TS/β-actin, 7.7; P TS gene expression levels in primary tumor according to histologic cell type revealed that small-cell carcinoma showed highest TS expression (mean TS/β-actin, 13.8) and that squamous cell carcinoma showed higher TS expression as compared with adenocarcinoma (mean TS/β-actin, 4.3 and 2.3, respectively; P TS gene expression was significantly increased along with a decrease in the grade of tumor cell differentiation. There was no significant difference in TS gene expression according to any other patient characteristics including tumor progression. Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

  17. Genome-wide analysis of the cellulose synthase-like (Csl) gene family in bread wheat (Triticum aestivum L.).

    Science.gov (United States)

    Kaur, Simerjeet; Dhugga, Kanwarpal S; Beech, Robin; Singh, Jaswinder

    2017-11-03

    Hemicelluloses are a diverse group of complex, non-cellulosic polysaccharides, which constitute approximately one-third of the plant cell wall and find use as dietary fibres, food additives and raw materials for biofuels. Genes involved in hemicellulose synthesis have not been extensively studied in small grain cereals. In efforts to isolate the sequences for the cellulose synthase-like (Csl) gene family from wheat, we identified 108 genes (hereafter referred to as TaCsl). Each gene was represented by two to three homeoalleles, which are named as TaCslXY_ZA, TaCslXY_ZB, or TaCslXY_ZD, where X denotes the Csl subfamily, Y the gene number and Z the wheat chromosome where it is located. A quarter of these genes were predicted to have 2 to 3 splice variants, resulting in a total of 137 putative translated products. Approximately 45% of TaCsl genes were located on chromosomes 2 and 3. Sequences from the subfamilies C and D were interspersed between the dicots and grasses but those from subfamily A clustered within each group of plants. Proximity of the dicot-specific subfamilies B and G, to the grass-specific subfamilies H and J, respectively, points to their common origin. In silico expression analysis in different tissues revealed that most of the genes were expressed ubiquitously and some were tissue-specific. More than half of the genes had introns in phase 0, one-third in phase 2, and a few in phase 1. Detailed characterization of the wheat Csl genes has enhanced the understanding of their structural, functional, and evolutionary features. This information will be helpful in designing experiments for genetic manipulation of hemicellulose synthesis with the goal of developing improved cultivars for biofuel production and increased tolerance against various stresses.

  18. Evolutionary Insights Based on SNP Haplotypes of Red Pericarp, Grain Size and Starch Synthase Genes in Wild and Cultivated Rice

    Directory of Open Access Journals (Sweden)

    Nisha Singh

    2017-06-01

    Full Text Available The origin and domestication of rice has been a subject of considerable debate in the post-genomic era. Rice varieties have been categorized based on isozyme and DNA markers into two broad cultivar groups, Indica and Japonica. Among other well-known cultivar groups Aus varieties are closer to Indica and Aromatic varieties including Basmati are closer to Japonica, while deep-water rice varieties share kinship to both Indica and Japonica cultivar groups. Here, we analyzed haplotype networks and phylogenetic relationships in a diverse set of genotypes including Indian Oryza nivara/Oryza rufipogon wild rice accessions and representative varieties of four rice cultivar groups based on pericarp color (Rc, grain size (GS3 and eight different starch synthase genes (GBSSI, SSSI, SSIIa, SSIIb, SSIIIa, SSIIIb, SSIVa, and SSIVb. Aus cultivars appear to have the most ancient origin as they shared the maximum number of haplotypes with the wild rice populations, while Indica, Japonica and Aromatic cultivar groups showed varying phylogenetic origins of these genes. Starch synthase genes showed higher variability in cultivated rice than wild rice populations, suggesting diversified selection during and after domestication. O. nivara/O. rufipogon wild rice accessions belonging to different sub-populations shared common haplotypes for all the 10 genes analyzed. Our results support polyphyletic origin of cultivated rice with a complex pattern of migration of domestication alleles from wild to different rice cultivar groups. The findings provide novel insights into evolutionary and domestication history of rice and will help utilization of wild rice germplasm for genetic improvement of rice cultivars.

  19. Evolutionary Insights Based on SNP Haplotypes of Red Pericarp, Grain Size and Starch Synthase Genes in Wild and Cultivated Rice

    Science.gov (United States)

    Singh, Nisha; Singh, Balwant; Rai, Vandna; Sidhu, Sukhjeet; Singh, Ashok K.; Singh, Nagendra K.

    2017-01-01

    The origin and domestication of rice has been a subject of considerable debate in the post-genomic era. Rice varieties have been categorized based on isozyme and DNA markers into two broad cultivar groups, Indica and Japonica. Among other well-known cultivar groups Aus varieties are closer to Indica and Aromatic varieties including Basmati are closer to Japonica, while deep-water rice varieties share kinship to both Indica and Japonica cultivar groups. Here, we analyzed haplotype networks and phylogenetic relationships in a diverse set of genotypes including Indian Oryza nivara/Oryza rufipogon wild rice accessions and representative varieties of four rice cultivar groups based on pericarp color (Rc), grain size (GS3) and eight different starch synthase genes (GBSSI, SSSI, SSIIa, SSIIb, SSIIIa, SSIIIb, SSIVa, and SSIVb). Aus cultivars appear to have the most ancient origin as they shared the maximum number of haplotypes with the wild rice populations, while Indica, Japonica and Aromatic cultivar groups showed varying phylogenetic origins of these genes. Starch synthase genes showed higher variability in cultivated rice than wild rice populations, suggesting diversified selection during and after domestication. O. nivara/O. rufipogon wild rice accessions belonging to different sub-populations shared common haplotypes for all the 10 genes analyzed. Our results support polyphyletic origin of cultivated rice with a complex pattern of migration of domestication alleles from wild to different rice cultivar groups. The findings provide novel insights into evolutionary and domestication history of rice and will help utilization of wild rice germplasm for genetic improvement of rice cultivars. PMID:28649256

  20. Functional genomic analysis supports conservation of function among cellulose synthase-like a gene family members and suggests diverse roles of mannans in plants

    DEFF Research Database (Denmark)

    Liepman, Aaron H; Nairn, C Joseph; Willats, William G T

    2007-01-01

    from Arabidopsis (Arabidopsis thaliana), guar (Cyamopsis tetragonolobus), and Populus trichocarpa catalyze beta-1,4-mannan and glucomannan synthase reactions in vitro. Mannan polysaccharides and homologs of CslA genes appear to be present in all lineages of land plants analyzed to date. In many plants...... in insect cells, and each CslA protein catalyzed mannan and glucomannan synthase reactions in vitro. Microarray mining and quantitative real-time reverse transcription-polymerase chain reaction analysis demonstrated that transcripts of Arabidopsis and loblolly pine (Pinus taeda) CslA genes display tissue...

  1. Firefly luciferase gene contains a cryptic promoter

    Czech Academy of Sciences Publication Activity Database

    Vopálenský, V.; Mašek, T.; Horváth, Ondřej; Vicenová, B.; Mokrejš, M.; Pospíšek, M.

    2008-01-01

    Roč. 14, č. 9 (2008), s. 1720-1729 ISSN 1355-8382 Grant - others:GAČR(CZ) GA204/03/1487; GAČR(CZ) GA301/07/0607; Mšk(CZ) LC06066 Program:LC Institutional research plan: CEZ:AV0Z50520514 Keywords : luciferase * cryptic promoter * hepatitis C virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.018, year: 2008

  2. Root parasitic plant Orobanche aegyptiaca and shoot parasitic plant Cuscuta australis obtained Brassicaceae-specific strictosidine synthase-like genes by horizontal gene transfer

    Science.gov (United States)

    2014-01-01

    Background Besides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found. Results A thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species. Conclusions Our study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts

  3. Root parasitic plant Orobanche aegyptiaca and shoot parasitic plant Cuscuta australis obtained Brassicaceae-specific strictosidine synthase-like genes by horizontal gene transfer.

    Science.gov (United States)

    Zhang, Dale; Qi, Jinfeng; Yue, Jipei; Huang, Jinling; Sun, Ting; Li, Suoping; Wen, Jian-Fan; Hettenhausen, Christian; Wu, Jinsong; Wang, Lei; Zhuang, Huifu; Wu, Jianqiang; Sun, Guiling

    2014-01-13

    Besides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found. A thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species. Our study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts.

  4. Fractal Topology of Gene Promoter Networks at Phase Transitions

    Directory of Open Access Journals (Sweden)

    Preston R. Aldrich

    2010-07-01

    Full Text Available Much is known regarding the structure and logic of genetic regulatory networks. Less understood is the contextual organization of promoter signals used during transcription initiation, the most pivotal stage during gene expression. Here we show that promoter networks organize spontaneously at a dimension between the 1-dimension of the DNA and 3-dimension of the cell. Network methods were used to visualize the global structure of E. coli sigma (σ recognition footprints using published promoter sequences (RegulonDB. Footprints were rendered as networks with weighted edges representing bp-sharing between promoters (nodes. Serial thresholding revealed phase transitions at positions predicted by percolation theory, and nuclei denoting short steps through promoter space with geometrically constrained linkages. The network nuclei are fractals, a power-law organization not yet described for promoters. Genome-wide promoter abundance also scaled as a power-law. We propose a general model for the development of a fractal nucleus in a transcriptional grammar.

  5. Identification and characterization of a class III chitin synthase gene of Moniliophthora perniciosa, the fungus that causes witches' broom disease of cacao.

    Science.gov (United States)

    Souza, Catiane S; Oliveira, Bruno M; Costa, Gustavo G L; Schriefer, Albert; Selbach-Schnadelbach, Alessandra; Uetanabaro, Ana Paula T; Pirovani, Carlos P; Pereira, Gonçalo A G; Taranto, Alex G; Cascardo, Júlio Cézar de M; Góes-Neto, Aristóteles

    2009-08-01

    Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches' broom disease of cacao.

  6. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation.

    Science.gov (United States)

    Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

    2015-01-01

    The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process.

  7. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation

    Science.gov (United States)

    Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

    2015-01-01

    The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

  8. Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents

    OpenAIRE

    George Seghal Kiran; Joseph eSelvin; Sathiyanarayanan eGanesan; Lipton eAnuj Nishanth; Naif eAbdullah Al-Dhabi; Mariadhas eValan Arasu

    2016-01-01

    The important biological macromolecules such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was also explored. A unique feature of type II PKS genes is their high amino acid sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of var...

  9. Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

    DEFF Research Database (Denmark)

    Müller, Christian; Hansen, K.; Szabo, Peter

    2003-01-01

    The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3...... broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations. (C) 2003 Wiley Periodicals, Inc....

  10. Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene

    DEFF Research Database (Denmark)

    Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.

    2005-01-01

    A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...... composition in rosette leaves was shown to vary naturally within this ecotype. Relatively modest environmental changes had a strong effect on the wild type level of flavonoids and some sinapate esters but much less effect on the glucosinolates. Potentially a reduction in the level of flavonoids could...

  11. Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Bandara, Nadeeka; Gurusinghe, Saliya; Chen, Haiying; Chen, Shuangfeng; Wang, Le-Xin; Lim, Shiang Y; Strappe, Padraig

    2016-04-01

    Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction. Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant

  12. Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-epi-5-epi-valiolone synthase gene in Streptomyces albus ATCC 21838.

    Science.gov (United States)

    Choi, Woo Sik; Wu, Xiumei; Choeng, Yong-Hoon; Mahmud, Taifo; Jeong, Byeong Chul; Lee, Sang Hee; Chang, Yong Keun; Kim, Chang-Joon; Hong, Soon-Kwang

    2008-09-01

    The cyclization of sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone, catalyzed by the 2-epi-5-epi-valiolone synthases, is the first committed step in the biosynthesis of C( 7 )N-aminocyclitol-containing natural products, such as validamycin and acarbose. These natural products contain in their structures a valienamine unit, which is important for their biological activity. The same core unit is also found in salbostatin, a related pseudodisaccharide that has strong trehalase inhibitory activity. In silico analysis of the putative biosynthetic gene cluster of salbostatin from Streptomyces albus ATCC 21838 revealed 20 open reading frames, including an acbC homolog gene (salQ), which is believed to be involved in the biosynthesis of salbostatin. The salQ gene was overexpressed in Escherichia coli and the catalytic function of the recombinant protein was confirmed to be a 2-epi-5-epi-valiolone synthase. In addition, SalF, SalL, SalM, SalN, SalO, and SalR were found to be homologous to AcbR, AcbM, AcbL, AcbN, AcbO, and AcbP from the acarbose pathway, respectively, which suggests that the biosynthesis of C(7)N-aminocyclitol moiety of salbostatin may be very similar to that of acarbose.

  13. Thymidylate synthase and methylenetetrahy-drofolate reductase gene polymorphisms and gastric cancer susceptibility in a population of Northern Brazil.

    Science.gov (United States)

    Araújo, M D; Borges, B N; Rodrigues-Antunes, S; Burbano, R M R; Harada, M L

    2015-08-21

    The folate metabolic pathway, which is involved in DNA synthesis and methylation, is associated with individual susceptibility to several diseases, including gastric tumors. In this study, we investigated four polymorphisms [thymidylate synthase enhancer region, single nucleotide polymorphism thymidylate synthase 5' (TS5'), TS3' untranslated region, and methylenetetrahydrofolate reductase (MTHFR) 677C> T] in 2 genes related to the folate pathway, TS and MTHFR, and their possible association with the risk gastric cancer development in a population from Pará state, Brazil. For the TS enhancer region, TS3' untranslated region, and single nucleotide polymorphism TS5' polymorphisms, no significant results were obtained. For the MTHFR 677C>T polymorphism, TT genotype carriers had a higher risk of developing tumors in the antrum (P = 0.19 vs CC and P = 0.02 vs CT) and intestine (odds ratio = 4.18, 95% confidence interval = 0.66-26.41; P = 0.252 vs CC and odds ratio = 2.25, 95% confidence interval = 0.32-15.75; P = 0.725 vs CT). Those carrying at least 1 T allele had an increased risk of lymph node metastasis (odds ratio = 3.00, 95% confidence interval = 0.88-10.12; P = 0.133). Our results suggest that polymorphisms in MTHFR affect the susceptibility to gastric tumors in the Brazilian population and may be a factor causing poor prognosis in such patients.

  14. Endothelial Nitric Oxide Synthase Gene Polymorphism (G894T and Diabetes Mellitus (Type II among South Indians

    Directory of Open Access Journals (Sweden)

    T. Angeline

    2011-01-01

    Full Text Available The objective of the study is to find out whether the endothelial nitric oxide synthase (eNOS G894T single-nucleotide polymorphism is associated with type 2 diabetes mellitus in South Indian (Tamil population. A total number of 260 subjects comprising 100 type 2 diabetic mellitus patients and 160 healthy individuals with no documented history of diabetes were included for the study. DNA was isolated, and eNOS G894T genotyping was performed using the polymerase chain reaction followed by restriction enzyme analysis using Ban II. The genotype distribution in patients and controls were compatible with the Hardy-Weinberg expectations (P>0.05. Odds ratio indicates that the occurrence of mutant genotype (GT/TT was 7.2 times (95% CI = 4.09–12.71 more frequent in the cases than in controls. Thus, the present study demonstrates that there is an association of endothelial nitric oxide synthase gene (G894T polymorphism with diabetes mellitus among South Indians.

  15. Relationship between single nucleotide polymorphism of glycogen synthase gene of Pacific oyster Crassostrea gigas and its glycogen content

    Science.gov (United States)

    Liu, Siwei; Li, Qi; Yu, Hong; Kong, Lingfeng

    2017-02-01

    Glycogen is important not only for the energy supplementary of oysters, but also for human consumption. High glycogen content can improve the stress survival of oyster. A key enzyme in glycogenesis is glycogen synthase that is encoded by glycogen synthase gene GYS. In this study, the relationship between single nucleotide polymorphisms (SNPs) in coding regions of Crassostrea gigas GYS (Cg-GYS) and individual glycogen content was investigated with 321 individuals from five full-sib families. Single-strand conformation polymorphism (SSCP) procedure was combined with sequencing to confirm individual SNP genotypes of Cg-GYS. Least-square analysis of variance was performed to assess the relationship of variation in glycogen content of C. gigas with single SNP genotype and SNP haplotype. As a consequence, six SNPs were found in coding regions to be significantly associated with glycogen content ( P haplotypes due to linkage disequilibrium. Furthermore, the most effective haplotype H2 (GAGGAT) had extremely significant relationship with high glycogen content ( P < 0.0001). These findings revealed the potential influence of Cg-GYS polymorphism on the glycogen content and provided molecular biological information for the selective breeding of good quality traits of C. gigas.

  16. Alfalfa Cellulose synthase gene expression under abiotic stress: a Hitchhiker's guide to RT-qPCR normalization.

    Directory of Open Access Journals (Sweden)

    Gea Guerriero

    Full Text Available Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L., no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress at various time points (e.g. 0, 24, 72 and 96 h. We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots, under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.

  17. Parallel evolution of the glycogen synthase 1 (muscle) gene Gys1 between Old World and New World fruit bats (Order: Chiroptera).

    Science.gov (United States)

    Fang, Lu; Shen, Bin; Irwin, David M; Zhang, Shuyi

    2014-10-01

    Glycogen synthase, which catalyzes the synthesis of glycogen, is especially important for Old World (Pteropodidae) and New World (Phyllostomidae) fruit bats that ingest high-carbohydrate diets. Glycogen synthase 1, encoded by the Gys1 gene, is the glycogen synthase isozyme that functions in muscles. To determine whether Gys1 has undergone adaptive evolution in bats with carbohydrate-rich diets, in comparison to insect-eating sister bat taxa, we sequenced the coding region of the Gys1 gene from 10 species of bats, including two Old World fruit bats (Pteropodidae) and a New World fruit bat (Phyllostomidae). Our results show no evidence for positive selection in the Gys1 coding sequence on the ancestral Old World and the New World Artibeus lituratus branches. Tests for convergent evolution indicated convergence of the sequences and one parallel amino acid substitution (T395A) was detected on these branches, which was likely driven by natural selection.

  18. Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae

    DEFF Research Database (Denmark)

    Müller, Christian; Hjort, C.M.; Hansen, K.

    2002-01-01

    In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence...

  19. Coordinated responses of phytochelatin synthase and metallothionein genes in black mangrove, Avicennia germinans, exposed to cadmium and copper

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez-Mendoza, Daniel [Departamento de Recursos del Mar, Cinvestav-Unidad Merida, Merida, Yucatan (Mexico); Moreno, Adriana Quiroz [Unidad de biotecnologia, CICY, Merida, Yucatan (Mexico); Zapata-Perez, Omar [Departamento de Recursos del Mar, Cinvestav-Unidad Merida, Merida, Yucatan (Mexico)]. E-mail: ozapata@mda.cinvestav.mx

    2007-08-01

    To evaluate the role of phytochelatins and metallothioneins in heavy metal tolerance of black mangrove Avicennia germinans, 3-month-old seedlings were exposed to cadmium or copper for 30 h, under hydroponic conditions. Degenerate Mt2 and PCS primers were synthesized based on amino acid and nucleotide alignment sequences reported for Mt2 and PCS in other plant species found in GenBank. Total RNA was isolated from A. germinans leaves and two partial fragments of metallothionein and phytochelatin synthase genes were isolated. Gene expression was evaluated with reverse transcripatase-polymerase chain reaction (RT-PCR) amplification technique. Temporal analysis showed that low Cd{sup 2+} and Cu{sup 2+} concentrations caused a slight (but not significant) increase in AvMt2 expression after a 16 h exposure time, while AvPCS expression showed a significant increase under the same conditions but only after 4 h. Results strongly suggest that the rapid increase in AvPCS expression may contribute to Cd{sup 2+} and Cu{sup 2+} detoxification. Moreover, we found that A. germinans has the capacity to over-express both genes (AvMt2 and AvPCS), which may constitute a coordinated detoxification response mechanism targeting non-essential metals. Nonetheless, our results confirm that AvPCS was the most active gene involved in the regulation of essential metals (e.g., Cu{sup 2+}) in A. germinans leaves.

  20. Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

    Directory of Open Access Journals (Sweden)

    Takashi Kadono

    2015-08-01

    Full Text Available Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

  1. Thymidylate synthase gene amplification predicts pemetrexed resistance in patients with advanced non-small cell lung cancer.

    Science.gov (United States)

    Shimizu, T; Nakagawa, Y; Takahashi, N; Hashimoto, S

    2016-01-01

    Thymidylate synthase (TYMS) expression in lung cancer tissue affects the therapeutic efficacy of pemetrexed (PMT). TYMS protein expression is primarily assessed using immunohistochemistry (IHC), but this method is not suitable for accurate quantitative analysis. It is not known whether the analysis of TYMS gene copy number using fluorescence in situ hybridization (FISH) is a useful method for assessment of TYMS expression. The participants were patients with chemo-naïve advanced NSCLC treated with PMT plus carboplatin (CBDCA) in prospective clinical phase II study. TYMS expression was evaluated in 40 patients by gene copy number and protein expression using FISH and IHC. Therapeutic efficacy was evaluated by investigating the response rate (RR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). TYMS gene amplification was detected in 8 patients (32 %) among 25 patients who could be evaluated for TYMS gene copy number. There were no patients with complete or partial response in the TYMS amplified group. RR and DCR were lower in the TYMS amplified group compared with the TYMS unamplified group (0 versus 35.3 %, p = 0.0539, 62.5 versus 94.1 %, p = 0.0443). PFS and OS were reduced in the TYMS amplified group. The analysis of TYMS gene copy number had higher sensitivity and specificity compared with TYMS protein expression (76.2 versus 50.0 %, 75.0 versus 66.7 %). The analysis of TYMS gene copy number is more suitable than TYMS protein expression for assessment of TYMS expression. TYMS gene amplification predicts outcome of patients receiving PMT with advanced NSCLC.

  2. Molecular cloning and characterization of the trichome specificchrysanthemyl diphosphate/chrysanthemol synthase promoter fromTanacetum cinerariifolium

    NARCIS (Netherlands)

    Sultana, S.; Hu, H.; Gao, L.; Mao, J.; Luo, J.; Jongsma, M.A.; Wang, C.

    2015-01-01

    Natural pyrethrins accumulate to high concentrations in the flower achenes of pyrethrum (Tanacetumcinerariifolium). They are extracted from dried flowers and widely used as a natural pesticide. Chrysanthe-mol synthase (CHS) is the first key enzyme in the biosynthesis of pyrethrins. In this work, a

  3. Expression of Terpenoids 1, a glandular trichome-specific transcription factor from tomato that activates the terpene synthase 5 promoter

    NARCIS (Netherlands)

    Spyropoulou, E.A.; Haring, M.A.; Schuurink, R.C.

    2014-01-01

    Terpene biosynthesis in tomato glandular trichomes has been well studied, with most if not all terpene synthases (TPSs) being identified. However, transcription factors (TFs) that regulate TPSs have not yet been discovered from tomato. In order to unravel the transcriptional regulation of the

  4. Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells.

    Science.gov (United States)

    Li, Huige; Xia, Ning; Brausch, Isolde; Yao, Ying; Förstermann, Ulrich

    2004-09-01

    Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.

  5. Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P. carinii pneumonia

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Benfield, Thomas; Eugen-Olsen, Jesper

    1999-01-01

    BACKGROUND: Sulpha drugs are widely used for the treatment and long-term prophylaxis of Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals. Sulpha resistance in many microorganisms is caused by point mutations in dihydropteroate synthase (DHPS), an enzyme that is essential...... for folate biosynthesis. We assessed whether mutations in the DHPS gene of P. carinii were associated with exposure to sulpha drugs and influenced outcome from PCP. METHODS: We studied bronchoalveolar samples collected in 1989-99 from a prospective cohort of HIV-1-infected patients who had PCP. In 144...... predictor of mortality (hazard ratio 3.1 [95% CI 1.2-8.1]). DHPS mutations were significantly more common in patients who had previous exposure to sulpha drugs (18 of 29 [62%]) than in those who had no exposure (13 of 123 [10.5%]; pDHPS mutations (p...

  6. Allele specific CAPS marker development and characterization of chalcone synthase gene in Indian mulberry (Morus spp., family Moraceae.

    Directory of Open Access Journals (Sweden)

    Vivek Arora

    Full Text Available Chalcone synthase (CHS is an essential enzyme in the phenylpropanoid pathway that catalyzes the first step in flavonoid biosynthesis in plants under diverse environmental stress. We have used CHS as a candidate gene in mulberry and developed Single Nucleotide Polymorphism (SNP based co-dominant Cleaved Amplified Polymorphic Sequence (CAPS marker associated with the CHS locus. The segregation pattern of the marker was studied in an F1 population derived from a hybridization program between two mulberry genotypes showing polymorphism for the CHS locus. Differential CHS activity of the recombinants has been correlated with the segregation pattern of the marker. Homology modelling and docking studies are performed for both the identified CHS alleles and correlated with respective CHS activity. Phenotyping of Powdery Mildew infected F1 population indicated a probable association with the CAPS marker.

  7. A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification.

    Science.gov (United States)

    Szemiako, Kasjan; Śledzińska, Anna; Krawczyk, Beata

    2017-08-01

    Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

  8. The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils

    Science.gov (United States)

    2012-01-01

    Background The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS), the key enzymes in constructing terpene carbon skeletons. Results Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita). Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (−)-(E)-β-caryophyllene (MrTPS1), (+)-germacrene A (MrTPS3), (E)-β-ocimene (MrTPS4) and (−)-germacrene D (MrTPS5). A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (−)-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. Conclusions The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+)-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils. PMID:22682202

  9. The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils.

    Science.gov (United States)

    Irmisch, Sandra; Krause, Sandra T; Kunert, Grit; Gershenzon, Jonathan; Degenhardt, Jörg; Köllner, Tobias G

    2012-06-08

    The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS), the key enzymes in constructing terpene carbon skeletons. Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita). Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (-)-(E)-β-caryophyllene (MrTPS1), (+)-germacrene A (MrTPS3), (E)-β-ocimene (MrTPS4) and (-)-germacrene D (MrTPS5). A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (-)-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+)-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils.

  10. The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils

    Directory of Open Access Journals (Sweden)

    Irmisch Sandra

    2012-06-01

    Full Text Available Abstract Background The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS, the key enzymes in constructing terpene carbon skeletons. Results Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita. Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (−-(E-β-caryophyllene (MrTPS1, (+-germacrene A (MrTPS3, (E-β-ocimene (MrTPS4 and (−-germacrene D (MrTPS5. A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (−-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. Conclusions The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils.

  11. Gene expression profiles of inducible nitric oxide synthase and cytokines in Leishmania major-infected macrophage-like RAW 264.7 cells treated with gallic acid

    NARCIS (Netherlands)

    Radtke, O.A.; Kiderlen, A.F.; Kayser, Oliver; Kolodziej, H

    2004-01-01

    The effects of gallic acid on the gene expressions of inducible nitric oxide synthase (iNOS) and the cytokines interleukin (IL)-1, IL-10, IL-12, IL-18, TNF-alpha, and interferon (IFN)-gamma were investigated by reverse-transcription polymerase chain reaction (RT-PCR). The experiments were performed

  12. Aldosterone synthase gene polymorphism in alimentary obesity, metabolic syndrome components, some secondary forms of arterial hypertension, pathology of the adrenals glands core (literature review

    Directory of Open Access Journals (Sweden)

    S.N. Koval

    2017-08-01

    Full Text Available Hormonal factors of adrenal origin belong to the pathophysiological mechanisms of the formation and progression of arterial hypertension (AH and should be consi­dered while developing differentiated approaches to the treatment and prevention of hypertensive states, their primary, secondary and resistant forms. The first thing we should point up is aldosterone (AL, enzyme aldosterone synthase (AS, which takes a direct part in the formation of this hormone, as well as gene polymorphisms of AS, which have not only molecular genetic, but also differential diagnostic and therapeutic significance for secondary forms of arterial hypertension, abdominal obesity (AO, metabolic syndrome (MS, adrenal pathology and other endocrine disorders. AL is a steroid (mineralocorticoid hormone of the adrenal cortex, which is synthesized from cholesterol (CH, mainly in the glomerular zone of the adrenal glands, is released under the action of angiotensin II (A II and potassium ions (K+. AL acti­vity is mediated through the corresponding mineralocorticoid receptors (MKR. The particular importance in AH and MS development belongs to AL activation and MKR density in adipocytes, this phenomenon is accompanied by increased expression of pro-inflammatory cytokines, leptin, an adipogenic effect, and the inhibition of MCR activity is accompanied by increased production of adiponectin, which is more pronounced in patients with AH. Aldosterone synthase, a mitochondrial human enzyme encoded by the CYP11B2 gene (cytochrome P450, family 11, subfamily B, polypeptide 2 is located on the 8th chromosome. AS belongs to the superfamily of cytochrome P450 and regulates the synthesis of AL hormone. The CYP11B2 gene encodes the key enzyme for the synthesis of AL 18-hydroxylase. In scientific papers, single nucleotide polymorphism (SNP of AS gene is often studied, such as 5312T, Intron 2, Lys-173/Arg; T-344C, 3097 C/A. 227 SNP of the AS gene were identified in different

  13. Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.

    Science.gov (United States)

    Tirakarn, Srisuda; Riangrungroj, Pinpunya; Kongsaeree, Palangpon; Imwong, Mallika; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2012-06-01

    Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Germacrene A Synthase in Yarrow (Achillea millefolium Is an Enzyme with Mixed Substrate Specificity: Gene Cloning, Functional Characterization and Expression Analysis

    Directory of Open Access Journals (Sweden)

    Leila ePazouki

    2015-03-01

    Full Text Available Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5 residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS. The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3, functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP, while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP. Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

  15. [Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum from three endemic malaria regions in Colombia].

    Science.gov (United States)

    Galindo, John Alexander; Cristiano, Fabio Aníbal; Knudson, Angélica; Nicholls, Rubén Santiago; Guerra, Angela Patricia

    2010-01-01

    Plasmodium falciparum has the ability to counter the antiparasitic activity of sulphadoxine-pyrimethamine by progressively accumulating mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. These mutations gradually increase the resistance of the parasite to these drugs and lead to therapeutic failure. To determine the frequency of mutations associated with resistance to sulphadoxine and pyrimethamine in the dhfr and dhps genes of P. falciparum in samples from patients in three endemic zones of Colombia -La Carpa, Guaviare; Casuarito, Vichada; and Tierralta and Puerto Libertador, Córdoba. Forty samples were selected from patients with uncomplicated P. falciparum malaria. The frequency profiles of the 108, 59 and 164 alleles of dhfr were obtained by application of an allele-specific polymerase chain reaction, whereas the other alleles (alleles 51 of the dhfr gene and 436, 437 and 540 of dhps) were obtained by polymerase chain reaction and restriction fragment length polymorphism. The 108N and 51I mutations in the dhfr gene were found in all of the 40 samples. No mutant alleles were found in the 59 and 164 codons of the dhfr gene, or in the 436 codon of the dhps gene. The 437G mutation was observed in 36 samples and the wild-type allele was present in 3 from Tierralta and one from La Carpa. The 540E mutation was only detected in two samples from Casuarito. The 108N, 51I and 437G mutations prevail in the populations of P. falciparum, indicating a cumulative effect of mutations and the need to continue surveillance for other changes which can lead to the total loss of the efficacy of sulphadoxine-pyrimethamine.

  16. Interleukin-18 gene promoter polymorphisms and recurrent spontaneous abortion.

    Science.gov (United States)

    Naeimi, Sirous; Ghiam, Alireza Fotouhi; Mojtahedi, Zahra; Dehaghani, Alamtaj Samsami; Amani, Dawar; Ghaderi, Abbas

    2006-01-01

    IL-18 is a multifunctional cytokine capable of inducing either Th1 or Th2 polarization depending on the immunologic milieu. IL-18 is detected at the materno-fetal interface very soon in early pregnancy. Two polymorphisms in the promoter region of the IL-18 gene at positions of -607 and -137 appear to have functional impacts. This study attempts to evaluate the frequency of these two polymorphisms in the IL-18 gene promoter in patients with recurrent spontaneous abortion (RSA) and normal pregnant women. One hundred and two RSA patients and 103 healthy pregnant women were enrolled in this study. Single nucleotide polymorphisms of the IL-18 gene at positions -607 (C/A) and -137 (G/C) were analyzed by the sequence-specific PCR method. There was no significant association between the allele, genotype, and haplotype frequencies of the two single nucleotide polymorphisms (SNPs) in the IL-18 gene promoter and RSA. The results of this study showed that IL-18 gene promoter polymorphisms at positions -607 and -137 did not confer susceptibility to RSA in southern Iranian patients.

  17. Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana.

    Science.gov (United States)

    Yang, Xiao; Beres, Zachery T; Jin, Lin; Parrish, Jason T; Zhao, Wanying; Mackey, David; Snow, Allison A

    2017-01-01

    Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide's target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines.

  18. Citrate-release-mediated aluminum resistance is coupled to the inducible expression of mitochondrial citrate synthase gene in Paraserianthes falcataria.

    Science.gov (United States)

    Osawa, Hiroki; Kojima, Katsumi

    2006-05-01

    Aluminum (Al) resistance in some leguminous plants is achieved by enhanced citrate release from roots. Enhancement requires several hours for complete activation and is postulated to involve Al-responsive genes or components. We examined the mechanism of Al-induced citrate release by studying the relationship between citrate release and expression of the mitochondrial citrate synthase (mCS) gene in three leguminous trees. Root elongation in Leucaena leucocephala (Lam.) de Wit was arrested within 24 h by 30 microM Al, whereas root elongation in Paraserianthes falcataria (L.) Neilson and Acacia mangium Willd. was inhibited mangium maintained enhanced release and accumulation of citrate for at least 28 days in response to Al treatment. Aluminum increased the accumulation of mCS transcripts in P. falcataria roots, but not in L. leucocephala roots, and thus up-regulation decreased following removal of Al. Lanthanum did not alter the expression level of mCS. Aluminum increased mCS activity concomitantly with enhanced mCS gene expression in P. falcataria, whereas it did not affect mCS activity in L. leucocephala. Aluminum content in root apices of P. falcataria was increased by cycloheximide, supporting the idea that de novo synthesis of proteins is a prerequisite for Al resistance. Our findings suggest that Al-inducible expression of mCS coupled with enhanced citrate release mediates Al resistance in P. falcataria.

  19. Negative feedback regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by heme oxygenase-1 induction in macrophages.

    Science.gov (United States)

    Ashino, Takashi; Yamanaka, Rieko; Yamamoto, Masayuki; Shimokawa, Hiroaki; Sekikawa, Kenji; Iwakura, Yoichiro; Shioda, Seiji; Numazawa, Satoshi; Yoshida, Takemi

    2008-04-01

    Heme oxygenase-1 (HO-1) is induced under infectious diseases in macrophages. We performed experiments using various gene deficient mouse-derived macrophages to determine a detailed induction mechanism of HO-1 by lipopolysaccharide (LPS) and the functional role of HO-1 induction in macrophages. LPS (1 microg/mL) maximally induced inducible nitric oxide synthase (iNOS) and HO-1 mRNAs in wild-type (WT) macrophages at 6h and 12h after treatment, respectively, and liberated tumor necrosis factor alpha (TNFalpha) from WT macrophages. LPS also induced iNOS and HO-1 in TNFalpha(-/-) macrophages, but not in iNOS(-/-) macrophages. Interestingly, although LPS strongly induced iNOS, it failed to induce HO-1 almost completely in nuclear-factor erythroid 2-related factor 2 (Nrf2)(-/-) macrophages. The LPS-induced iNOS gene expression was suppressed by pretreatment with HO-1 inducers, hemin and Co-protoporphyrin (CoPP), but not with HO-1 inhibitor, Sn-protoporphyrin in WT macrophages. In the Nrf2(-/-) macrophages, the ability of CoPP to induce HO-1 and its inhibitory effect on the LPS-induced iNOS gene expression were lower than seen in WT macrophages. The present findings suggest that HO-1 is induced via NO-induced nuclear translocation of Nrf2, and the enzymatic function of HO-1 inhibits the overproduction of NO in macrophages.

  20. Molecular cloning and characterization of two β-ketoacyl-acyl carrier protein synthase I genes from Jatropha curcas L.

    Science.gov (United States)

    Xiong, Wangdan; Wei, Qian; Wu, Pingzhi; Zhang, Sheng; Li, Jun; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2017-07-01

    The β-ketoacyl-acyl carrier protein synthase I (KASI) is involved in de novo fatty acid biosynthesis in many organisms. Two putative KASI genes, JcKASI-1 and JcKASI-2, were isolated from Jatropha curcas. The deduced amino acid sequences of JcKASI-1 and JcKASI-2 exhibit around 83.8% and 72.5% sequence identities with AtKASI, respectively, and both contain conserved Cys-His-Lys-His-Phe catalytic active sites. Phylogenetic analysis indicated that JcKASI-2 belongs to a clade with several KASI proteins from dicotyledonous plants. Both JcKASI genes were expressed in multiple tissues, most strongly in filling stage seeds of J. curcas. Additionally, the JcKASI-1 and JcKASI-2 proteins were both localized to the plastids. Expressing JcKASI-1 in the Arabidopsis kasI mutant rescued the mutant's phenotype and restored the fatty acid composition and oil content in seeds to wild-type, but expressing JcKASI-2 in the Arabidopsis kasI mutant resulted in only partial rescue. This implies that JcKASI-1 and JcKASI-2 exhibit partial functional redundancy and KASI genes play a universal role in regulating fatty acid biosynthesis, growth, and development in plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

  1. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  2. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  3. Hemorrhagic Shock-Induced Vascular Hyporeactivity in the Rat: Relationship to Gene Expression of Nitric Oxide Synthase, Endothelin-1, and Select Cytokines in Corresponding Organs

    Science.gov (United States)

    2005-01-01

    Parvathaneni, L. S., Bourlier, V., Sauter, C., Laubach, V. E., and Cobb, J. P. iNOS gene expression modu- lates microvascular responsiveness in endotoxin...Burtrum, D., and Silerstein, F. S. Cerebral hypoxia-ischemia stimulates cytokine gene expression in peri- natal rats. Stroke 26: 1093, 1995. 22...Mattson, D. L., and Wu, F. Nitric oxide synthase activity and isoforms in rat renal vasculature. Hypertension 35: 337, 2000. 23. Thiemermann, C., Szabó, C

  4. Sex-specific differences in natriuretic peptide and nitric oxide synthase expression in ANP gene-disrupted mice.

    Science.gov (United States)

    Wong, Philip G; Armstrong, David W J; Tse, M Yat; Brander, Emily P A; Pang, Stephen C

    2013-02-01

    Sex-specific differences in hormone-mediated gene regulation may influence susceptibility to cardiac hypertrophy, a primary risk factor for cardiovascular disease. Under hormonal influence, natriuretic peptide (NP) and nitric oxide synthase (NOS) systems modulate cardio-protective gene programs through common downstream production of cyclic guanosine 3'-5' monophosphate (cGMP). Ablation of either system can adversely affect cardiac adaptation to stresses and insults. This study elucidates sex-specific differences in cardiac NP and NOS system gene expression and assesses the impact of the estrous cycle on these systems using the atrial natriuretic peptide gene-disrupted (ANP(-/-)) mouse model. Left ventricular expression of the NP and NOS systems was analyzed using real-time quantitative polymerase chain reaction in 13- to 16-week-old male, proestrous and estrous female ANP(+/+) and ANP(-/-) mice. Left ventricular and plasma cGMP levels were measured to assess the convergent downstream effects of the NP and NOS systems. Regardless of genotype, males had higher expression of the NP system while females had higher expression of the NOS system. In females, transition from proestrus to estrus lowered NOS system expression in ANP(+/+) mice while the opposite was observed in ANP(-/-) mice. No significant changes in left ventricular cGMP levels across gender and genotype were observed. Significantly lower plasma cGMP levels were observed in ANP(-/-) mice compared to ANP(+/+) mice. Regardless of genotype, sex-specific differences in cardiac NP and NOS system expression exist, each sex enlisting a predominant system to conserve downstream cGMP. Estrous cycle-mediated alterations in NOS system expression suggests additional hormone-mediated gene regulation in females.

  5. Endothelial nitric oxide synthase gene intron 4, 27 bp repeat polymorphism and essential hypertension in the Kazakh Chinese population.

    Science.gov (United States)

    Deng, Fengmei; Hu, Qinghua; Tang, Bin; He, Fang; Guo, Shuxia; Chen, Jiang; Li, Feng; Wu, Xuehua; Zhang, Jun; Zhang, Huimin; Zhao, Juan; Zhong, Hua; He, Ling; Li, Jun; Zhang, Le; Wang, Shuren

    2007-05-01

    To investigate the relationship between 27 bp repeat polymorphism in intron 4 in the endothelial nitric oxide synthase (eNOS4) gene and essential hypertension in the Kazakh Chinese population, 151 patients with essential hypertension and 138 healthy people were selected from the Boertonggu countryside of Shawan region in the Xinjiang uygur autonomous region of China in 2006. The polymorphism of eNOS in the two groups was detected with polymerase chain reaction assays and the genotype frequencies in each group were calculated following the Hardy-Weinberg law. Four and five tandem 27 bp repeats were designated as "a" and "b", respectively. It was found that the frequencies of b/b, b/a and a/a genotypes of the eNOS4 gene were 84.06%, 15.22% and 0.72% in the control group, and 81.46%, 15.89% and 2.65% in the hypertension group, respectively. The frequencies of gene "a" and "b" were 91.67% and 8.33% in the control group and 89.40% and 10.60% in the hypertension group, respectively. It was found that plasma eNOS activity was not associated with genotypes and alleles of eNOS gene. Plasma eNOS activity in the hypertension group was significantly decreased compared with the control group (P<0.01). The results suggest that eNOS4 gene polymorphisms are unlikely to be the major genetic susceptibility factors for essential hypertension in the Xinjiang Kazakh population. However, a positive association between plasma eNOS activity and essential hypertension has been revealed.

  6. Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum.

    Science.gov (United States)

    Matsui, K; Miwa, K; Sano, K

    1987-01-01

    A 5-fluorotryptophan-resistant mutant, termed 1041, was isolated from Brevibacterium lactofermentum AJ12036. The anthranilate synthase of 1041 was insensitive to feedback inhibition by tryptophan, and the specific activities of the anthranilate synthase and anthranilate phosphoribosyltransferase of 1041 were 29- and 23-fold higher than those in parental strain AJ12036, respectively. A single-base change (adenine to cytosine) that resulted in a Ser-to-Arg substitution was found in the trpE structural gene of 1041. This substitution was identified as the cause of the desensitization to feedback inhibition by tryptophan of anthranilate synthase in 1041. Another substitution (guanine to adenine) was found at a position in which a mutation would destabilize the rho-independent terminator structure within the putative attenuator. The enhanced synthesis of tryptophan enzymes in 1041 could be caused by this substitution in the attenuator. PMID:3667535

  7. Cobalamin-independent methionine synthase (MetE: a face-to-face double barrel that evolved by gene duplication.

    Directory of Open Access Journals (Sweden)

    Robert Pejchal

    2005-02-01

    Full Text Available Cobalamin-independent methionine synthase (MetE catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH, both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (betaalpha(8 barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys(3Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E.Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  8. Gene identification and functional analysis of methylcitrate synthase in citric acid-producing Aspergillus niger WU-2223L.

    Science.gov (United States)

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2013-01-01

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

  9. Molecular cloning and expression profile of β-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.

    Directory of Open Access Journals (Sweden)

    Long Hongxu

    2015-01-01

    Full Text Available Tung tree (Vernicia fordii is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole α-eleostearic acid (9 cis, 11 trans, 13 trans octadecatrienoic acid. Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a multienzyme complex including β-ketoacyl-acyl-carrier-protein synthase (KAS. Little is known about KAS in tung tree. The objective of this study was to clone KAS genes and analyze their expression profiles in tung tree. A full-length cDNA encoding KAS III and a partial cDNA encoding KAS II were isolated from tung tree by PCR cloning using degenerate primers and rapid amplification of cDNA ends system. The full-length cDNA of VfKAS III was 1881 bp in length with an open reading frame of 1212 bp. VfKAS III genomic DNA was also isolated and sequenced, which contained 8 exons in 5403 bp length. The deduced VfKAS III protein shared approximately 80% identity with homologous KAS IIIs from other plants. Quantitative PCR analysis revealed that KAS II and KAS III were expressed in all of the tissues and organs tested but exhibited different expression patterns in tung tree. The expression levels of KAS II in young tissues were much lower than those in mature tissues, whereas the highest expression levels of KAS III were observed in young stem and young leaf. These results should facilitate further studies on the regulation of tung oil biosynthesis by KAS in tung tree.

  10. Cobalamin-Independent Methionine Synthase (MetE): A Face-to-Face Double Barrel that Evolved by Gene Duplication

    Energy Technology Data Exchange (ETDEWEB)

    Pejcha, Robert; Ludwig, Martha L. (Michigan)

    2010-03-08

    Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two ({beta}{alpha}){sub 8} barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys){sub 3}Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E {center_dot} Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

  11. Tissue-specific production of limonene in Camelina sativa with the Arabidopsis promoters of genes BANYULS and FRUITFULL.

    Science.gov (United States)

    Borghi, Monica; Xie, De-Yu

    2016-02-01

    Arabidopsis promoters of genes BANYULS and FRUITFULL are transcribed in Camelina. They triggered the transcription of limonene synthase and induced higher limonene production in seeds and fruits than CaMV 35S promoter. Camelina sativa (Camelina) is an oilseed crop of relevance for the production of biofuels and the plant has been target of a recent and intense program of genetic manipulation aimed to increase performance, seed yield and to modify the fatty acid composition of the oil. Here, we have explored the performance of two Arabidopsis thaliana (Arabidopsis) promoters in triggering transgene expression in Camelina. The promoters of two genes BANYULS (AtBAN pro ) and FRUITFULL (AtFUL pro ), which are expressed in seed coat and valves of Arabidopsis, respectively, have been chosen to induce the expression of limonene synthase (LS) from Citrus limon. In addition, the constitutive CaMV 35S promoter was utilized to overexpress LS in Camelina . The results of experiments revealed that AtBAN pro and AtFUL pro are actively transcribed in Camelina where they also retain specificity of expression in seeds and valves as previously observed in Arabidopsis. LS induced by AtBAN pro and AtFUL pro leads to higher limonene production in seeds and fruits than when the CaMV 35S was used to trigger the expression. In conclusion, the results of experiments indicate that AtBAN pro and AtFUL pro can be successfully utilized to induce the expression of the transgenes of interest in seeds and fruits of Camelina.

  12. Interleukin 10 gene promoter polymorphism and risk of diffuse large ...

    African Journals Online (AJOL)

    Roba M. Talaat

    2013-10-09

    Oct 9, 2013 ... Abstract. Purpose: Given the importance of understanding the genetic variations involved in the pathogen- esis of non-Hodgkin's lymphoma (NHL), this work was designed to study the impact of IL-10. (А1082 G/A; rs1800896 and А819 C/T; rs1800871) gene promoter polymorphism on susceptibility.

  13. Interleukin 10 gene promoter polymorphism and risk of diffuse large ...

    African Journals Online (AJOL)

    Purpose: Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin's lymphoma (NHL), this work was designed to study the impact of IL-10 (1082 G/A; rs1800896 and 819 C/T; rs1800871) gene promoter polymorphism on susceptibility of Egyptians to diffuse large B cell ...

  14. Cloning and analysis of the ascorbate peroxidase gene promoter ...

    African Journals Online (AJOL)

    enoh

    2012-03-22

    Mar 22, 2012 ... (Promega) according to the manufacturer's instructions. The actual value was normalized by protein content of each sample and expressed as the relative luminescence units (RLU)/mg of protein. Analysis of BnAPX gene promoter by light. To understand whether BnAPX contribute to photoprotection or not,.

  15. IL6 gene promoter polymorphisms and type 2 diabetes

    DEFF Research Database (Denmark)

    Huth, Cornelia; Heid, Iris M; Vollmert, Caren

    2006-01-01

    Several lines of evidence indicate a causal role of the cytokine interleukin (IL)-6 in the development of type 2 diabetes in humans. Two common polymorphisms in the promoter of the IL-6 encoding gene IL6, -174G>C (rs1800795) and -573G>C (rs1800796), have been investigated for association with typ...

  16. DNA methylation of PTEN gene promoter region is not correlated ...

    African Journals Online (AJOL)

    Yomi

    2012-02-23

    Feb 23, 2012 ... Tumor suppressor gene PTEN plays an important role in cell cycle. Disorder of PTEN protein can cause cell growth and division in an uncontrolled way, which can lead to the formation of tumors. It has been proven that epigenetic mechanisms, such as promoter hypermethylation, may account for ...

  17. DNA methylation of PTEN gene promoter region is not correlated ...

    African Journals Online (AJOL)

    Tumor suppressor gene PTEN plays an important role in cell cycle. Disorder of PTEN protein can cause cell growth and division in an uncontrolled way, which can lead to the formation of tumors. It has been proven that epigenetic mechanisms, such as promoter hypermethylation, may account for inactivation of PTEN in a ...

  18. Chemical analysis of a genome wide polyketide synthase gene deletion library in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Larsen, Thomas Ostenfeld; Klejnstrup, Marie Louise; Nielsen, Jakob Blæsbjerg

    type strains by culturing on different complex media to provoke induction of the secondary metabolism; ii) over expression of transcription factors encoding genes that are present in PKS gene clusters; iii) modification of chromatin structure regulation by knock out of histone H3 lysine methylation; iv...

  19. Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Kentaro Wakasa

    Full Text Available Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

  20. Genes encoding chavicol/eugenol synthase from the creosote bush Larrea tridentata

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Norman G.; Davin, Laurence B.; Kim, Sung -Jin; Vassao, Daniel Giddings; Patten, Ann M.; Eichinger, Dietmar

    2015-09-15

    Particular aspects provide novel methods for redirecting carbon allocation in plants or cell culture from lignification to inherently more useful and tractable materials, and to facilitate the generation of, e.g., biofuels from the remaining plant ro culture biomass. Particular aspects provided novel methods for converting monolignols into allyl/propenyl phenols, and for chavicol/eugenol formation or production. Additional aspects relate to the discovery of novel chavicol/eugenol synthases that convert p-coumaryl/coniferyl alcohol esters into chavicol/eugenol, and to novel compositions (e.g., novel proteins and nucleic acids encoding same), and novel methods using same for producing or forming chavicol/eugenol and other derivatives in cell culture and/or genetically modified plants, and for re-engineering the composition of plant biomass. Particular aspects provide novel methods for generation in culture or in planta of liquid/combustible allyl/propenyl phenols, and these phenolic products are utilized for (non-ethanol) biofuel/bioenergy purposes, while the remaining plant biomass facilitates the generation of other biofuels.

  1. In silico identification and analysis of phytoene synthase genes in plants.

    Science.gov (United States)

    Han, Y; Zheng, Q S; Wei, Y P; Chen, J; Liu, R; Wan, H J

    2015-08-14

    In this study, we examined phytoene synthetase (PSY), the first key limiting enzyme in the synthesis of carotenoids and catalyzing the formation of geranylgeranyl pyrophosphate in terpenoid biosynthesis. We used known amino acid sequences of the PSY gene in tomato plants to conduct a genome-wide search and identify putative candidates in 34 sequenced plants. A total of 101 homologous genes were identified. Phylogenetic analysis revealed that PSY evolved independently in algae as well as monocotyledonous and dicotyledonous plants. Our results showed that the amino acid structures exhibited 5 motifs (motifs 1 to 5) in algae and those in higher plants were highly conserved. The PSY gene structures showed that the number of intron in algae varied widely, while the number of introns in higher plants was 4 to 5. Identification of PSY genes in plants and the analysis of the gene structure may provide a theoretical basis for studying evolutionary relationships in future analyses.

  2. Epigenomic elements enriched in the promoters of autoimmunity susceptibility genes.

    Science.gov (United States)

    Dozmorov, Mikhail G; Wren, Jonathan D; Alarcón-Riquelme, Marta E

    2014-02-01

    Genome-wide association studies have identified a number of autoimmune disease-susceptibility genes. Whether or not these loci share any regulatory or functional elements, however, is an open question. Finding such common regulators is of considerable research interest in order to define systemic therapeutic targets. The growing amount of experimental genomic annotations, particularly those from the ENCODE project, provide a wealth of opportunities to search for such commonalities. We hypothesized that regulatory commonalities might not only delineate a regulatory landscape predisposing to autoimmune diseases, but also define functional elements distinguishing specific diseases. We further investigated if, and how, disease-specific epigenomic elements can identify novel genes yet to be associated with the diseases. We evaluated transcription factors, histone modifications, and chromatin state data obtained from the ENCODE project for statistically significant over- or under-representation in the promoters of genes associated with Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), and Systemic Sclerosis (SSc). We identified BATF, BCL11A, IRF4, NFkB, PAX5, and PU.1 as transcription factors over-represented in SLE- and RA-susceptibility gene promoters. H3K4me1 and H3K4me2 epigenomic marks were associated with SLE susceptibility genes, and H3K9me3 was common to both SLE and RA. In contrast to a transcriptionally active signature in SLE and RA, SSc-susceptibility genes were depleted in activating epigenomic elements. Using epigenomic elements enriched in SLE and RA, we identified additional immune and B cell signaling-related genes with the same elements in their promoters. Our analysis suggests common and disease-specific epigenomic elements that may define novel therapeutic targets for controlling aberrant activation of autoimmune susceptibility genes.

  3. Gene promoter hypermethylation in leukoplakia of the oral mucosa

    Directory of Open Access Journals (Sweden)

    Mingli Liu

    2010-07-01

    Full Text Available Mingli Liu1, Lei Feng2, Ximing Tang3, Shanchun Guo41Department of Physics, Tufts University School of Medicine, Boston, Massachussetts; 2Department of Thoracic/Head and Neck Medical Oncology, 3Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4Sylvester Cancer Center, University of Miami School of Medicine, Florida, USAAbstract: To examine whether aberrant DNA methylation in the promoter region might occur earlier in tumorigenesis, particularly in premalignant lesions, we examined biopsies from 111 participants in a chemoprevention trial aimed at reversal of oral leukoplakia, using methylation-specific polymerase chain reaction for the promoter regions of the tumor suppressor gene CDKN2A (p16, the putative metastasis suppressor gene for death-associated protein kinase (DAP-K, the DNA repair gene O6-methyguanine-DNA-methyltransferase (MGMT, and the detoxification gene glutathione S-transferase p1(GSTP1. p16 promoter hypermethylation was detected in 21 of 82 (25.6%, DAP-K hypermethylation in 28 of 87 (32.2%, and MGMT hypermethylation in 32 of 106 (30.2% oral leukoplakia lesions analyzed. No aberrant methylation was found at the GSTP1 gene in 110 lesions examined. Among 68 biopsies analyzed for all three genes (p16, DAP-K, MGMT, 17 biopsies were detected with an abnormal methylation pattern at only one gene, 15 at two genes, and 8 at all three genes. Among clinical characteristics and their correlation with methylation, only alcohol consumption was correlated with DAP-K methylation (P = 0.027, while MGMT methylation was more frequent in females (P = 0.003 and nonsmokers (P = 0.0005. A significant correlation was found between p16 and DAP-K hypermethylation; p16 promoter was methylated in 14 (56% of 25 lesions with DAP-K methylation, and only 5 (11.1% of 45 DAP-K methylation-negative lesions (P = 0.0001. DAP-K aberrant methylation was also significantly correlated with MGMT methylation (16 of 31 in MGMT methylation

  4. Phylogenetically distinct cellulose synthase genes support secondary wall thickening in arabidopsis shoot trichomes and cotton fiber.

    Science.gov (United States)

    Betancur, Lissete; Singh, Bir; Rapp, Ryan A; Wendel, Jonathan F; Marks, M David; Roberts, Alison W; Haigler, Candace H

    2010-02-01

    Abstract Through exploring potential analogies between cotton seed trichomes (or cotton fiber) and arabidopsis shoot trichomes we discovered that CesAs from either the primary or secondary wall phylogenetic clades can support secondary wall thickening. CesA genes that typically support primary wall synthesis, AtCesA1,2,3,5, and 6, underpin expansion and secondary wall thickening of arabidopsis shoot trichomes. In contrast, apparent orthologs of CesA genes that support secondary wall synthesis in arabidopsis xylem, AtCesA4,7, and 8, are up-regulated for cotton fiber secondary wall deposition. These conclusions arose from: (a) analyzing the expression of CesA genes in arabidopsis shoot trichomes; (b) observing birefringent secondary walls in arabidopsis shoot trichomes with mutations in AtCesA4, 7, or 8; (c) assaying up-regulated genes during different stages of cotton fiber development; and (d) comparing genes that were co-expressed with primary or secondary wall CesAs in arabidopsis with genes up-regulated in arabidopsis trichomes, arabidopsis secondary xylem, or cotton fiber during primary or secondary wall deposition. Cumulatively, the data show that: (a) the xylem of arabidopsis provides the best model for secondary wall cellulose synthesis in cotton fiber; and (b) CesA genes within a "cell wall toolbox" are used in diverse ways for the construction of particular specialized cell walls.

  5. Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis.

    Science.gov (United States)

    Ma, Shisong; Snyder, Michael; Dinesh-Kumar, Savithramma P

    2017-07-17

    Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

  6. Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

    Directory of Open Access Journals (Sweden)

    Wei Lei

    2010-01-01

    Full Text Available A cDNA encoding chalcone synthase (CHS, the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE. The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767, was 1649 bp with a 1170 bp open reading frame (ORF that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.

  7. DNA sequence and expression variation of hop (Humulus lupulus) valerophenone synthase (VPS), a key gene in bitter acid biosynthesis.

    Science.gov (United States)

    Castro, Consuelo B; Whittock, Lucy D; Whittock, Simon P; Leggett, Grey; Koutoulis, Anthony

    2008-08-01

    The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, 'Symphony' and 'Ember', which typically have high bitter acid levels. In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development.

  8. 2C-Methyl- D- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene.

    Science.gov (United States)

    Kumar, Hitesh; Singh, Kashmir; Kumar, Sanjay

    2012-12-01

    Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5' diphospho)-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100 μM) down-regulated the expression of SrMDS at 4 h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.

  9. Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1

    Directory of Open Access Journals (Sweden)

    Liuqing Wang

    2015-07-01

    Full Text Available Ochratoxin A (OTA, a potentially carcinogenic mycotoxin which contaminates grains, is produced by several Aspergillus species. A comparative sequence analysis of the OTA-producing Aspergillus ochraceus fc-1 strain and other Aspergillus species was performed. Two new OTA-related polyketide synthase (PKS (AoOTApks genes were identified. The predicted amino acid sequence of AoOTApks-1 displayed high similarity to previously identified PKSs from OTA-producing A. carbonarius ITEM 5010 (67%; [PI] No. 173482 and A. niger CBS 513.88 (62%; XP_001397313. However, the predicted amino acid sequence of AoOTApks-2 displayed lower homology with A. niger CBS 513.88 (38% and A. carbonarius ITEM 5010 (28%. A phylogenetic analysis of the β-ketosynthase and acyl-transferase domains of the AoOTApks proteins indicated that they shared a common origin with other OTA-producing species, such as A. carbonarius, A. niger, and A. westerdijkiae. A real-time reverse-transcription PCR analysis showed that the expression of AoOTApks-1 and -2 was positively correlated with the OTA concentration. The pks gene deleted mutants ∆AoOTApks-1 and ∆AoOTApks-2 produced nil and lesser OTA than the wild-type strain, respectively. Our study suggests that AoOTApks-1 could be involved in OTA biosynthesis, while AoOTApks-2 might be indirectly involved in OTA production.

  10. Gene transfer of neuronal nitric oxide synthase to carotid body reverses enhanced chemoreceptor function in heart failure rabbits.

    Science.gov (United States)

    Li, Yu-Long; Li, Yi-Fan; Liu, Dongmei; Cornish, Kurtis G; Patel, Kaushik P; Zucker, Irving H; Channon, Keith M; Schultz, Harold D

    2005-08-05

    Our previous studies showed that decreased nitric oxide (NO) production enhanced carotid body (CB) chemoreceptor activity in chronic heart failure (CHF) rabbits. In the present study, we investigated the effects of neuronal NO synthase (nNOS) gene transfer on CB chemoreceptor activity in CHF rabbits. The nNOS protein expression and NO production were suppressed in CBs (Pchemoreceptors in CHF versus sham rabbits. Ad.nNOS decreased the baseline discharge (4.5+/-0.3 versus 7.3+/-0.4 imp/s at 105+/-1.9 mm Hg) and the response to hypoxia (18.3+/-1.2 imp/s versus 35.6+/-1.1 at 40+/-2.1 mm Hg) from CB chemoreceptors in CHF rabbits (Ad.nNOS CB versus contralateral noninfected CB respectively, Pactivity in CHF rabbits. In addition, nNOS gene transfer to the CBs also significantly blunted the baseline renal sympathetic nerve activity (RSNA) and the response of RSNA to hypoxia in CHF rabbits (Pactivity in the CB plays an important role in the enhanced activity of the CB chemoreceptors and peripheral chemoreflex function in CHF rabbits.

  11. The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules

    Science.gov (United States)

    Barraza, Aarón; Contreras-Cubas, Cecilia; Estrada-Navarrete, Georgina; Reyes, José L.; Juárez-Verdayes, Marco A.; Avonce, Nelson; Quinto, Carmen; Díaz-Camino, Claudia; Sanchez, Federico

    2016-01-01

    Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant. PMID:27847509

  12. RNAi-mediated down-regulation of a melanin polyketide synthase (pks1) gene in the fungus Slafractonia leguminicola.

    Science.gov (United States)

    Alhawatema, Mohammad S; Gebril, Sayed; Cook, Daniel; Creamer, Rebecca

    2017-09-20

    The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.

  13. The Class II trehalose 6-phosphate synthase gene PvTPS9 modulates trehalose metabolism in Phaseolus vulgaris nodules.

    Directory of Open Access Journals (Sweden)

    Aarón Barraza

    2016-11-01

    Full Text Available Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS of common bean (Phaseolus vulgaris, was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1% of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

  14. Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    ML Rossi

    2009-06-01

    Full Text Available Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs and impaired bioavailabilty of nitric oxide (NO are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5’ nuclease assays (TaqMan™ PCRs to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA and 15 with acute coronary syndromes (ACS without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001 in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype.

  15. DHA Production in Escherichia coli by Expressing Reconstituted Key Genes of Polyketide Synthase Pathway from Marine Bacteria.

    Science.gov (United States)

    Peng, Yun-Feng; Chen, Wen-Chao; Xiao, Kang; Xu, Lin; Wang, Lian; Wan, Xia

    2016-01-01

    The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10-15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea.

  16. Overexpression of Populus×canescens isoprene synthase gene in Camelina sativa leads to alterations in its growth and metabolism.

    Science.gov (United States)

    Rossi, Lorenzo; Borghi, Monica; Yang, Jinfen; Xie, De-Yu

    2017-08-01

    Isoprene (2-methyl-1,3-butadiene) is a hemiterpene molecule. It has been estimated that the plant kingdom emits 500-750 million tons of isoprene in the environment, half of which results from tropical broadleaf trees and the remainder from shrubs. Camelina (Camelina sativa (L.) Crantz) is an emerging bioenergy plant for biodiesel. In this study, we characterized isoprene formation following a diurnal/nocturnal cycle in wild-type Camelina plants. To understand the potential effects of isoprene emission on this herbaceous plant, a gray poplar Populus×canescens isoprene synthase gene (PcISPS) was overexpressed in Camelina. Transgenic plants showed increased isoprene production, and the emissions were characterized by a diurnal/nocturnal cycle. Measurements of the expression of six genes of the plastidial 2-C-methyl-d-erythriol-4-phosphate (MEP) pathway revealed that the expression patterns of three key genes were associated with isoprene formation dynamics in the three genotypic plants. Conversely, dissimilar gene expression levels existed in different genotypes, indicating that dynamics and variations occurred among plants. Moreover, transgenic plants grew shorter and developed smaller leaves than the wild-type and empty vector control transgenic plants. Photosynthetic analysis showed that the CO2 assimilation rate, intracellular CO2 concentration, mesophyll conductance and contents of chlorophylls a and b were similar among PcISPS transgenic, empty-vector control transgenic, and wild-type plants, indicating that the transgene did not negatively affect photosynthesis. Based on these results, we suggest that the reduced biomass was likely a trade-off consequence of the increased isoprene emission. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Delineating the structural, functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses

    Directory of Open Access Journals (Sweden)

    Khalil Zaynali

    2010-06-01

    Full Text Available Abstract Background Sucrose phosphate synthase (SPS is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII. Results The expression in bread wheat over the course of development of various sucrose biosynthesis genes monitored on an Affymetrix array showed that the SPS genes were regulated over time and space. SPSII homoeologue-specific assays were used to show that the three homoeologues contributed differentially to the global expression of SPSII. Genetic mapping placed the set of homoeoloci on the short arms of the homoeologous group 3 chromosomes. A resequencing of the A and B genome copies allowed the detection of four haplotypes at each locus. The 3B copy includes an unspliced intron. A comparison of the sequences of the wheat SPSII orthologues present in the diploid progenitors einkorn, goatgrass and Triticum speltoides, as well as in the more distantly related species barley, rice, sorghum and purple false brome demonstrated that intronic sequence was less well conserved than exonic. Comparative sequence and phylogenetic analysis of SPSII gene showed that false purple brome was more similar to Triticeae than to rice. Wheat - rice synteny was found to be perturbed at the SPS region. Conclusion The homoeologue-specific assays will be suitable to derive associations between SPS functionality and key phenotypic traits. The amplicon sequences derived from the homoeologue-specific primers are informative regarding the evolution of SPSII in a polyploid context.

  18. The Lc3-synthase gene B3gnt5 is essential to pre-implantation development of the murine embryo

    Directory of Open Access Journals (Sweden)

    Cinelli Paolo

    2008-11-01

    Full Text Available Abstract Background Glycosphingolipids (GSL are integral components of mammalian cell membranes that are involved in cell adhesion and cell signaling processes. GSL are subdivided into structural series, like ganglio-, lacto/neolacto-, globo- and isoglo-series, which are defined by distinct trisaccharide cores. The β1,3 N-acetylglucosaminyltransferase-V (B3gnt5 enzyme catalyzes the formation of the Lc3 structure, which is the core of lactoseries derived GSL. Results The biological significance of the glycoconjugates produced by the B3gnt5 enzyme was investigated by inactivating the B3gnt5 gene in the mouse germline. The disruption of the B3gnt5 protein-coding region in mouse embryonic stem cells resulted in reduced Lc3-synthase activity, supporting its specific contribution to lactoseries derived GSL synthesis. Breeding of heterozygous mutant mice failed to produce any viable progeny homozygous for the B3gnt5-null allele. The genotypic examination of embryos from heterozygous crosses showed that the disruption of the B3gnt5 gene leads to pre-implantation lethality. This finding was compatible with the expression pattern of the B3gnt5 gene in the pre-implantation embryo as shown by in situ hybridization. The analysis of GSL profiles in embryonic stem cells heterozygous for the B3gnt5-null allele confirmed the reduced levels of lactoseries derived GSL levels and of other GSL species. Conclusion The disruption of the B3gnt5 gene in mice affected the expression of lactoseries derived GLS and possibly of protein-bound β3GlcNAc-linked glycans, thereby demonstrating an essential contribution of these glycoconjugates in early embryonic development, and supporting the importance of these glycoconjugates in cell differentiation and adhesion processes.

  19. Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed

    Directory of Open Access Journals (Sweden)

    Michelle F. Valentine

    2017-05-01

    Full Text Available Soybean [Glycine max (L. Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2, to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets.

  20. Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters

    Directory of Open Access Journals (Sweden)

    Avinash Mishra

    2017-05-01

    Full Text Available Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile, and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters (NHX, SOS, HKT, VTPase, ion channels (Cl−, Ca2+, aquaporins, antioxidant encoding genes (APX, CAT, GST, BADH, SOD and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes. It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.

  1. Sense oligonucleotide competition for gene promoter binding and activation.

    Science.gov (United States)

    Cutroneo, Kenneth R; Chiu, Jen-Fu

    2003-01-01

    Considerable evidence has ensued on the importance of growth factors during regeneration both for cell replication and for stimulation of reparative cells to synthesize and secrete extracellular matrix components. During the healing process if the growth factor concentration is too high because of over-expression, abnormal wound healing and tissue fibrosis will occur. The growth factor concentration at the wound site may be controlled by gene therapy and the titration of gene dosage. However, if there is a narrow window between the beneficial effects and adverse effects of gene therapy, oligonucleotide approaches may be used concurrently with gene therapy to control growth factor concentration(s) at the wound site. Antisense oligos offer a method to control the concentration of growth factors at the level of translation. A novel method using sense oligos to the proalpha1 (I) collagen gene to inhibit gene transcription and collagen synthesis has recently been reported. The exogenous modified oligodeoxynucleotide competes with the cis-element (i.e. the transforming growth factor-beta (TGF-beta) element) in the distal 5'-flanking region of the proalpha1 (I) collagen gene for the trans-acting factor (i.e. the TGF-beta activator protein complex), thereby down regulating promoter activity of the proalpha1 (I) collagen gene and inhibiting type I collagen synthesis. The oligonucleotide approaches, both antisense and sense therapies, may be used to regulate over-expression of growth factors and thereby either eliminate or lessen the potential adverse effects of gene therapy.

  2. The comparative analysis of the potential relationship between resveratrol and stilbene synthase gene family in the development stages of grapes (Vitis quinquangularis and Vitis vinifera).

    Science.gov (United States)

    Shi, Jiangli; He, Mingyang; Cao, Jiangling; Wang, Huan; Ding, Jiahua; Jiao, Yuntong; Li, Ruimin; He, Jing; Wang, Dan; Wang, Yuejin

    2014-01-01

    Resveratrol is positively correlated with grapevine disease resistance and its consumption is also highly beneficial to human health. HPLC analyses showed that resveratrol content was significantly higher in most wild Chinese grapevines than in most European Vitis vinifera grapevine cvs. Fruit of the wild Chinese genotype Vitis quinquangularis Danfeng-2 contains much higher levels of resveratrol than some others. Because stilbene synthase is responsible for resveratrol biosynthesis, 41 full-length stilbene synthase genes were isolated from Danfeng-2 using the RACE method. A neighbor-joining tree of the STS family displayed high similarity between Danfeng-2 and V. vinifera cv. Pinot Noir. The content of the endogenous stilbene synthase family in tissues and the expression levels induced by powdery mildew were both higher in Danfeng-2 than in Pinot Noir. Moreover, expression in the berry was significantly higher than in the leaves. Our results demonstrated that resveratrol accumulation was consistent with endogenous STS gene expressions, and that both were higher in Danfeng-2 than in Pinot Noir. Therefore, STS genes and producing resveratrol from V. quinquangularis played more important role in Vitis resistance. Otherwise, the gene VqSTS6 was markedly higher than the other VqSTS genes in the six tissues/organs assayed by Real-time PCR, which will offer a useful basis for commercial application of resveratrol from Chinese wild grapes. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams).

    Science.gov (United States)

    Pua, Eng-Chong; Chandramouli, Sumana; Han, Ping; Liu, Pei

    2003-01-01

    Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.

  4. Chrysanthemum expressing a linalool synthase gene 'smells good', but 'tastes bad' to western flower thrips.

    Science.gov (United States)

    Yang, Ting; Stoopen, Geert; Thoen, Manus; Wiegers, Gerrie; Jongsma, Maarten A

    2013-09-01

    Herbivore-induced plant volatiles are often involved in direct and indirect plant defence against herbivores. Linalool is a common floral scent and found to be released from leaves by many plants after herbivore attack. In this study, a linalool/nerolidol synthase, FaNES1, was overexpressed in the plastids of chrysanthemum plants (Chrysanthemum morifolium). The volatiles of FaNES1 chrysanthemum leaves were strongly dominated by linalool, but they also emitted small amount of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. Four nonvolatile linalool glycosides in methanolic extracts were found to be significantly increased in the leaves of FaNES1 plants compared to wild-type plants. They were putatively identified by LC-MS-MS as two linalool-malonyl-hexoses, a linalool-pentose-hexose and a glycoside of hydroxy-linalool. A leaf-disc dual-choice assay with western flower thrips (WFT, Frankliniella occidentalis) showed, initially during the first 15 min of WFT release, that FaNES1 plants were significantly preferred. This gradually reversed into significant preference for the control, however, at 20-28 h after WFT release. The initial preference was shown to be based on the linalool odour of FaNES1 plants by olfactory dual-choice assays using paper discs emitting pure linalool at similar rates as leaf discs. The reversal of preference into deterrence could be explained by the initial nonvolatile composition of the FaNES1 plants, as methanolic extracts were less preferred by WFT. Considering the common occurrence of linalool and its glycosides in plant tissues, it suggests that plants may balance attractive fragrance with 'poor taste' using the same precursor compound. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter.

    Science.gov (United States)

    Wang, Zhi; Zhu, Yan; Wang, Lili; Liu, Xia; Liu, Yongxiu; Phillips, Jonathan; Deng, Xin

    2009-11-01

    Accumulation of compatible osmolytes, such as soluble sugars, in plants is an important osmoprotective mechanism. Sugars play a role in osmotic adjustment and are associated with stabilization of proteins and cell structures, reactive oxygen species scavenging, signaling functions or induction of adaptive pathways. Galactinol is the galactosyl donor for the synthesis of raffinose family oligosaccharides (RFOs) and its synthesis by galactinol synthase (GolS) is the first committed step of the RFOs biosynthetic pathway. GolS genes are induced by a variety of stresses in both stress-sensitive and tolerant-plant species; however, the mechanism of transcriptional regulation is not fully established. In this paper, we characterized a GolS gene (BhGolS1) that was dehydration and ABA-inducible in the resurrection plant Boea hygrometrica and conferred dehydration tolerance in a transgenic tobacco system. Four W-box cis-elements were identified in the BhGolS1 promoter and shown to be bound by an early dehydration and ABA-inducible WRKY gene (BhWRKY1). These data suggest a mechanism where BhWRKY1 is likely to function in an ABA-dependent signal pathway to regulate BhGolS1 expression, which leads to the accumulation of RFOs in desiccation-tolerant B. hygrometrica leaves.

  6. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  7. Polymorphisms in thymidylate synthase gene and susceptibility to breast cancer in a Chinese population: a case-control analysis

    Directory of Open Access Journals (Sweden)

    Liu Jiyong

    2006-05-01

    Full Text Available Abstract Background Accumulative evidence suggests that low folate intake is associated with increased risk of breast cancer. Polymorphisms in genes involved in folate metabolism may influence DNA methylation, nucleotide synthesis, and thus individual susceptibility to cancer. Thymidylate synthase (TYMS is a key enzyme that participates in folate metabolism and catalyzes the conversion of dUMP to dTMP in the process of DNA synthesis. Two potentially functional polymorphisms [a 28-bp tandem repeat in the TYMS 5'-untranslated enhanced region (TSER and a 6-bp deletion/insertion in the TYMS 3'-untranslated region (TS 3'-UTR] were suggested to be correlated with alteration of thymidylate synthase expression and associated with cancer risk. Methods To test the hypothesis that polymorphisms of the TYMS gene are associated with risk of breast cancer, we genotyped these two polymorphisms in a case-control study of 432 incident cases with invasive breast cancer and 473 cancer-free controls in a Chinese population. Results We found that the distribution of TS3'-UTR (1494del6 genotype frequencies were significantly different between the cases and controls (P = 0.026. Compared with the TS3'-UTR del6/del6 wild-type genotype, a significantly reduced risk was associated with the ins6/ins6 homozygous variant genotype (adjusted OR = 0.58, 95% CI = 0.35–0.97 but not the del6/ins6 genotype (OR = 1.09, 95% CI = 0.82–1.46. Furthermore, breast cancer risks associated with the TS3'-UTR del6/del6 genotype were more evident in older women, postmenopausal subjects, individuals with a younger age at first-live birth and individuals with an older age at menarche. However, there was no evidence for an association between the TSER polymorphism and breast cancer risks. Conclusion These findings suggest that the TS3'-UTR del6 polymorphism may play a role in the etiology of breast cancer. Further larger population-based studies as well as functional evaluation of the

  8. Integration of Physical, Genetic, and Cytogenetic Mapping Data for Cellulose Synthase (CesA Genes in Flax (Linum usitatissimum L.

    Directory of Open Access Journals (Sweden)

    Olga Y. Yurkevich

    2017-08-01

    Full Text Available Flax, Linum usitatissimum L., is a valuable multi-purpose plant, and currently, its genome is being extensively investigated. Nevertheless, mapping of genes in flax genome is still remaining a challenging task. The cellulose synthase (CesA multigene family involving in the process of cellulose synthesis is especially important for metabolism of this fiber crop. For the first time, fluorescent in situ hybridization (FISH-based chromosomal localization of the CesA conserved fragment (KF011584.1, 5S, and 26S rRNA genes was performed in landrace, oilseed, and fiber varieties of L. usitatissimum. Intraspecific polymorphism in chromosomal distribution of KF011584.1 and 5S DNA loci was revealed, and the generalized chromosome ideogram was constructed. Using BLAST analysis, available data on physical/genetic mapping and also whole-genome sequencing of flax, localization of KF011584.1, 45S, and 5S rRNA sequences on genomic scaffolds, and their anchoring to the genetic map were conducted. The alignment of the results of FISH and BLAST analyses indicated that KF011584.1 fragment revealed on chromosome 3 could be anchored to linkage group (LG 11. The common LG for 45S and 5S rDNA was not found probably due to the polymorphic localization of 5S rDNA on chromosome 1. Our findings indicate the complexity of integration of physical, genetic, and cytogenetic mapping data for multicopy gene families in plants. Nevertheless, the obtained results can be useful for future progress in constructing of integrated physical/genetic/cytological maps in L. usitatissimum which are essential for flax breeding.

  9. RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say).

    Science.gov (United States)

    Shi, Ji-Feng; Mu, Li-Li; Chen, Xu; Guo, Wen-Chao; Li, Guo-Qing

    2016-01-01

    Dietary introduction of bacterially expressed double-stranded RNA (dsRNA) has great potential for management of Leptinotarsa decemlineata. Identification of the most attractive candidate genes for RNA interference (RNAi) is the first step. In the present paper, three complete chitin synthase cDNA sequences (LdChSAa, LdChSAb and LdChSB) were cloned. LdChSAa and LdChSAb, two splicing variants of LdChSA gene, were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells. Feeding bacterially expressed dsChSA (derived from a common fragment of LdChSAa and LdChSAb), dsChSAa, dsChSAb and dsChSB in the second- and fourth-instar larvae specifically knocked down their target mRNAs. RNAi of LdChSAa+LdChSAb and LdChSAa lowered chitin contents in whole body and integument samples, and thinned tracheal taenidia. The resulting larvae failed to ecdyse, pupate, or emerge as adults. Comparably, knockdown of LdChSAb mainly affected pupal-adult molting. The LdChSAb RNAi pupae did not completely shed the old larval exuviae, which caused failure of adult emergence. In contrast, silencing of LdChSB significantly reduced foliage consumption, decreased chitin content in midgut sample, damaged midgut peritrophic matrix, and retarded larval growth. As a result, the development of the LdChSB RNAi hypomorphs was arrested. Our data reveal that these LdChSs are among the effective candidate genes for an RNAi-based control strategy against L. decemlineata.

  10. Prevalence of polymorphisms in glucose-6-phosphate dehydrogenase, sickle haemoglobin and nitric oxide synthase genes and their relationship with incidence of uncomplicated malaria in Iganga, Uganda.

    Science.gov (United States)

    Lwanira, Catherine Nassozi; Kironde, Fred; Kaddumukasa, Mark; Swedberg, Göte

    2017-08-09

    Host genetics play an important role in Plasmodium falciparum malaria susceptibility. However, information on host genetic factors and their relationships with malaria in the vaccine trial site of Iganga, Uganda is limited. The main objective of this study was to determine the prevalence of selected host genetic markers and their relationship to malaria incidence in the vaccine trial site of Iganga, Uganda. In a 1-year longitudinal cohort study, 423 children aged below 9 years were recruited and their malaria episodes were investigated. Host genetic polymorphisms were assessed by PCR-RFLP, haemoglobin electrophoresis and DNA sequencing. Using a multivariate negative binomial regression model, estimates of the impact of human genetic polymorphisms on malaria incidence were performed. In all statistical tests, a P value of sickle cell haemoglobin trait, G6PD c.202 G>A (rs 1050828) and NOS2 -954 G>C (rs 1800482) variants were 26.6, 22.7 and 17.3%, respectively. Inducible nitric oxide synthase 2 (NOS2 -954 G>C; rs 1800482) heterozygosity was associated with lower incidence of malaria in all age groups {Adjusted incident rates ratio (aIRR) 0.59; 95% CI [0.386-0.887]; P = 0.012)}. About 4% of study subjects had co-existence of sickle cell Hb trait and G6PD deficiency. Sickle cell Hb heterozygotes (Hb AS) aged less than 1 year experienced significantly more malaria episodes annually than children with normal haemoglobin (Hb AA) {aIRR = 1.98; 95% CI [1.240-3.175]; P = 0.004}. There was no significant influence of the sickle cell trait on malaria incidence among older children of 1-9 years. Mutation (NOS2 -954 G>C; rs 1800482) of nitric oxide synthase 2 gene promoter was associated with a lower incidence of acute malaria. The normal haemoglobin (wild genotype; HbAA) was associated with reduced malaria incidence rates during the first year of life. More understanding of the interplay between host genetics and malaria susceptibility is required.

  11. Organophosphate Pesticide Exposures, Nitric Oxide Synthase Gene Variants, and Gene-Pesticide Interactions in a Case-Control Study of Parkinson's Disease, California (USA).

    Science.gov (United States)

    Paul, Kimberly C; Sinsheimer, Janet S; Rhodes, Shannon L; Cockburn, Myles; Bronstein, Jeff; Ritz, Beate

    2016-05-01

    Nitric oxide synthase (NOS) genes are candidates for Parkinson's disease (PD) because NOS enzymes produce nitric oxide (NO), a pro-oxidant that can damage neurons. Widely used organophosphate (OP) pesticides can induce oxidative stress and are reported to increase PD risk. Additionally, two single nucleotide polymorphisms (SNPs) from the PON1 (paraoxonase 1) gene influence the ability to metabolize OPs. Here, we investigated contributions of NOS genes and OP pesticides to PD risk, controlling for PON1 status. In 357 incident PD cases and 495 population controls, we investigated eight NOS SNPs and interactions with both household and ambient agricultural OP exposures assessed with geographic information system (GIS). In comparing PD in homozygous variant carriers of NOS2A rs1060826 versus homozygous wild-type or heterozygotes, we estimate an adjusted odds ratio (OR) of 1.51 (95% CI: 0.95, 2.41). When considering interactions between NOS1 rs2682826 and OP exposure from household use, the OR for frequent OP use alone was 1.30 (95% CI: 0.72, 2.34) and for the CT+TT genotype alone was 0.89 (95% CI: 0.58, 1.39), and for frequent OP use combined with the CT+TT genotype the OR was 2.84 (95% CI: 1.49, 5.40) (interaction p-value 0.04). Similar results were seen for ambient OP exposure. Interactions between OP exposure and three other NOS1 SNPs and a genetic risk score combining all NOS1 SNPs reached statistical significance. We found that OP pesticides were more strongly associated with PD among participants with variant genotypes in NOS1, consistent with the importance of oxidative stress-inducing mechanisms. Our data provide evidence for NOS1 modifying PD risk in OP exposed populations. Paul KC, Sinsheimer JS, Rhodes SL, Cockburn M, Bronstein J, Ritz B. 2016. Organophosphate pesticide exposures, nitric oxide synthase gene variants, and gene-pesticide interactions in a case-control study of Parkinson's disease, California (USA). Environ Health Perspect 124:570-577;

  12. Cloning and characterization of ATP synthase CF1 α gene from ...

    African Journals Online (AJOL)

    ajl yemi

    2011-12-19

    Dec 19, 2011 ... et al., 2009a,b; Trapnell et al., 2009) at Galaxy's web platform allowing only one base mismatch. All clean tags corresponding to. atpA gene gave a raw expression level in different tissues, and then an empirical approach described by edgeR package (Robinson et al., 2010) was used to normalize the ...

  13. Citric acid production and citrate synthase genes in distinct strains of ...

    African Journals Online (AJOL)

    Citric acid is an important organic acid, multifunctional with a wide array of uses. The objectives of this study were the isolation and selection strains of the genus Aspergillus, investigating the solubilization of phosphate of these isolates, verifying the expression rate of genes involved in the identification of isolates, and ...

  14. Differential accumulation of β-carotene and tissue specific expression of phytoene synthase (MaPsy) gene in banana (Musa sp) cultivars.

    Science.gov (United States)

    Dhandapani, R; Singh, V P; Arora, A; Bhattacharya, R C; Rajendran, Ambika

    2017-12-01

    An experiment was conducted with twelve major Indian banana cultivars to investigate the molecular relationship between the differential accumulation of β-carotene in peel and pulp of the banana fruit and carotenoid biosynthetic pathway genes. The high performance liquid chromatography showed that all banana cultivars accumulated two-three fold more β-carotene in non-edible portion of the banana fruit. However, Nendran, a famous orange fleshed cultivar of South India, had high β-carotene content (1362 µg/100 g) in edible pulp. The gene encoding Musa accuminata phytoene synthase (MaPsy) was successfully amplified using a pair of degenerate primers designed from Oncidium orchid. The deduced amino acid sequences shared a high level of identity to phytoene synthase gene from other plants. Gene expression analysis confirmed the presence of two isoforms (MaPsy1 and MaPsy2) of MaPsy gene in banana fruits. Presence of two isoforms of MaPsy gene in peel and one in pulp confirmed the differential accumulation of β-carotene in banana fruits. However, Nendran accumulated more β-carotene in edible pulp due to presence of both the isoforms of MaPsy gene. Thus, carotenoid accumulation is a tissue specific process strongly dependent on differential expression pattern of two isoforms of MaPsy gene in banana.

  15. Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments.

    Science.gov (United States)

    Lanfranconi, Mariana P; Alvarez, Adrián F; Alvarez, Héctor M

    2015-12-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest.

  16. Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck).

    Science.gov (United States)

    Tao, Nengguo; Hu, Zhiyong; Liu, Qin; Xu, Juan; Cheng, Yunjiang; Guo, Linlin; Guo, Wenwu; Deng, Xiuxin

    2007-06-01

    Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of "Washington" navel orange and its red-fleshed mutant "Cara Cara". Results showed that phytoene was exclusively accumulated in peel and pulp of "Cara Cara". Although phytoene was observed accumulating with fruit ripening of "Cara Cara", the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in "Cara Cara" and "Washington" genomes. During "Cara Cara" and "Washington" fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of "Cara Cara" was higher than that in "Washington", as evidenced by phytoene accumulation in the peel.

  17. Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene.

    Science.gov (United States)

    Welsch, Ralf; Arango, Jacobo; Bär, Cornelia; Salazar, Bertha; Al-Babili, Salim; Beltrán, Jesús; Chavarriaga, Paul; Ceballos, Hernan; Tohme, Joe; Beyer, Peter

    2010-10-01

    Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

  18. Mitochondrially-Encoded Adenosine Triphosphate Synthase 6 Gene Haplotype Variation among World Population during 2003-2013

    Directory of Open Access Journals (Sweden)

    Steven Steven

    2016-08-01

    Full Text Available Background: Adaptation and natural selection serve as an important part of evolution. Adaptation in molecular level can lead to genetic drift which causes mutation of genetic material; one of which is polymorphism of mitochondrial DNA (mtDNA. The aim of this study is to verify the polymorphism of mitochondrially-encoded Adenosine Triphosphate synthase6gene (MT-ATP6 as one of mtDNA building blocks among tropic, sub-tropic, and polar areas. Methods: This descriptive quantitative research used 3,210 mtDNA sequences, taken from GenBank, as secondary data from 27 different populations. The data were grouped into 3 population groups based on the climates of their location. After grouping, the sequences were then aligned and trimmed using Unipro EUGENE, and analyzed by Arlequin and MitoTool. Results: Results demonstrated 21 haplotypes distributed among 3 populations with variations between each climate population. In the tropic and sub-tropic populations, the dominant haplotype is h1 while h6 is dominant in the polar population. Conclusions: There is a variation of haplotype polymorphism between tropic, sub-tropic, and polar climate population.

  19. Cloning and Characterisation of the Gene Encoding 3-Hydroxy-3-Methylglutaryl-CoA Synthase in Tripterygium wilfordii

    Directory of Open Access Journals (Sweden)

    Yu-Jia Liu

    2014-11-01

    Full Text Available Tripterygium wilfordii is a traditional Chinese medical plant used to treat rheumatoid arthritis and cancer. The main bioactive compounds of the plant are diterpenoids and triterpenoids. 3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS catalyses the reaction of acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, which is the first committed enzyme in the mevalonate (MVA pathway. The sequence information of HMGS in Tripterygium wilfordii is a basic resource necessary for studying the terpenoids in the plant. In this paper, full-length cDNA encoding HMGS was isolated from Tripterygium wilfordii (abbreviated TwHMGS, GenBank accession number: KM978213. The full length of TwHMGS is 1814 bp, and the gene encodes a protein with 465 amino acids. Sequence comparison revealed that TwHMGS exhibits high similarity to HMGSs of other plants. The tissue expression patterns revealed that the expression level of TwHMGS is highest in the stems and lowest in the roots. Induced expression of TwHMGS can be induced by MeJA, and the expression level is highest 4 h after induction. The functional complement assays in the YML126C knockout yeast demonstrated that TwHMGS participates in yeast terpenoid biosynthesis.

  20. Selective AR Modulators that Distinguish Proliferative from Differentiative Gene Promoters

    Science.gov (United States)

    2016-08-01

    inhibitor into an oncoprotein following P21 loss (20). Moreover, in prostate cancer, P53 negatively regulates AR when over-expressed (21). These...Characterization of the androgen receptor in a benign prostate tissue-derived human prostate epithelial cell line: RC-165N/human telomerase reverse...dependent kinase inhibitor p21 gene through an androgen response element in the proximal promoter. Molecular endocrinology 13, 376 (Mar, 1999). 9

  1. Gene activation regresses atherosclerosis, promotes health, and enhances longevity

    Directory of Open Access Journals (Sweden)

    Luoma Pauli V

    2010-07-01

    Full Text Available Abstract Background Lifestyle factors and pharmacological compounds activate genetic mechanisms that influence the development of atherosclerotic and other diseases. This article reviews studies on natural and pharmacological gene activation that promotes health and enhances longevity. Results Living habits including healthy diet and regular physical activity, and pharmacotherapy, upregulate genes encoding enzymes and apolipoprotein and ATP-binding cassette transporters, acting in metabolic processes that promote health and increase survival. Cytochrome P450-enzymes, physiological factors in maintaining cholesterol homeostasis, generate oxysterols for the elimination of surplus cholesterol. Hepatic CTP:phosphocholine cytidylyltransferase-α is an important regulator of plasma HDL-C level. Gene-activators produce plasma lipoprotein profile, high HDL-C, HDL2-C and HDL-C/cholesterol ratio, which is typical of low risk of atherosclerotic disease, and also of exceptional longevity together with reduced prevalence of cardiovascular, metabolic and other diseases. High HDL contributes to protection against inflammation, oxidation and thrombosis, and associates with good cognitive function in very old people. Avoiding unhealthy stress and managing it properly promotes health and increases life expectancy. Conclusions Healthy living habits and gene-activating xenobiotics upregulate mechanisms that produce lipoprotein pattern typical of very old people and enhance longevity. Lipoprotein metabolism and large HDL2 associate with the process of living a very long life. Major future goals for health promotion are the improving of commitment to both wise lifestyle choices and drug therapy, and further the developing of new and more effective and well tolerated drugs and treatments.

  2. Risk Factors for Normal and High-Tension Glaucoma in Poland in Connection with Polymorphisms of the Endothelial Nitric Oxide Synthase Gene.

    Directory of Open Access Journals (Sweden)

    Ewa Kosior-Jarecka

    Full Text Available The purpose of this study was to evaluate the influence of polymorphisms of the eNOS gene on the clinical status of patients with normal and high tension glaucoma.266 Polish Caucasian patients with primary open angle glaucoma were studied. Of the 266, 156 had normal tension glaucoma (NTG and 110 high tension glaucoma (HTG. DNA material was isolated from peripheral venous blood using commercial kits. Real-time PCR reaction was used to amplify the promoter site of the endothelial nitric oxide synthase (eNOS gene, including the single nucleotide polymorphism (SNP site T-786C and part of the 7th exon of eNOS, including G894T SNP. Genotypes were determined with TaqMan SNP Genotyping Assays.There were no significant differences in frequencies of the allelic variants of both polymorphisms. In G894T SNP, however, the wild GG form was more common in the HTG group. The SNP of the eNOS gene did not significantly influence the progression rate in either of the groups studied. There were no differences in variants of the eNOS gene regarding the necessity for and success of surgery and the progression of the disease. In the NTG group, no statistical correlation was observed between G894T, T786C polymorphism variants, and risk factors such as optic disc haemorrhages, optic disc notches, and peripapillary atrophy. Mean diastolic and systolic pressure during the day and night were lowest in NTG patients with the CC variant of the T786C polymorphism. No statistical correlation was observed between the G894T and T786C polymorphisms and capillaroscopic examination results.Genotype frequencies are similar for both the eNOS G894T and T-786C polymorphisms in NTG and HTG patients. These polymorphisms do not correlate with risk factors and do not influence the state of the capillary system in NTG patients. Systolic blood pressure is lower in NTG patients with mutated alleles of both polymorphisms.

  3. Risk Factors for Normal and High-Tension Glaucoma in Poland in Connection with Polymorphisms of the Endothelial Nitric Oxide Synthase Gene

    Science.gov (United States)

    Kosior-Jarecka, Ewa; Łukasik, Urszula; Wróbel-Dudzińska, Dominika; Kocki, Janusz; Bartosińska, Joanna; Witczak, Agnieszka; Chodorowska, Grażyna; Mosiewicz, Jerzy; Żarnowski, Tomasz

    2016-01-01

    Aim The purpose of this study was to evaluate the influence of polymorphisms of the eNOS gene on the clinical status of patients with normal and high tension glaucoma. Methods 266 Polish Caucasian patients with primary open angle glaucoma were studied. Of the 266, 156 had normal tension glaucoma (NTG) and 110 high tension glaucoma (HTG). DNA material was isolated from peripheral venous blood using commercial kits. Real-time PCR reaction was used to amplify the promoter site of the endothelial nitric oxide synthase (eNOS) gene, including the single nucleotide polymorphism (SNP) site T-786C and part of the 7th exon of eNOS, including G894T SNP. Genotypes were determined with TaqMan SNP Genotyping Assays. Results There were no significant differences in frequencies of the allelic variants of both polymorphisms. In G894T SNP, however, the wild GG form was more common in the HTG group. The SNP of the eNOS gene did not significantly influence the progression rate in either of the groups studied. There were no differences in variants of the eNOS gene regarding the necessity for and success of surgery and the progression of the disease. In the NTG group, no statistical correlation was observed between G894T, T786C polymorphism variants, and risk factors such as optic disc haemorrhages, optic disc notches, and peripapillary atrophy. Mean diastolic and systolic pressure during the day and night were lowest in NTG patients with the CC variant of the T786C polymorphism. No statistical correlation was observed between the G894T and T786C polymorphisms and capillaroscopic examination results. Conclusions Genotype frequencies are similar for both the eNOS G894T and T-786C polymorphisms in NTG and HTG patients. These polymorphisms do not correlate with risk factors and do not influence the state of the capillary system in NTG patients. Systolic blood pressure is lower in NTG patients with mutated alleles of both polymorphisms. PMID:26807726

  4. How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

    Science.gov (United States)

    Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

    2013-07-01

    A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

  5. β-catenin promoter ChIP-chip reveals potential schizophrenia and bipolar disorder gene network.

    Science.gov (United States)

    Pedrosa, Erika; Shah, Abhishek; Tenore, Christopher; Capogna, Michael; Villa, Catalina; Guo, Xingyi; Zheng, Deyou; Lachman, Herbert M

    2010-12-01

    Therapeutic concentrations of lithium salts inhibit glycogen synthase kinase 3 beta (GSK3β) and phosphoinositide (PI) signaling suggesting that abnormal activation of these pathways could be a factor in the pathophysiology of bipolar disorder (BD). Involvement of these pathways is also supported by recent genome-wide association studies (GWASs). One way investigators have investigated the molecular basis of BD and the therapeutic action of lithium is by microarray expression studies, since both GSK3β- and PI-mediated signal transduction pathways are coupled to transcriptional activation and inhibition. However, expression profiling has some limitations and investigators cannot use the approach to analyze fetal brain tissue, arguably the most relevant biological structure related to the development of genetically based psychiatric disorders. To address these shortcomings, the authors have taken a novel approach using chromatin immunoprecipitation-enriched material annealed to microarrays (ChIP-chip) targeting genes in fetal brain tissue bound by β-catenin, a transcription factor that is directly regulated by GSK3β. The promoters for 640 genes were found to be bound by β-catenin, many of which are known schizophrenia (SZ), autism spectrum disorder (ASD), and BD candidates, including CACNA1B, NRNG, SNAP29, FGFR1, PCDH9, and nine others identified in recently published GWASs and genome-wide searches for copy number variants (CNVs). The findings suggest that seemingly disparate candidate genes for SZ and BD can be incorporated into a common molecular network revolving around GSK3β/β-catenin signaling. In addition, the finding that a putative lithium-responsive pathway may influence a subgroup of SZ and ASD candidate genes could have therapeutic implications.

  6. A Genome-Wide Association Study for Culm Cellulose Content in Barley Reveals Candidate Genes Co-Expressed with Members of the CELLULOSE SYNTHASE A Gene Family

    Science.gov (United States)

    Houston, Kelly; Burton, Rachel A.; Sznajder, Beata; Rafalski, Antoni J.; Dhugga, Kanwarpal S.; Mather, Diane E.; Taylor, Jillian; Steffenson, Brian J.; Waugh, Robbie; Fincher, Geoffrey B.

    2015-01-01

    Cellulose is a fundamentally important component of cell walls of higher plants. It provides a scaffold that allows the development and growth of the plant to occur in an ordered fashion. Cellulose also provides mechanical strength, which is crucial for both normal development and to enable the plant to withstand both abiotic and biotic stresses. We quantified the cellulose concentration in the culm of 288 two – rowed and 288 six – rowed spring type barley accessions that were part of the USDA funded barley Coordinated Agricultural Project (CAP) program in the USA. When the population structure of these accessions was analysed we identified six distinct populations, four of which we considered to be comprised of a sufficient number of accessions to be suitable for genome-wide association studies (GWAS). These lines had been genotyped with 3072 SNPs so we combined the trait and genetic data to carry out GWAS. The analysis allowed us to identify regions of the genome containing significant associations between molecular markers and cellulose concentration data, including one region cross-validated in multiple populations. To identify candidate genes we assembled the gene content of these regions and used these to query a comprehensive RNA-seq based gene expression atlas. This provided us with gene annotations and associated expression data across multiple tissues, which allowed us to formulate a supported list of candidate genes that regulate cellulose biosynthesis. Several regions identified by our analysis contain genes that are co-expressed with CELLULOSE SYNTHASE A (HvCesA) across a range of tissues and developmental stages. These genes are involved in both primary and secondary cell wall development. In addition, genes that have been previously linked with cellulose synthesis by biochemical methods, such as HvCOBRA, a gene of unknown function, were also associated with cellulose levels in the association panel. Our analyses provide new insights into the

  7. Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS gene expression patterns

    Directory of Open Access Journals (Sweden)

    Abercrombie Jason M

    2011-07-01

    Full Text Available Abstract Background A number of innovations underlie the origin of rapid reproductive cycles in angiosperms. A critical early step involved the modification of an ancestrally short and slow-growing pollen tube for faster and longer distance transport of sperm to egg. Associated with this shift are the predominantly callose (1,3-β-glucan walls and septae (callose plugs of angiosperm pollen tubes. Callose synthesis is mediated by callose synthase (CalS. Of 12 CalS gene family members in Arabidopsis, only one (CalS5 has been directly linked to pollen tube callose. CalS5 orthologues are present in several monocot and eudicot genomes, but little is known about the evolutionary origin of CalS5 or what its ancestral function may have been. Results We investigated expression of CalS in pollen and pollen tubes of selected non-flowering seed plants (gymnosperms and angiosperms within lineages that diverged below the monocot/eudicot node. First, we determined the nearly full length coding sequence of a CalS5 orthologue from Cabomba caroliniana (CcCalS5 (Nymphaeales. Semi-quantitative RT-PCR demonstrated low CcCalS5 expression within several vegetative tissues, but strong expression in mature pollen. CalS transcripts were detected in pollen tubes of several species within Nymphaeales and Austrobaileyales, and comparative analyses with a phylogenetically diverse group of sequenced genomes indicated homology to CalS5. We also report in silico evidence of a putative CalS5 orthologue from Amborella. Among gymnosperms, CalS5 transcripts were recovered from germinating pollen of Gnetum and Ginkgo, but a novel CalS paralog was instead amplified from germinating pollen of Pinus taeda. Conclusion The finding that CalS5 is the predominant callose synthase in pollen tubes of both early-diverging and model system angiosperms is an indicator of the homology of their novel callosic pollen tube walls and callose plugs. The data suggest that CalS5 had transient expression

  8. Identification and expression of the trehalose-6-phosphate synthase gene family members in tomato exposed to different light spectra

    Directory of Open Access Journals (Sweden)

    Chen Zexiong

    2017-01-01

    Full Text Available Light is the source of energy for plants. Light wavelengths, densities and irradiation periods act as signals directing morphological and physiological characteristics during plant growth and development. To evaluate the effects of light wavelengths on tomato growth and development, Solanum lycopersicum (cv. micro-Tom seedlings were exposed to different light-quality environments, including white light and red light supplemented with blue light (at ratios of 3:1 and 8;1, respectively. Tomatoes grown under red light supplemented with blue light displayed significantly shorter stem length, a higher number of flower buds and rate of fruit set, but an extremely late flowering compared to white-light-grown plants. To illustrate the mechanism underlying the inhibition of stem growth and floral transition mediated by red/blue light, 10 trehalose-6-phosphate synthase (TPS genes were identified in tomato, and bioinformatics analysis was performed. qRT-PCR analysis showed that SlTPSs were expressed widely throughout plant development and SlTPS1 was expressed at extremely high levels in stems and buds. Further analysis of several flowering-associated genes and microRNAs showed that the expressions of SlTPS1, SlFT and miR172 were significantly downregulated in tomato grown under red and blue light compared with those grown under white light, whereas miR156 transcript levels were increased. A regulatory model underlying vegetative growth and floral transition regulated by light qualities is presented. Our data provide evidence that light quality strongly affects plant growth and phase transition, most likely via the TPS1-T6P signaling pathway.

  9. Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328.

    Science.gov (United States)

    Kirimura, Kohtaro; Watanabe, Shotaro; Kobayashi, Keiichi

    2016-05-13

    Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr_8_2: 2978617-2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni(2+)-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Association of endothelial nitric oxide synthase gene polymorphisms with coronary artery disease: an updated meta-analysis and systematic review.

    Directory of Open Access Journals (Sweden)

    Himanshu Rai

    Full Text Available Several association studies of endothelial nitric oxide synthase (NOS3 gene polymorphisms with respect to coronary artery disease (CAD have been published in the past two decades. However, their association with the disease, especially among different ethnic subgroups, still remains controversial. This prompted us to conduct a systematic review and an updated structured meta-analysis, which is the largest so far (89 articles, 132 separate studies, and a sample size of 69,235, examining association of three polymorphic forms of the NOS3 gene (i.e. Glu298Asp, T786-C and 27 bp VNTR b/a with CAD. In a subgroup analysis, we tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries. The pooled analysis confirmed the association of all the three selected SNP with CAD in three different genetic models transcending all ancestries worldwide. The Glu298Asp polymorphism showed strongest association (OR range = 1.28-1.52, and P<0.00001 for all comparisons, followed by T786-C (OR range = 1.34-1.42, and P<0.00001 for all comparisons and 4b/a, (OR range = 1.19-1.41, and P ≤ 0.002 for all comparisons in our pooled analysis. Subgroup analysis revealed that Glu298Asp (OR range = 1.54-1.87, and P<0.004 for all comparisons and 4b/a (OR range = 1.71-3.02, and P<0.00001 for all comparisons have highest degree of association amongst the Middle Easterners. On the other hand, T786-C and its minor allele seem to carry a highest risk for CAD among subjects of Asian ancestry (OR range = 1.61-1.90, and P ≤ 0.01 for all comparisons.

  11. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  12. The Terpene Synthase Gene Family of Carrot (Daucus carota L.: Identification of QTLs and Candidate Genes Associated with Terpenoid Volatile Compounds

    Directory of Open Access Journals (Sweden)

    Jens Keilwagen

    2017-11-01

    Full Text Available Terpenes are an important group of secondary metabolites in carrots influencing taste and flavor, and some of them might also play a role as bioactive substances with an impact on human physiology and health. Understanding the genetic and molecular basis of terpene synthases (TPS involved in the biosynthesis of volatile terpenoids will provide insights for improving breeding strategies aimed at quality traits and for developing specific carrot chemotypes possibly useful for pharmaceutical applications. Hence, a combination of terpene metabolite profiling, genotyping-by-sequencing (GBS, and genome-wide association study (GWAS was used in this work to get insights into the genetic control of terpene biosynthesis in carrots and to identify several TPS candidate genes that might be involved in the production of specific monoterpenes. In a panel of 85 carrot cultivars and accessions, metabolite profiling was used to identify 31 terpenoid volatile organic compounds (VOCs in carrot leaves and roots, and a GBS approach was used to provide dense genome-wide marker coverage (>168,000 SNPs. Based on this data, a total of 30 quantitative trait loci (QTLs was identified for 15 terpenoid volatiles. Most QTLs were detected for the monoterpene compounds ocimene, sabinene, β-pinene, borneol and bornyl acetate. We identified four genomic regions on three different carrot chromosomes by GWAS which are both associated with high significance (LOD ≥ 5.91 to distinct monoterpenes and to TPS candidate genes, which have been identified by homology-based gene prediction utilizing RNA-seq data. In total, 65 TPS candidate gene models in carrot were identified and assigned to known plant TPS subfamilies with the exception of TPS-d and TPS-h. TPS-b was identified as largest subfamily with 32 TPS candidate genes.

  13. Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers.

    Science.gov (United States)

    Aros, Danilo; Gonzalez, Veronica; Allemann, Rudolf K; Müller, Carsten T; Rosati, Carlo; Rogers, Hilary J

    2012-04-01

    Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar 'Sweet Laura' is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. 'Sweet Laura' with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. 'Sweet Laura' and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. 'Sweet Laura' placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R(28)(R)X(8)W and D(321)DXXD are the putative Mg(2+)-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. 'Sweet Laura' flowers.

  14. [The G894T mutation of the endothelial nitric oxide synthase gene is associated with coronary atherosclerotic heart disease in Chinese].

    Science.gov (United States)

    Wei, Danhong; Shan, Jiang; Chen, Zhimei; Shi, Yuping

    2002-12-01

    To investigate the association of the endothelial nitric oxide synthase (eNOS) gene polymorphism with coronary atherosclerotic heart disease (CHD) in Chinese Han nationality. For 106 patients with CHD and 108 unrelated health individuals, the G894T mutation at exon 7 of the endothelial nitric oxide synthase gene was studied by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. (1) Among the normal subjects of Chinese Han nationality, the frequencies of the eNOS/GG, GT and TT genotypes were 0.9095, 0.0883 and 0.0021, respectively. The G and T allele frequencies were 0.9537 and 0.0463. (2) The authors assumed the effects of the T allele to be dominant (GT and TT combined vs GG). The GT+TT genotype frequencies in CHD and myocardial infarction (MI) subgroup were 0.2219 and 0.2387, respectively. The frequencies of eNOS/GT+TT genotypes in CHD patients, as well as MI subgroup were significantly higher than that of the normal subjects (P0.05). The G894T mutation of the endothelial nitric oxide synthase gene may be a marker for genetical predisposition of CHD in Chinese Han population.

  15. Large-scale phylogenetic classification of fungal chitin synthases and identification of a putative cell-wall metabolism gene cluster in Aspergillus genomes.

    Directory of Open Access Journals (Sweden)

    Jose Ramon Pacheco-Arjona

    Full Text Available The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp enzymes. There are seven classes of chitin synthase genes (CHS and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%. Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome.

  16. Polymorphisms in the leptin gene promoter in Brazilian beef herds.

    Science.gov (United States)

    Guimarães, R C; Azevedo, J S N; Corrêa, S C; Campelo, J E G; Barbosa, E M; Gonçalves, E C; Silva Filho, E

    2016-12-02

    Brazil is the world's largest producer of beef cattle; however, the quality of its herds needs to be improved. The use of molecular markers as auxiliary tools in selecting animals for reproduction with high pattern for beef production would significantly improve the quality of the final beef product in Brazil. The leptin gene has been demonstrated to be an excellent candidate gene for bovine breeding. The objective of this study was to sequence and compare the leptin gene promoter of Brazil's important cattle breeds in order to identify polymorphisms in it. Blood samples of the Nellore, Guzerat, Tabapuã, and Senepol breeds were collected for genomic DNA extraction. The genomic DNA was used as a template for polymerase chain reaction (PCR) to amplify a 1575-bp fragment, which in turn was sequenced, aligned, and compared between animals of different breeds. Twenty-three single nucleotide polymorphic sites, including transitions and transversions, were detected at positions -1457, -1452, -1446, -1397, -1392, -1361, -1238, -963,-901, -578, -516, -483, -478, -470, -432, -430, -292, -282, -272, -211, -202, -170, and -147. Additionally, two insertion sites at positions -680 and -416 and two deletion sites at positions -1255 and -1059 were detected. As the promoter region of the leptin gene has been demonstrated to vary among breeds, these variations must be tested for their use as potential molecular markers for artificial selection of animals for enhanced beef production in different systems of bovine production in Brazil.

  17. Long noncoding RNA XIST is a prognostic factor in colorectal cancer and inhibits 5-fluorouracil-induced cell cytotoxicity through promoting thymidylate synthase expression.

    Science.gov (United States)

    Xiao, Yang; Yurievich, Usenko Alexander; Yosypovych, Smorzhevskyi Valentyn

    2017-10-10

    A major reason for the failure of advanced colorectal cancer (CRC) treatment is the occurrence of chemoresistance to 5-fluorouracil (5FU)-based treatment. Recent studies have shown that long non-coding RNAs (lncRNAs) are critical regulators in chemoresistance. By using the next generation HiSeq sequencing assay, we identified lncRNAs showing differential expression levels in 5FU resistant and non-resistant CRC patients. RT-qPCR was then performed for validation in tissues and serum samples, and lncRNA XIST was verified to be up-regulated in non-responding patients and have considerable diagnostic potential to identify responding patients from non-responding patients. In addition, increased serum XIST level was associated with poor response and lower survival rate in CRC patients receiving 5FU-based treatment. Subsequently, the 5FU resistant (5FU-R) cell lines were established, and lncRNA XIST was significantly up-regulated HT29 5FU-R and HCT116 5FU-R cells. Furthermore, knockdown of XIST reversed 5FU resistance while enhanced XIST could restrained the 5FU-induced cell cytotoxcity in both CRC cell lines. Western blotting and immunofluorescence analysis indicated that XIST promoted the expression of thymidylate synthase, a critical 5FU-targetd enzyme. In conclusion, our integrated approach demonstrates that increased expression of lncRNA XIST3 in CRC confers a potent poor therapeutic efficacy, and that lncRNA XIST participated in 5FU resistance through promoting the expression of thymidylate synthase. Thus, specific silence oflncRNA XIST could be a future direction to develop a novel therapeutic strategy to overcome 5FU resistance of CRC patients.

  18. Characterization of the Granule-Bound Starch Synthase I Gene in Chenopodium

    Directory of Open Access Journals (Sweden)

    Douglass C. Brown

    2015-03-01

    Full Text Available L. is a relatively under-studied genus that includes the cultivated seed crop quinoa ( Willd.. Quinoa is an allotetraploid (2 = 4 = 36, AABB genomes that is cultivated by subsistence farmers and commercial growers in the Andean regions of South America. Approximately 60% of a quinoa seed is starch, a glucose polymer that is an important carbohydrate energy source in the human diet. Seed starch is normally composed of amylose and amylopectin in a 1:3 ratio. The accumulation of the amylose fraction of starch is controlled by a single dominant gene in quinoa, . We report the sequencing and characterization of the gene in 18 accessions of , including Andean quinoa and the related Mesoamerican chenopod domesticate, subsp. Saff. Two distinct homeologs ( and were identified in the tetraploid accessions, and 19 different alleles were identified, including three null mutants—one in an accession of quinoa and two in a waxy landrace of subsp. . Expression analysis of the null mutants revealed that and were both strongly expressed late in seed development. sequences were used to analyze the phylogenetic relationships between quinoa and other members of the genus. This study and the discovery of null-mutants will assist in the development of new crops with novel starches.

  19. Silencing of CHD5 gene by promoter methylation in leukemia.

    Directory of Open Access Journals (Sweden)

    Rui Zhao

    Full Text Available Chromodomain helicase DNA binding protein 5 (CHD5 was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2 as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter.

  20. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

  1. Association between recurrent aphthous stomatitis and inheritance of a single-nucleotide polymorphism of the NOS2 gene encoding inducible nitric oxide synthase.

    Science.gov (United States)

    Karasneh, Jumana A; Darwazeh, Azmi M G; Hassan, Ahmad F; Thornhill, Martin

    2011-10-01

     Recurrent aphthous stomatitis is a common ulcerative disease of the oral mucosa. Recurrent oral aphthous ulceration is also a feature of the more serious and systemic Behçet's disease. Nitric oxide is a free radical synthesized by one of a family of nitric oxide synthase (NOS) enzymes and is an important regulator of inflammation and immunity. Association of NOS3 gene polymorphisms encoding endothelial nitric oxide synthase has been reported in Behçet's disease but not recurrent aphthous stomatitis. The aim of this study was to investigate any association between NOS2 gene polymorphisms that encode inducible nitric oxide synthase and recurrent aphthous stomatitis.  This is a case control association study. Eighty-three Jordanian recurrent aphthous stomatitis patients and 83 age, gender and ethnically matched controls were genotyped for three NOS2 single-nucleotide polymorphisms, rs10459953, rs1060822 and rs2297518. Chi-squared analysis was used to compare the allele frequencies and genotypes.  There was a significant association between recurrent aphthous stomatitis and inheritance of single-nucleotide polymorphism rs2297518 (P = 0.006). Although no direct association was demonstrated between rs10459953 or rs1060822 and recurrent aphthous stomatitis, a strong linkage disequilibrium was identified between rs1060822 and rs2297518.  Inheritence of a NOS2 single-nucleotide polymorphism rs2297518 is associated with increased risk of recurrent aphthous stomatitis in a Jordanian population. Confirmatory studies in other populations and investigation of other NOS2 gene polymorphisms will enhance our understanding of the functional basis of this association and help elucidate the role of inducible nitric oxide synthase in recurrent aphthous stomatitis. © 2011 John Wiley & Sons A/S.

  2. Engineering the robustness of Saccharomyces cerevisiae by introducing bifunctional glutathione synthase gene.

    Science.gov (United States)

    Qiu, Zhiqi; Deng, Zujun; Tan, Hongming; Zhou, Shining; Cao, Lixiang

    2015-04-01

    Robust, high-yielding Saccharomyces cerevisiae is highly desirable for cost-effective cellulosic ethanol production. In this study, the bifunctional glutathione (GSH) synthetase genes GCSGS at high copy number was integrated into ribosomal DNA of S. cerevisiae by Cre-LoxP system. Threefold higher GSH contents (54.9 μmol/g dry weight) accumulated in the engineered strain BY-G compared to the reference strain. Tolerance of BY-G to H2O2 (3 mM), temperature (40 °C), furfural (10 mM), hydroxymethylfurfural (HMF, 10 mM) and 0.5 mM Cd(2+) increased compared to reference strain. Twofold higher ethanol concentration was obtained by BY-G in simultaneous saccharification and fermentation of corn stover compared to the reference strain. The results showed that intracellular GSH content of S. cerevisiae has an influence on robustness. The strategy is used to engineer S. cerevisiae strains adaptive to a combination of tolerance to inhibitors and raised temperature that may occur in high solid simultaneous saccharification and fermentation of lignocellulosic feedstocks.

  3. Novel quadruple mutations in dihydropteroate synthase genes of Plasmodium falciparum in West Bengal, India.

    Science.gov (United States)

    Das, Sabyasachi; Chakraborty, Subhankari P; Tripathy, Satyajit; Hati, Amiyakumar; Roy, Somenath

    2012-11-01

    To evaluate the anti-folate (sulphadoxine)-resistant pattern in Kolkata, one of the malaria endemic zones of Eastern India. At first, 107 P. falciparum suspected cases were enrolled in this study. Ninety isolates (84.11%) of 107 suspected cases were analysed, as they had mono-infection with P. falciparum. In vitro susceptibility assays were performed in all 90 isolates. Parasitic DNA was isolated by phenol-chloroform extraction method and polymerase chain reaction was followed by restriction fragment length polymorphism analysis of different codons of the pfdhps gene (436, 437, 540, 581 and 613). Among 90 isolates from Kolkata, dhps mutant isolates at codons 436, 437, 540, 581 and 613 were found in 53.33%, 67.78%, 46.66%, 15.56% and 45.55%, respectively. In vitro sulphadoxine resistance was found in 49 isolates (54.44%). Interestingly we found 33 isolates (36.67%) with quadruple AGEAT mutant allele, of which 32 isolates (96.97%) were highly sulphadoxine resistant (P < 0.01) in vitro. Our present findings implicate that because of enormous drug (sulphfadoxine) pressure, novel AGEAT mutation was highly correlated (P < 0.01) with sulphadoxine resistance. © 2012 Blackwell Publishing Ltd.

  4. A novel 3-base deletion (IVS3+2_4delTGG of the hydroxymethylbilane synthase gene in a Brazilian patient with acute intermittent porphyria

    Directory of Open Access Journals (Sweden)

    Georgina Severo Ribeiro

    2007-01-01

    Full Text Available Acute intermittent porphyria (AIP, OMIM 176000 is an autosomal dominant metabolic disease caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS; EC 4.3.1.8; formely named porphobilinogen deaminase, PBGD, mapped to chromosome 11q23.3. We describe a novel mutation of the HMBS gene, a de novo 3-base deletion in the splicing donor site of intron 3 (IVS3+2_4delTGG in a woman affected by AIP. RT-PCR analysis revealed an abnormal HMBS mRNA, compatible with exon 3 skipping.

  5. Promoter Methylation Analysis of IDH Genes in Human Gliomas.

    Science.gov (United States)

    Flanagan, Simon; Lee, Maggie; Li, Cheryl C Y; Suter, Catherine M; Buckland, Michael E

    2012-01-01

    Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a "toxic gain-of-function" to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

  6. Promoter methylation analysis of IDH genes in human gliomas

    Directory of Open Access Journals (Sweden)

    Simon eFlanagan

    2012-12-01

    Full Text Available Mutations in isocitrate dehydrogenase (IDH -1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132 or IDH2 (R172. But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a ‘toxic gain of function’ to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumour suppressor gene. As most, if not all, tumour suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumours, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumour suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumours, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumours examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumours. These findings do not support a tumour suppressor role for IDH genes in human gliomas.

  7. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  8. Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion.

    Directory of Open Access Journals (Sweden)

    Robert Schnell

    Full Text Available The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity.

  9. RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

    Science.gov (United States)

    Baumgardt, Kathrin; Charoenpanich, Pornsri; McIntosh, Matthew; Schikora, Adam; Stein, Elke; Thalmann, Sebastian; Kogel, Karl-Heinz; Klug, Gabriele; Becker, Anke; Evguenieva-Hackenberg, Elena

    2014-04-01

    Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels. The ectopic rne expression led to a higher level of rne mRNA and a lower level of sinI mRNA independently of the presence of ExpR, the AHL receptor, and AHLs. In line with this, IPTG (isopropyl-β-D-thiogalactopyranoside)-induced overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. Moreover, using translational sinI-egfp fusions, we found that sinI expression is specifically decreased upon induced overexpression of rne, independently of the presence of the global posttranscriptional regulator Hfq. The 28-nucleotide 5' untranslated region (UTR) of sinI mRNA was sufficient for this effect. Random amplification of 5' cDNA ends (5'-RACE) analyses revealed a potential RNase E cleavage site at position +24 between the Shine-Dalgarno site and the translation start site. We postulate therefore that RNase E-dependent degradation of sinI mRNA from the 5' end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin quorum-sensing system to respond rapidly to changes in transcriptional control of AHL production.

  10. Shear stress induction of the endothelial nitric oxide synthase gene is calcium-dependent but not calcium-activated.

    Science.gov (United States)

    Xiao, Z; Zhang, Z; Ranjan, V; Diamond, S L

    1997-05-01

    Arterial levels of shear stress (25 dynes/cm2) can elevate constitutive endothelial nitric oxide synthase (eNOS) gene expression in cultured endothelial cells (Ranjan et al., 1995). By PhosphorImaging of Northern blots, we report that the eNOS/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio in bovine aortic endothelial cells (BAEC) increased by 4.8- and 7.95-fold after 6-hr shear stress exposure of 4 and 25 dynes/cm2, respectively. Incubation of BAEC with dexamethasone (1 microM) had no effect on shear stress induction of eNOS mRNA. Buffering of intracellular calcium in BAEC with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, stimulation of BAEC with ionomycin (0.1-1.0 microM) for 6-24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indicated that the shear stress induction of eNOS mRNA was a calcium-dependent, but not calcium-activated, process. Shear stress was a very potent and rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinases with genistein (10 microM) or tyrphostin B46 (10 microM) or inhibition of G-protein signaling with guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) (600 microM, 6-hr preincubation) did not block the shear stress elevation of eNOS mRNA.

  11. Overexpression of 3-deoxy-7-phosphoheptulonate synthase gene from Gossypium hirsutum enhances Arabidopsis resistance to Verticillium wilt.

    Science.gov (United States)

    Yang, Jun; Ji, Lianlian; Wang, Xingfen; Zhang, Yan; Wu, Lizhu; Yang, Yingna; Ma, Zhiying

    2015-08-01

    Expression of DHS1 in cotton is induced upon infection by Verticillium dahliae , and overexpression of GhDHS1 endows transgenic Arabidopsis plants excellent Verticillium resistance. Verticillium wilt is caused by a soil-borne fungus Verticillium dahliae. Resistance in most cotton cultivars is either scarce or unavailable, making Verticillium wilt a major obstacle in cotton production. Here, we identified a 3-deoxy-7-phosphoheptulonate synthase (DHS, EC 4.1.2.15) gene from Gossypium hirsutum, named GhDHS1. Its 1620 bp open reading frame encodes a putative 59.4 kDa protein. Phylogenetic analysis indicated that GhDHS1 is clustered in a clade with potato and tomato DHSs that can be induced by wounding and elicitors, respectively. Expression analysis demonstrated that GhDHS1 is constitutively expressed in cotton roots and stems, but transcripts are rare or non-existent in the leaves. Subcellular localization showed that GhDHS1 occurs in the plastids. When plants of three cultivars were inoculated with V. dahliae, DHS1 expression was more significantly up-regulated in the roots of resistant G. barbadense cv. Pima90-53 and G. hirsutum cv. Jimian20 than in the susceptible G. hirsutum cv. Han208. This suggested that DHS1 is involved in the cotton resistance to Verticillium wilt. Furthermore, GhDHS1 overexpressing transgenic lines of Arabidopsis were developed via Agrobacterium tumefaciens-mediated transformation. Compared with the untransformed WT (wild type), these transgenic plants showed excellent Verticillium wilt resistance with a significantly lower disease index. The overexpressing transgenic lines also had significantly longer primary roots and greatly increased xylem areas under V. dahliae infection. Overall, our results indicate that GhDHS1 performs a role in the cotton resistance to V. dahliae and would be potential to breeding cottons of Verticillium wilt resistance.

  12. Analysis of MaACS2, a stress-inducible ACC Synthase Gene in Musa acuminata AAA Group Cultivar Pisang Ambon

    Directory of Open Access Journals (Sweden)

    Resnanti Utami Handayani

    2014-07-01

    Full Text Available Ethylene has an important function in plant growth and development. Ethylene production generally increases in response to pathogen attacks and other environmental stress conditions. The synthesis of this phytohormone is regulated by two enzymes, ACC synthase (ACS and ACC oxidase (ACO. ACC synthase is encoded by a multigene that regulates the production of ACC, after which this precursor is converted into ethylene by ACO. Pisang Ambon (Musa sp. AAA group, a banana cultivar originating from Indonesia, has nine ACS genes (MaACS 1-9 and one ACO gene (MaACO. One of the banana ACS genes, MaACS2, is stress-inducible. In this research, we have investigated the expression profile of MaACS2 in the roots and leaf tissues of infected tissue culture plants. Quantification of gene expression was analyzed using Real-Time PCR (qPCR using Ma18srRNA and MaGAPDH as reference genes. The results showed nine-to ten fold higher MaACS2 expression levels in the infected roots tissues compared to the uninfected roots tissues. However, MaACS2 expression in the leaves was only detected in infected tissue.

  13. Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast.

    Science.gov (United States)

    Niedzwiecka, Katarzyna; Tisi, Renata; Penna, Sara; Lichocka, Malgorzata; Plochocka, Danuta; Kucharczyk, Roza

    2018-01-01

    The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. SNP in Chalcone Synthase gene is associated with variation of 6-gingerol content in contrasting landraces of Zingiber officinale.Roscoe.

    Science.gov (United States)

    Ghosh, Subhabrata; Mandi, Swati Sen

    2015-07-25

    Zingiber officinale, medicinally the most important species within Zingiber genus, contains 6-gingerol as the active principle. This compound obtained from rhizomes of Z.officinale, has immense medicinal importance and is used in various herbal drug formulations. Our record of variation in content of this active principle, viz. 6-gingerol, in land races of this drug plant collected from different locations correlated with our Gene expression studies exhibiting high Chalcone Synthase gene (Chalcone Synthase is the rate limiting enzyme of 6-gingerol biosynthesis pathway) expression in high 6-gingerol containing landraces than in the low 6-gingerol containing landraces. Sequencing of Chalcone Synthase cDNA and subsequent multiple sequence alignment revealed seven SNPs between these contrasting genotypes. Converting this nucleotide sequence to amino acid sequence, alteration of two amino acids becomes evident; one amino acid change (asparagine to serine at position 336) is associated with base change (A→G) and another change (serine to leucine at position 142) is associated with the base change (C→T). Since asparagine at position 336 is one of the critical amino acids of the catalytic triad of Chalcone Synthase enzyme, responsible for substrate binding, our study suggests that landraces with a specific amino acid change viz. Asparagine (found in high 6-gingerol containing landraces) to serine causes low 6-gingerol content. This is probably due to a weak enzyme substrate association caused by the absence of asparagine in the catalytic triad. Detailed study of this finding could also help to understand molecular mechanism associated with variation in 6-gingerol content in Z.officinale genotypes and thereby strategies for developing elite genotypes containing high 6-gingerol content. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

    Science.gov (United States)

    Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

    2018-02-01

    R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Differential gene expression signatures for cell wall integrity found in chitin synthase II (chs2Δ and myosin II (myo1Δ deficient cytokinesis mutants of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Rodríguez-Medina José R

    2009-05-01

    Full Text Available Abstract Background Myosin II-dependent contraction of the cytokinetic ring and primary septum formation by chitin synthase II are interdependent processes during cytokinesis in Saccharomyces cerevisiae. Hence, null mutants of myosin II (myo1Δ and chitin synthase II (chs2Δ share multiple morphological and molecular phenotypes. To understand the nature of their interdependent functions, we will seek to identify genes undergoing transcriptional regulation in chs2Δ strains and to establish a transcription signature profile for comparison with myo1Δ strains. Results A total of 467 genes were commonly regulated between myo1Δ and chs2Δ mutant strains (p ≤ 0.01. Common regulated biological process categories identified by Gene Set Enrichment Analysis (GSEA in both gene expression profiles were: protein biosynthesis, RNA processing, and stress response. Expression of 17/20 genes in the main transcriptional fingerprint for cell wall stress was confirmed in the chs2Δ strain versus 5/20 for the myo1Δ strain. One of these genes, SLT2/MPK1, was up-regulated in both strains and both strains accumulated the hyperphosphorylated form of Slt2p thereby confirming that the PKC1 cell wall integrity pathway (CWIP was activated by both mutations. The SLT2/MPK1 gene, essential for myo1Δ strains, was not required in the chs2Δ strain. Conclusion Comparison of the chs2Δ and myo1Δ gene expression profiles revealed similarities in the biological process categories that respond to the chs2Δ and myo1Δ gene mutations. This supports the view that these mutations affect a common function in cytokinesis. Despite their similarities, these mutants exhibited significant differences in expression of the main transcriptional fingerprint for cell wall stress and their requirement of the CWIP for survival.

  17. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms with Coronary Artery Disease: An Updated Meta-Analysis and Systematic Review

    Science.gov (United States)

    Parveen, Farah; Kapoor, Aditya; Sinha, Nakul

    2014-01-01

    Several association studies of endothelial nitric oxide synthase (NOS3) gene polymorphisms with respect to coronary artery disease (CAD) have been published in the past two decades. However, their association with the disease, especially among different ethnic subgroups, still remains controversial. This prompted us to conduct a systematic review and an updated structured meta-analysis, which is the largest so far (89 articles, 132 separate studies, and a sample size of 69,235), examining association of three polymorphic forms of the NOS3 gene (i.e. Glu298Asp, T786-C and 27bp VNTR b/a) with CAD. In a subgroup analysis, we tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries. The pooled analysis confirmed the association of all the three selected SNP with CAD in three different genetic models transcending all ancestries worldwide. The Glu298Asp polymorphism showed strongest association (OR range = 1.28–1.52, and P<0.00001 for all comparisons), followed by T786-C (OR range = 1.34–1.42, and P<0.00001 for all comparisons) and 4b/a, (OR range = 1.19–1.41, and P≤0.002 for all comparisons) in our pooled analysis. Subgroup analysis revealed that Glu298Asp (OR range = 1.54–1.87, and P<0.004 for all comparisons) and 4b/a (OR range = 1.71–3.02, and P<0.00001 for all comparisons) have highest degree of association amongst the Middle Easterners. On the other hand, T786-C and its minor allele seem to carry a highest risk for CAD among subjects of Asian ancestry (OR range = 1.61–1.90, and P≤0.01 for all comparisons). PMID:25409023

  18. Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328

    Energy Technology Data Exchange (ETDEWEB)

    Kirimura, Kohtaro, E-mail: kkohtaro@waseda.jp; Watanabe, Shotaro; Kobayashi, Keiichi

    2016-05-13

    Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.

  19. Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats.

    Directory of Open Access Journals (Sweden)

    Gracia Zabala

    Full Text Available We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-r(m is homozygous for a mutable allele (r(m that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-r(m line had a 13 kb CACTA subfamily transposon insertion (designated TgmR* at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983. Although the MYB encoded by R was expressed at only very low levels in older seed coats of the black revertant RM30-R* line, it upregulated expression of anthocyanidin synthase genes (ANS2, ANS3 to promote the synthesis of anthocyanins. Surprisingly, the RM30-R* revertant also carried the 13 kb TgmR* insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13 kb TgmR* element. As determined by whole genome methylation sequencing, we demonstrate that the TgmR* sequence was relatively more methylated in RM30-R* than in the mutable RM55-r(m progenitor line. The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

  20. Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

    Directory of Open Access Journals (Sweden)

    Tao Zhou

    2015-01-01

    Full Text Available The incorporation pattern of biosynthetic precursors into two structurally unique polyketides, akaeolide and lorneic acid A, was elucidated by feeding experiments with 13C-labeled precursors. In addition, the draft genome sequence of the producer, Streptomyces sp. NPS554, was performed and the biosynthetic gene clusters for these polyketides were identified. The putative gene clusters contain all the polyketide synthase (PKS domains necessary for assembly of the carbon skeletons. Combined with the 13C-labeling results, gene function prediction enabled us to propose biosynthetic pathways involving unusual carbon-carbon bond formation reactions. Genome analysis also indicated the presence of at least ten orphan type I PKS gene clusters that might be responsible for the production of new polyketides.

  1. Sensitivity of Aspergillus nidulans to the cellulose synthase inhibitor dichlobenil: insights from wall-related genes' expression and ultrastructural hyphal morphologies.

    Directory of Open Access Journals (Sweden)

    Gea Guerriero

    Full Text Available The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes' expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h, revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.

  2. A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved in the Biosynthesis of Ochratoxin A in Aspergillus niger.

    Science.gov (United States)

    Zhang, Jian; Zhu, Liuyang; Chen, Haoyu; Li, Min; Zhu, Xiaojuan; Gao, Qiang; Wang, Depei; Zhang, Ying

    2016-12-28

    The polyketide synthase gene An15g07920 was known in Aspergillus niger CBS 513.88 as putatively involved in the production of ochratoxin A (OTA). Genome resequencing analysis revealed that the gene An15g07920 is also present in the ochratoxin-producing A. niger strain 1062. Disruption of An15g07920 in A. niger 1062 removed its capacity to biosynthesize ochratoxin β (OTβ), ochratoxin α (OTα), and OTA. These results indicate that the polyketide synthase encoded by An15g07920 is a crucial player in the biosynthesis of OTA, in the pathway prior to the phenylalanine ligation step. The gene An15g07920 reached its maximum transcription level before OTA accumulation reached its highest level, confirming that gene transcription precedes OTA production. These findings will not only help explain the mechanism of OTA production in A. niger but also provide necessary information for the development of effective diagnostic, preventive, and control strategies to reduce the risk of OTA contamination in foods.

  3. Glycogen Metabolic Genes Are Involved in Trehalose-6-Phosphate Synthase-Mediated Regulation of Pathogenicity by the Rice Blast Fungus Magnaporthe oryzae

    Science.gov (United States)

    Wilson, Richard A.; Wang, Zheng-Yi; Kershaw, Michael J.; Talbot, Nicholas J.

    2013-01-01

    The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a Δagl1Δgph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae. PMID:24098112

  4. Inducible Nitric Oxide Synthase Promoter Haplotypes and Residential Traffic-Related Air Pollution Jointly Influence Exhaled Nitric Oxide Level in Children.

    Directory of Open Access Journals (Sweden)

    Muhammad T Salam

    Full Text Available Exhaled nitric oxide (FeNO, a biomarker of airway inflammation, predicts asthma risk in children. We previously found that the promoter haplotypes in inducible nitric oxide synthase (NOS2 and exposure to residential traffic independently influence FeNO level. Because NOS2 is inducible by environmental exposures such as traffic-related exposure, we tested the hypothesis that common NOS2 promoter haplotypes modulate the relationship between residential traffic-related exposure and FeNO level in children.In a cross-sectional population-based study, subjects (N = 2,457; 7-11 year-old were Hispanic and non-Hispanic white children who participated in the Southern California Children's Health Study and had FeNO measurements. For residential traffic, lengths of local roads within circular buffers (50m, 100m and 200m radii around homes around the subjects' homes were estimated using geographic information system (GIS methods. We interrogated the two most common NOS2 promoter haplotypes that were found to affect FeNO level.The relationship between local road lengths within 100m and 200m circular buffers and FeNO level varied significantly by one of the NOS2 promoter haplotypes (P-values for interaction between road length and NOS2 promoter haplotype = 0.02 and 0.03, respectively. In children who had ≤250m of local road lengths within 100m buffer around their homes, those with two copies of the haplotype had significantly lower FeNO (adjusted geometric mean = 11.74ppb; 95% confidence intervals (CI: 9.99 to 13.80 than those with no copies (adjusted geometric mean = 15.28ppb; 95% CI: 14.04 to 16.63 with statistically significant trend of lower FeNO level with increasing number of haplotype copy (P-value for trend = 0.002. In contrast, among children who had >250m of local road lengths within 100m buffer, FeNO level did not significantly differ by the haplotype copy-number (P-value for trend = 0.34. Similar interactive effects of this haplotype and local

  5. Inducible Nitric Oxide Synthase Promoter Haplotypes and Residential Traffic-Related Air Pollution Jointly Influence Exhaled Nitric Oxide Level in Children.

    Science.gov (United States)

    Salam, Muhammad T; Lin, Pi-Chu; Eckel, Sandrah P; Gauderman, W James; Gilliland, Frank D

    2015-01-01

    Exhaled nitric oxide (FeNO), a biomarker of airway inflammation, predicts asthma risk in children. We previously found that the promoter haplotypes in inducible nitric oxide synthase (NOS2) and exposure to residential traffic independently influence FeNO level. Because NOS2 is inducible by environmental exposures such as traffic-related exposure, we tested the hypothesis that common NOS2 promoter haplotypes modulate the relationship between residential traffic-related exposure and FeNO level in children. In a cross-sectional population-based study, subjects (N = 2,457; 7-11 year-old) were Hispanic and non-Hispanic white children who participated in the Southern California Children's Health Study and had FeNO measurements. For residential traffic, lengths of local roads within circular buffers (50m, 100m and 200m radii around homes) around the subjects' homes were estimated using geographic information system (GIS) methods. We interrogated the two most common NOS2 promoter haplotypes that were found to affect FeNO level. The relationship between local road lengths within 100m and 200m circular buffers and FeNO level varied significantly by one of the NOS2 promoter haplotypes (P-values for interaction between road length and NOS2 promoter haplotype = 0.02 and 0.03, respectively). In children who had ≤250m of local road lengths within 100m buffer around their homes, those with two copies of the haplotype had significantly lower FeNO (adjusted geometric mean = 11.74ppb; 95% confidence intervals (CI): 9.99 to 13.80) than those with no copies (adjusted geometric mean = 15.28ppb; 95% CI: 14.04 to 16.63) with statistically significant trend of lower FeNO level with increasing number of haplotype copy (P-value for trend = 0.002). In contrast, among children who had >250m of local road lengths within 100m buffer, FeNO level did not significantly differ by the haplotype copy-number (P-value for trend = 0.34). Similar interactive effects of this haplotype and local road

  6. Acute intermittent porphyria: A single-base deletion and a nonsense mutation in the human hydroxymethylbilane synthase gene, predicting truncations of the enzyme polypeptide

    Energy Technology Data Exchange (ETDEWEB)

    Lee, G.L.; Astrin, K.H.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States)

    1995-08-28

    Acute intermittent porphyria (AIP) is an autosomal-dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase (HMB-synthase). AIP is an ecogenetic condition, since the life-threatening acute attacks are precipitated by various factors, including drugs, alcohol, fasting, and certain hormones. Biochemical diagnosis is problematic, and the identification of mutations in the HMB-synthase gene provides accurate detection of presymptomatic heterozygotes, permitting avoidance of the acute precipitating factors. By direct solid-phase sequencing, two mutations causing AIP were identified, an adenine deletion at position 629 in exon 11(629delA), which alters the reading frame and predicts premature truncation of the enzyme protein after amino acid 255, and a nonsense mutation in exon 12 (R225X). These mutations were confirmed by either restriction enzyme analysis or family studies of symptomatic patients, permitting accurate presymptomatic diagnosis of affected relatives. 29 refs., 2 figs.

  7. Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

    Directory of Open Access Journals (Sweden)

    Ritland Carol

    2009-08-01

    Full Text Available Abstract Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs and full-length (FLcDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR and a cytochrome P450 (CYP720B4 from a non-arrayed genomic BAC library of white spruce (Picea glauca. Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR and 94 kbp (CYP720B4 long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs, high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene

  8. Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis.

    Science.gov (United States)

    Kato, Shota; Soshino, Mika; Takaichi, Shinichi; Ishikawa, Takahiro; Nagata, Noriko; Asahina, Masashi; Shinomura, Tomoko

    2017-07-17

    Photosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content. After cultivation for 7 days under continuous light at 920 μmol m-2 s-1, β-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 μmol m-2 s-1). The high-intensity light (920 μmol m-2 s-1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility

  9. The Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with the Susceptibility to Immunoglobulin a Nephropathy in Chinese Population

    Directory of Open Access Journals (Sweden)

    Jie Gao

    2017-09-01

    Full Text Available Background/Aims: Endothelial nitric oxide synthase (eNOS is one of the most important enzymes for producting nitric oxide (NO, which regulate the function of many organs and cells. The single nucleotide polymorphisms (SNPs of eNOS were found to be associated with many kidney diseases. However, it is lack of relevant studies to evaluate the associations between eNOS polymorphisms and immunoglobulin A nephropathy (IgAN. This case-control study aimed to evaluate the relationship between eNOS polymorphisms and IgAN. Methods: We recruited 351 IgAN patients and 310 age- and sex-matched healthy controls from Northwest China. Sequenom MassARRAY was used to detect the genotypes of two common eNOS SNPs (rs1799983 and rs2070744. Odds ratios (ORs and 95% confidence intervals (95% CIs were calculated by the Chi square test to evaluate the associations between eNOS and IgAN. Phase 2.1 was used to conduct haplotype analysis. Results: In the overall analysis, we found that the rs1799983 polymorphism was associated with a decreased risk of IgAN (G/T vs. G/G: OR=0.57, 95%CI=0.34–0.96; G/T+T/T vs. G/G: OR=0.52, 95%CI=0.31–0.86; G/T vs. G/G-T/T: OR=0.60, 95%CI=0.36–0.99; Log-additive model: OR=0.48, 95%CI=0.30–0.78. Haplotype analysis indicated that Trs1799983Crs2070744 is a protective factor against IgAN (OR=0.62, 95%CI=0.42––0.92. However, no significant differences were found between the two SNPs (rs1799983 and rs2070744 and clinical features (age, sex, blood pressure, and Lee’s grade of IgAN. Conclusion: The eNOS gene rs1799983 polymorphism and Trs1799983Crs2070744 haplotype may reduce the risk of IgAN in Chinese populations.

  10. Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation.

    Science.gov (United States)

    Keen, Eric C; Bliskovsky, Valery V; Malagon, Francisco; Baker, James D; Prince, Jeffrey S; Klaus, James S; Adhya, Sankar L

    2017-01-17

    Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. Bacteriophages (phages), viruses that infect bacteria, are the planet's most numerous biological entities

  11. Methylation loss at H19 imprinted gene correlates withmethylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples from infertile males

    OpenAIRE

    Rotondo, John C; Selvatici, Rita; Di Domenico, Maura; Marci, Roberto; Vesce, Fortunato; Tognon, Mauro; Martini, Fernanda

    2013-01-01

    Aberrant methylation at the H19 paternal imprinted gene has been identified in different cohorts of infertile males. The causes of H19 methylation errors are poorly understood. In this study, we investigated the methylation status of the H19 gene in semen DNA samples from infertile males affected by MTHFR gene promoter hypermethylation. DNA from normal and abnormal semen samples harbouring MTHFR gene promoter hypermethylated, hmMTHFR-nor and hmMTHFR-abn, and without MTHFR methylation, MTHFR-n...

  12. Molecular cloning and expression profile of ß-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.)

    Science.gov (United States)

    Tung tree (Vernicia fordii) is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid). Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a ...

  13. Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity.

    Science.gov (United States)

    Fattorini, L; Veloccia, A; Della Rovere, F; D'Angeli, S; Falasca, G; Altamura, M M

    2017-07-11

    Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. Altogether, results showed that IBA induced

  14. Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis M-135

    Science.gov (United States)

    Yoshikazu, Kawata; Shin-Ichi, Yano; Hiroyuki, Kojima

    1998-03-01

    An efficient and simple method for constructing a genomic DNA library using a TA cloning vector is presented. It is based on the sonicative cleavage of genomic DNA and modification of fragment ends with Taq DNA polymerase, followed by ligation using a TA vector. This method was applied for cloning of the phytoene synthase gene crt B from Spirulina platensis. This method is useful when genomic DNA cannot be efficiently digested with restriction enzymes, a problem often encountered during the construction of a genomic DNA library of cyanobacteria.

  15. Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases

    OpenAIRE

    Kim, Jaekwang; Suh, Hyunsuk; Kim, Songhee; Kim, Kiyoung; Ahn, Chiyoung; Yim, Jeongbin

    2006-01-01

    The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and...

  16. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    Science.gov (United States)

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. A functional cellulose synthase from ascidian epidermis

    OpenAIRE

    Matthysse, Ann G.; Deschet, Karine; Williams, Melanie; Marry, Mazz; White, Alan R.; Smith, William C.

    2004-01-01

    Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase ami...

  18. С(-344Т polymorphism of aldosterone synthase gene, risk of metabolic syndrome, and atrial fibrillation risk for inhabitants of the Northwest regions of Russia

    Directory of Open Access Journals (Sweden)

    I. Ma

    2016-01-01

    Full Text Available Objective: to estimate contribution of C(-344T version of aldosterone synthase CYP11B2 gene in risk of metabolic syndrome (MS and atrial fibrillation (AF for inhabitants of the Northwest region of Russia. Material and methods: 199 patients with MS, 103 from them with AF and 267 healthy persons. Identification of C-344T gene of CYP11B2 aldosterone sinthase has been performed by means of PCR method with restriction analysis. Results: in MS carriership of TT (-344 genotype met more often than in control: 33.2 % and 24.3 %, respectively (p = 0.04. Carriership of TT (-344 genotype increases risk of MS: OR = 1.54 (95 % CI 1.03-2.32 and was not associated with an increased risk of AF. Conclusion: association of TT(-344 genotype of CYP11B2 gene with risk of metabolic syndrome for inhabitants of the Northwest region ofRussia is revealed.

  19. MALAT1 is a prognostic factor in glioblastoma multiforme and induces chemoresistance to temozolomide through suppressing miR-203 and promoting thymidylate synthase expression.

    Science.gov (United States)

    Chen, Wei; Xu, Xin-Ke; Li, Jun-Liang; Kong, Kuan-Kei; Li, Hui; Chen, Cheng; He, Jing; Wang, Fangyu; Li, Ping; Ge, Xiao-Song; Li, Fang-Cheng

    2017-04-04

    Glioblastoma multiforme (GBM) is the most malignant brain tumor with limited therapeutic options. Temozolomide (TMZ) is a novel cytotoxic agent used as first-line chemotherapy for GBM, however, some individual cells can't be isolated for surgical resection and show treatment-resistance, thus inducing poor prognosis. By using the HiSeq sequencing and bioinformatics methods, we identified lncRNAs showing different expression levels in TMZ-resistant and non-resistant patients. RT-qPCR was then performed in tissues and serum samples, and lncRNA MALAT1 was finally identified to show considerable discriminating potential to identify responding patients from non-responding patients. Moreover, high serum MALAT1 expression was associated with poor chemoresponse and survival in GBM patients receiving TMZ treatment. Subsequently, the TMZ resistant cell lines were established, and the CCK8 assay showed that lncRNA MALAT1 knockdown significantly reversed TMZ resistance in GBM cells. The gain and loss-function experiments revealed that miR-203 was down-regulated by MALAT1 and this interaction has reciprocal effects. Besides, thymidylate synthase (TS) mRNA was identified as a direct target of miR-203. LncRNA MALAT1 inhibition re-sensitized TMZ resistant cells through up-regulating miR-203 and down-regulating TS expression. On the other hand, MALAT1 overexpression promoted resistance by suppressing miR-203 and promoting TS expression. In conclusion, our integrated approach demonstrates that enhanced expression of lncRNA MALAT1 confers a potent poor therapeutic efficacy and inhibition of MALAT1 levels could be a future direction to develop a novel therapeutic strategy to overcome TMZ resistance in GBM patients.

  20. A Mutant of Hepatitis B Virus X Protein (HBxΔ127 Promotes Cell Growth through A Positive Feedback Loop Involving 5-Lipoxygenase and Fatty Acid Synthase

    Directory of Open Access Journals (Sweden)

    Qi Wang

    2010-02-01

    Full Text Available Hepatocellular carcinoma (HCC is one of the most common malignant tumors worldwide. Hepatitis B virus X protein (HBx contributes to the development of HCC, whereas HBx with COOH-terminal deletion is a frequent event in the HCC tissues. Previously, we identified a natural mutant of HBx-truncated 27 amino acids at the COOH-terminal (termed HBxΔ127, which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. Accordingly, fatty acid synthase (FAS plays a crucial role in cancer cell survival and proliferation; thus, we examined the signaling pathways involving FAS. Our data showed that HBxΔ127 strongly increased the transcriptional activities of FAS in human hepatoma HepG2 and H7402 cells. Moreover, we found that 5-lipoxygenase (5-LOX was responsible for the up-regulation of FAS by using MK886 (an inhibitor of 5-LOX and 5-LOX small interfering RNA. We observed that HBxΔ127 could upregulate 5-LOX through phosphorylated extracellular signal-regulated protein kinases 1/2 and thus resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX by ELISA. The additional LTB4 could upregulate the expression of FAS in the cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS. Collectively, HBxΔ127 promotes cell growth through a positive feedback loop involving 5-LOX and FAS, in which released LTB4 is involved in the up-regulation of FAS. Thus, our finding provides a new insight into the mechanism involving the promotion of cell growth mediated by HBxΔ127.

  1. Genome-wide analysis of regions similar to promoters of histone genes

    KAUST Repository

    Chowdhary, Rajesh

    2010-05-28

    Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that

  2. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  3. [The methylation of ZHX2 gene promoter enhances AFP gene expression in hepatocellular carcinoma].

    Science.gov (United States)

    Lv, Zili; DU, Yangjun; Wen, Jianming

    2013-07-01

    To investigate the relationship between Zinc-fingers and homeoboxes 2 (ZHX2) promoter methylation and alpha-fetoprotein (AFP) gene expression, and analyze the mechanism of AFP gene expression. HepG2 cell line was cultured with 0.5, 1.0 or 5.0 μmol/L of 5-aza-deoxycytidine (5-Aza-Dc). RT-PCR and Western blotting were used to detect the expressions of ZHX2 and AFP in HepG2 cell line. Methylation-specific PCR was used to detect ZHX2 promoter methylation in 38 hepatocellular carcinoma tissues. The HepG2 cell line showed a low level of ZHX2 mRNA, negative expression of ZHX2 protein, but high expression of AFP at both mRNA and protein levels. After the HepG2 cells were treated with 1.0 or 5.0 μmol/L 5-Aza-Dc for 6 d, the expression of ZHX2 mRNA and protein increased and the expression of AFP mRNA and protein decreased. Among 38 hepatocellular carcinoma tissues, ZHX2 promoter methylation was found in 16 hepatocellular carcinoma tissues with AFP>25 ng/mL in serum. No methylation of ZHX2 promoter was found in 8 hepatocellular carcinoma tissues with AFPexpression.

  4. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.; Salentijn, E.M.J.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  5. Genome-wide analyses of chitin synthases identify horizontal gene transfers towards bacteria and allow a robust and unifying classification into fungi.

    Science.gov (United States)

    Gonçalves, Isabelle R; Brouillet, Sophie; Soulié, Marie-Christine; Gribaldo, Simonetta; Sirven, Catherine; Charron, Noémie; Boccara, Martine; Choquer, Mathias

    2016-11-24

    Chitin, the second most abundant biopolymer on earth after cellulose, is found in probably all fungi, many animals (mainly invertebrates), several protists and a few algae, playing an essential role in the development of many of them. This polysaccharide is produced by type 2 glycosyltransferases, called chitin synthases (CHS). There are several contradictory classifications of CHS isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate. A genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about 130 genomes. Phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences (e.g. highly variable regions, truncated or recombined sequences, long-branch attraction). This allowed us to revise and unify the fungal CHS classification and to study the evolutionary history of the CHS multigenic family. This update has the advantage of being user-friendly due to the development of a dedicated website ( http://wwwabi.snv.jussieu.fr/public/CHSdb ), and it includes any correspondences with previously published classifications and mutants. Concerning the evolutionary history of CHS, this family has mainly evolved via duplications and losses. However, it is likely that several horizontal gene transfers (HGT) also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria. This comprehensive multi-species analysis contributes to the classification of fungal CHS, in particular by optimizing its robustness, consensuality and accessibility. It also highlights the importance of HGT in the evolutionary history of CHS and describes bacterial chs genes for the first time. Many of the bacteria that have acquired a chitin synthase are plant pathogens (e.g. Dickeya spp; Pectobacterium spp; Brenneria spp; Agrobacterium vitis and Pseudomonas cichorii). Whether they are able to

  6. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  7. Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

    Energy Technology Data Exchange (ETDEWEB)

    Paez, David, E-mail: dpaez@santpau.cat [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Salazar, Juliana; Pare, Laia [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Pertriz, Lourdes [Department of Radiotherapy, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Targarona, Eduardo [Department of Surgery, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Rio, Elisabeth del [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Barnadas, Agusti; Marcuello, Eugenio [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Baiget, Montserrat [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain)

    2011-12-01

    Purpose: Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Methods and Materials: Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5 Prime UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. Results: The Asterisk-Operator 3/ Asterisk-Operator 3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in Asterisk-Operator 3/ Asterisk-Operator 3 vs. 35% in Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk-Operator 3; p = .013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the Asterisk-Operator 3/ Asterisk-Operator 3 patients and 84 months for the Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk

  8. Pharmacogenetic study in rectal cancer patients treated with preoperative chemoradiotherapy: polymorphisms in thymidylate synthase, epidermal growth factor receptor, GSTP1, and DNA repair genes.

    Science.gov (United States)

    Páez, David; Salazar, Juliana; Paré, Laia; Pertriz, Lourdes; Targarona, Eduardo; del Rio, Elisabeth; Barnadas, Agusti; Marcuello, Eugenio; Baiget, Montserrat

    2011-12-01

    Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5'UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. The ∗3/∗3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in ∗3/∗3 vs. 35% in ∗2/∗2 and ∗2/∗3; p=.013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the ∗3/∗3 patients and 84 months for the ∗2/∗2 and ∗2/∗3 patients (p=.039). For XRCC1 Arg399Gln SNP, the median progression-free survival was 101 months for the G/G, 78 months for the G/A, and 31 months for the A/A patients (p=.048). The thymidylate synthase genotype and XRCC1 Arg399Gln polymorphism might help to

  9. The Jasmonate-Activated Transcription Factor MdMYC2 Regulates ETHYLENE RESPONSE FACTOR and Ethylene Biosynthetic Genes to Promote Ethylene Biosynthesis during Apple Fruit Ripening[OPEN

    Science.gov (United States)

    Xu, Yaxiu; Zhang, Lichao; Ji, Yinglin; Tan, Dongmei; Yuan, Hui

    2017-01-01

    The plant hormone ethylene is critical for ripening in climacteric fruits, including apple (Malus domestica). Jasmonate (JA) promotes ethylene biosynthesis in apple fruit, but the underlying molecular mechanism is unclear. Here, we found that JA-induced ethylene production in apple fruit is dependent on the expression of MdACS1, an ACC synthase gene involved in ethylene biosynthesis. The expression of MdMYC2, encoding a transcription factor involved in the JA signaling pathway, was enhanced by MeJA treatment in apple fruits, and MdMYC2 directly bound to the promoters of both MdACS1 and the ACC oxidase gene MdACO1 and enhanced their transcription. Furthermore, MdMYC2 bound to the promoter of MdERF3, encoding a transcription factor involved in the ethylene-signaling pathway, thereby activating MdACS1 transcription. We also found that MdMYC2 interacted with MdERF2, a suppressor of MdERF3 and MdACS1. This protein interaction prevented MdERF2 from interacting with MdERF3 and from binding to the MdACS1 promoter, leading to increased transcription of MdACS1. Collectively, these results indicate that JA promotes ethylene biosynthesis through the regulation of MdERFs and ethylene biosynthetic genes by MdMYC2. PMID:28550149

  10. The Jasmonate-Activated Transcription Factor MdMYC2 Regulates ETHYLENE RESPONSE FACTOR and Ethylene Biosynthetic Genes to Promote Ethylene Biosynthesis during Apple Fruit Ripening.

    Science.gov (United States)

    Li, Tong; Xu, Yaxiu; Zhang, Lichao; Ji, Yinglin; Tan, Dongmei; Yuan, Hui; Wang, Aide

    2017-06-01

    The plant hormone ethylene is critical for ripening in climacteric fruits, including apple (Malus domestica). Jasmonate (JA) promotes ethylene biosynthesis in apple fruit, but the underlying molecular mechanism is unclear. Here, we found that JA-induced ethylene production in apple fruit is dependent on the expression of MdACS1, an ACC synthase gene involved in ethylene biosynthesis. The expression of MdMYC2, encoding a transcription factor involved in the JA signaling pathway, was enhanced by MeJA treatment in apple fruits, and MdMYC2 directly bound to the promoters of both MdACS1 and the ACC oxidase gene MdACO1 and enhanced their transcription. Furthermore, MdMYC2 bound to the promoter of MdERF3, encoding a transcription factor involved in the ethylene-signaling pathway, thereby activating MdACS1 transcription. We also found that MdMYC2 interacted with MdERF2, a suppressor of MdERF3 and MdACS1 This protein interaction prevented MdERF2 from interacting with MdERF3 and from binding to the MdACS1 promoter, leading to increased transcription of MdACS1 Collectively, these results indicate that JA promotes ethylene biosynthesis through the regulation of MdERFs and ethylene biosynthetic genes by MdMYC2. © 2017 American Society of Plant Biologists. All rights reserved.

  11. Molecular characterization of a cellulose synthase gene (AaxmCesA1) isolated from an Acacia auriculiformis x Acacia mangium hybrid.

    Science.gov (United States)

    Yong, Seok Yien Christina; Wickneswari, Ratnam

    2013-01-01

    Cellulose is the major component of plant cell walls, providing mechanical strength to the structural framework of plants. In association with lignin, hemicellulose, protein and pectin, cellulose forms the strong yet flexible bio-composite tissue of wood. Wood formation is an essential biological process and is of significant importance to the cellulosic private sector industry. Cellulose synthase genes encode the catalytic subunits of a large protein complex responsible for the biogenesis of cellulose in higher plants. The hybrid Acacia auriculiformis x Acacia mangium represents an important source of tree cellulose for forest-based product manufacturing, with enormous economic potential. In this work, we isolate the first cellulose synthase gene, designated AaxmCesA1, from this species. The isolated full-length AaxmCesA1 cDNA encodes a polypeptide of 1,064 amino acids. Sequence analyses revealed that AaxmCesA1 cDNA possesses the key motif characteristics of a CesA protein. AaxmCesA1 shares more than 75 % amino acid sequence identity with CesA proteins from other plant species. Subsequently, the full-length AaxmCesA1 gene of 7,389 bp with partial regulatory and 13 intron regions was also isolated. Relative gene expression analysis by quantitative PCR in different tissues of the Acacia hybrid, suggests the involvement of the AaxmCesA1 gene in primary cell wall synthesis of rapidly dividing young root cells. Similarity analyses using Blast algorithms also suggests a role in primary cell wall deposition in the Acacia hybrid. Southern analysis predicts that AaxmCesA1 is a member of a multigene family with at least two isoforms in the genome of the Acacia hybrid.

  12. Polyketide genes in the marine sponge Plakortis simplex: a new group of mono-modular type I polyketide synthases from sponge symbionts

    Science.gov (United States)

    Della Sala, Gerardo; Hochmuth, Thomas; Costantino, Valeria; Teta, Roberta; Gerwick, William; Gerwick, Lena; Piel, Jörn; Mangoni, Alfonso

    2013-01-01

    Summary Sponge symbionts are a largely unexplored source of new and unusual metabolic pathways. Insights into the distribution and function of metabolic genes of sponge symbionts are crucial to dissect and exploit their biotechnological potential. Screening of the metagenome of the marine sponge Plakortis simplex led to the discovery of the swf family, a new group of mono-modular type I polyketide synthase/fatty acid synthase (PKS/FAS) specifically associated with sponge symbionts. Two different examples of the swf cluster were present in the metagenome of P. simplex. A third example of the cluster is present in the previously sequenced genome of a poribacterium from the sponge Aplysina aerophoba but was formerly considered orthologous to the wcb/rkp cluster. The swf cluster was also found in six additional species of sponges. Therefore, the swf cluster represents the second group of mono-modular PKS, after the supA family, to be widespread in marine sponges. The putative swf operon consists of swfA (type I PKS/FAS), swfB (reductase and sulphotransferase domains) and swfC (radical S-adenosylmethionine, or radical SAM). Activation of the acyl carrier protein (ACP) domain of the SwfA protein to its holo-form by co-expression with Svp is the first functional proof of swf type genes in marine sponges. However, the precise biosynthetic role of the swf clusters remains unknown. PMID:24249289

  13. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

    DEFF Research Database (Denmark)

    Brinch-Pedersen, Henrik; Galili, G; Knudsen, S

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the...... as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.......In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance...... the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8...

  14. Fatty acid synthase 2 contributes to diapause preparation in a beetle by regulating lipid accumulation and stress tolerance genes expression

    OpenAIRE

    Tan, Qian-Qian; Liu, Wen; Zhu, Fen; Lei, Chao-Liang; Wang, Xiao-Ping

    2017-01-01

    Diapause, also known as dormancy, is a state of arrested development that allows insects to survive unfavorable environmental conditions. Diapause-destined insects store large amounts of fat when preparing for diapause. However, the extent to which these accumulated fat reserves influence diapause remains unclear. To address this question, we investigated the function of fatty acid synthase (FAS), which plays a central role in lipid synthesis, in stress tolerance, the duration of diapause pre...

  15. Relationship between Expression of Chalcone Synthase Genes and Chromones in Artificial Agarwood induced by Formic Acid Stimulation Combined with Fusarium sp. A2 Inoculation.

    Science.gov (United States)

    Chen, Xiaodong; Zhu, Xiaoling; Feng, Meirou; Zhong, Zhaojian; Zhou, Xin; Chen, Xiaoying; Ye, Wei; Zhang, Weimin; Gao, Xiaoxia

    2017-04-25

    Agarwood (gaharu) is a fragrant resin produced in the heartwood of resinous Gyrinops and Aquilaria species. Artificial agarwood samples were obtained from Aquilaria sinensis (Lour.) Gilg using formic acid (FA) stimulation combined with Fusarium sp. A2 inoculation. The relationship between the expression of chalcone synthase genes (CHS) and dynamic changes in chromone content was explored in resin-deposited parts of the trunks of A. sinensis. CHS gene expression levels were detected by qRT-PCR analysis. The chemical composition of agarwood obtained from the heartwood of A. sinensis before and within 1 year after induction was determined by GC-MS. After induction with FA stimulation combined with F. sp. A2 inoculation, the CHS1 gene showed relatively high expression, whereas the CHS2 gene showed low expression. The relative gene expression level of CHS1 peaked at 12 months, with a 153.1-fold increase, and the dominant period of the CHS2 gene expression was 10 months with a 14.13-fold increase. Moreover, chromones were not detected until after 2 months, and a large proportion of chromone compounds were detected after 4 months. Chromone content increased with time and peaked at 12 months. CHS1 gene expression was significantly correlated with 6-hydroxy-2-(2-phenylethyl)chromone accumulation, and CHS2 gene expression was significantly correlated with 5-hydroxy-6-methoxy-2-(2-phenylethyl)chromone accumulation. CHS gene expression was extremely sensitive to FA stimulation combined with F. sp. A2 inoculation and responded to late-onset injury. CHS genes expression also preceded the chromone accumulation. This work laid the foundation for studies on the mechanism by which genes regulate chromone biosynthesis pathways during the formation of agarwood resin in A. sinensis.

  16. First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

    Directory of Open Access Journals (Sweden)

    Patrick C Y Woo

    Full Text Available BACKGROUND: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes. METHODOLOGY/PRINCIPAL FINDINGS: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05. There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05. CONCLUSIONS/SIGNIFICANCE: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid

  17. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  18. An X11alpha/FSBP complex represses transcription of the GSK3beta gene promoter.

    LENUS (Irish Health Repository)

    Lau, Kwok-Fai

    2010-08-04

    X11alpha is a neuronal adaptor protein that interacts with the amyloid precursor protein (APP) through a centrally located phosphotyrosine binding domain to inhibit the production of Abeta peptide that is deposited in Alzheimer\\'s disease brains. X11alpha also contains two C-terminal postsynaptic density-95, large discs, zona occludens 1 (PDZ) domains, and we show here that through its PDZ domains, X11alpha interacts with a novel transcription factor, fibrinogen silencer binding protein. Moreover, we show that an X11alpha\\/fibrinogen silencer binding protein complex signals to the nucleus to repress glycogen synthase kinase-3beta promoter activity. Glycogen synthase kinase-3beta is a favoured candidate kinase for phosphorylating tau in Alzheimer\\'s disease. Our findings show a new function for X11alpha that may impact on Alzheimer\\'s disease pathogenesis.

  19. Transcriptional coupling of synaptic transmission and energy metabolism: role of nuclear respiratory factor 1 in co-regulating neuronal nitric oxide synthase and cytochrome c oxidase genes in neurons.

    Science.gov (United States)

    Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T

    2009-10-01

    Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.

  20. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  1. Functional analysis of Plasmodium vivax dihydrofolate reductase-thymidylate synthase genes through stable transformation of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Alyson M Auliff

    Full Text Available Mechanisms of drug resistance in Plasmodium vivax have been difficult to study partially because of the difficulties in culturing the parasite in vitro. This hampers monitoring drug resistance and research to develop or evaluate new drugs. There is an urgent need for a novel method to study mechanisms of P. vivax drug resistance. In this paper we report the development and application of the first Plasmodium falciparum expression system to stably express P. vivax dhfr-ts alleles. We used the piggyBac transposition system for the rapid integration of wild-type, single mutant (117N and quadruple mutant (57L/58R/61M/117T pvdhfr-ts alleles into the P. falciparum genome. The majority (81% of the integrations occurred in non-coding regions of the genome; however, the levels of pvdhfr transcription driven by the P. falciparum dhfr promoter were not different between integrants of non-coding and coding regions. The integrated quadruple pvdhfr mutant allele was much less susceptible to antifolates than the wild-type and single mutant pvdhfr alleles. The resistance phenotype was stable without drug pressure. All the integrated clones were susceptible to the novel antifolate JPC-2067. Therefore, the piggyBac expression system provides a novel and important tool to investigate drug resistance mechanisms and gene functions in P. vivax.

  2. Functional analysis of Plasmodium vivax dihydrofolate reductase-thymidylate synthase genes through stable transformation of Plasmodium falciparum.

    Science.gov (United States)

    Auliff, Alyson M; Balu, Bharath; Chen, Nanhua; O'Neil, Michael T; Cheng, Qin; Adams, John H

    2012-01-01

    Mechanisms of drug resistance in Plasmodium vivax have been difficult to study partially because of the difficulties in culturing the parasite in vitro. This hampers monitoring drug resistance and research to develop or evaluate new drugs. There is an urgent need for a novel method to study mechanisms of P. vivax drug resistance. In this paper we report the development and application of the first Plasmodium falciparum expression system to stably express P. vivax dhfr-ts alleles. We used the piggyBac transposition system for the rapid integration of wild-type, single mutant (117N) and quadruple mutant (57L/58R/61M/117T) pvdhfr-ts alleles into the P. falciparum genome. The majority (81%) of the integrations occurred in non-coding regions of the genome; however, the levels of pvdhfr transcription driven by the P. falciparum dhfr promoter were not different between integrants of non-coding and coding regions. The integrated quadruple pvdhfr mutant allele was much less susceptible to antifolates than the wild-type and single mutant pvdhfr alleles. The resistance phenotype was stable without drug pressure. All the integrated clones were susceptible to the novel antifolate JPC-2067. Therefore, the piggyBac expression system provides a novel and important tool to investigate drug resistance mechanisms and gene functions in P. vivax.

  3. Effect of thymidylate synthase gene polymorphism on the response to chemotherapy and clinical outcome of non-small cell lung cancer patients.

    Science.gov (United States)

    Dong, Honglin; Bao, Dengke; Guo, Xu; Hu, Jie; Li, Xiaofei; Wan, Shaogui; Xing, Jinliang

    2015-09-01

    Genetic polymorphisms of thymidylate synthase (TYMS) gene have been reported to be associated with development or prognosis of several cancers. However, the association between polymorphisms of TYMS gene and clinical outcomes of non-small cell lung cancer (NSCLC) patients are still unknown. In the present study, we investigated the associations between single nucleotide polymorphisms (SNPs) of TYMS gene and response to chemotherapy as well as clinical outcomes in NSCLC patients. Five SNPs in TYMS gene were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 500 NSCLC patients, and their associations with NSCLC outcomes were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant, and recessive models). Our data showed that there was no significant association between individual SNP and overall survival of NSCLC patients. However, SNP rs2847153 was significantly associated with NSCLC recurrence under recessive model. We further identified a significant interaction between rs2847153 and chemotherapy in modifying clinical outcome of patients. Our data showed that individuals carrying GG/GA genotypes of rs2847153 had a significantly better response to chemotherapy when comparing to those carrying AA genotype. Conclusively, our data suggest that SNPs rs2847153 in TYMS gene may be a potential biomarker for predicting clinical outcome and personalized treatment in NSCLC patients.

  4. Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

    Science.gov (United States)

    Zhang, Lu; Wang, Huijuan; Chen, Jianyi; Shen, Qida; Wang, Shigui; Xu, Hongxing; Tang, Bin

    2017-01-01

    RNA interference has been used to study insects' gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates' conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  5. RNA interference of a trehalose-6-phosphate synthase gene reveals its roles during larval-pupal metamorphosis in Bactrocera minax (Diptera: Tephritidae).

    Science.gov (United States)

    Xiong, Ke-Cai; Wang, Jia; Li, Jia-Hao; Deng, Yu-Qing; Pu, Po; Fan, Huan; Liu, Ying-Hong

    2016-01-01

    Trehalose is the major blood sugar in insects, which plays a crucial role as an instant source of energy and the starting substrate for chitin biosynthesis. In insects, trehalose is synthesized by catalysis of an important enzyme, trehalose-6-phosphate synthase (TPS). In the present study, a trehalose-6-phosphate synthase gene from Bactrocera minax (BmTPS) was cloned and characterized. BmTPS contained an open reading frame of 2445 nucleotides encoding a protein of 814 amino acids with a predicted molecular weight of 92.05kDa. BmTPS was detectable in all developmental stages of Bactrocera minax and expressed higher in the final- (third-) instar larvae. Tissue-specific expression patterns of BmTPS showed that it was mainly expressed in the fat body. The 20-hydroxyecdysone (20E) induced the expression of BmTPS and three genes in the chitin biosynthesis pathway. Moreover, injection of double-stranded RNA into third-instar larvae successfully silenced the transcription of BmTPS in B. minax, and thereby decreased the activity of TPS and trehalose content. Additionally, silencing of BmTPS inhibited the expression of three key genes in the chitin biosynthesis pathway and exhibited 52% death and abnormal phenotypes. The findings demonstrate that BmTPS is indispensable for larval-pupal metamorphosis. Besides, the establishment of RNAi experimental system in B. minax would lay a solid foundation for further investigation of molecular biology and physiology of this pest. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae

    Directory of Open Access Journals (Sweden)

    Kang-Kang Xu

    2013-08-01

    Full Text Available Chitin synthase (CHS, a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2 was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

  7. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang

    2011-01-01

    element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked...

  8. Plant Growth-Promoting Genes can Switch to be Virulence Factors via Horizontal Gene Transfer.

    Science.gov (United States)

    Stritzler, Margarita; Soto, Gabriela; Ayub, Nicolás

    2018-02-23

    There are increasing evidences that horizontal gene transfer (HGT) is a critical mechanism of bacterial evolution, while its complete impact remains unclear. A main constraint of HGT effects on microbial evolution seems to be the conservation of the function of the horizontally transferred genes. From this perspective, inflexible nomenclature and functionality criteria have been established for some mobile genetic elements such as pathogenic and symbiotic islands. Adhesion is a universal prerequisite for both beneficial and pathogenic plant-microbe interactions, and thus, adhesion systems (e.g., the Lap cluster) are candidates to have a dual function depending on the genomic background. In this study, we showed that the virulent factor Lap of the phytopathogen Erwinia carotovora SCRI1043, which is located within a genomic island, was acquired by HGT and probably derived from Pseudomonas. The transformation of the phytopathogen Erwinia pyrifoliae Ep1/96 with the beneficial factor Lap from the plant growth-promoting bacterium Pseudomonas fluorescens Pf-5 significantly increased its natural virulence, experimentally recapitulating the beneficial-to-virulence functional switch of the Lap cluster via HGT. To our knowledge, this is the first report of a functional switch of an individual gene or a cluster of genes mediated by HGT.

  9. Analysis of the gene polymorphism of aldosterone synthase (CYP11B2) and atrial natriuretic peptide (ANP) in women with preeclampsia.

    Science.gov (United States)

    Bogacz, Anna; Bartkowiak-Wieczorek, Joanna; Procyk, Danuta; Seremak-Mrozikiewicz, Agnieszka; Majchrzycki, Marian; Dziekan, Karolina; Bienert, Agnieszka; Czerny, Bogusław

    2016-02-01

    Preeclampsia (PE) is a major cause of mortality of mothers, fetuses and newborns around the world. The etiology of preeclampsia has not yet been clarified, but many studies indicate a multifactorial basis of PE. Aldosterone synthase (CYP11B2) is responsible for synthesis of aldosterone responsible for regulating blood pressure. Similarly, natriuretic peptide (ANP) regulates blood pressure through a variety of mechanisms affecting the sodium concentration and the amount of extracellular fluid. Currently, attention is paid to the role of the polymorphisms in the expression level of these genes. The aim of the study was to determine the frequencies of genotypes and alleles for polymorphisms of -344C>T CYP11B2 gene and 2238T>C ANP gene in women with preeclampsia and healthy pregnant women from the Caucasian population. The study included a group of 165 pregnant women (59 women with preeclampsia and 109 healthy pregnant women). DNA was extracted from peripheral blood. Determination of the polymorphism of -344C>T CYP11B2 gene and 2238T>C ANP gene was performed by PCR-RFLP method. The results showed that the frequencies of the TC and CC genotypes of 2238T>C polymorphism in ANP gene were significantly higher in patients with PE compared to control group. For -344C>T polymorphism of CYP11B2 gene, the frequency of TT genotype was significantly higher in patients with hypertension than in controls (32.2% vs. 23.58%). Our findings showed that gene polymorphism of CYP11B2 (-344C>T) and ANP (2238T>C) may be associated with developing PE during pregnancy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Structure and expression profile of the sucrose synthase gene family in the rubber tree: indicative of roles in stress response and sucrose utilization in the laticifers.

    Science.gov (United States)

    Xiao, Xiaohu; Tang, Chaorong; Fang, Yongjun; Yang, Meng; Zhou, Binhui; Qi, Jiyan; Zhang, Yi

    2014-01-01

    Sucrose synthase (Sus, EC 2.4.1.13) is widely recognized as a key enzyme in sucrose metabolism in plants. However, nothing is known about this gene family in Hevea brasiliensis (para rubber tree). Here, we identified six Sus genes in H. brasiliensis that comprise the entire Sus family in this species. Analysis of the gene structure and phylogeny of the Sus genes demonstrates evolutionary conservation in the Sus families across Hevea and other plant species. The expression of Sus genes was investigated via Solexa sequencing and quantitative PCR in various tissues, at various phases of leaf development, and under abiotic stresses and ethylene treatment. The Sus genes exhibited distinct but partially redundant expression profiles. Each tissue has one abundant Sus isoform, with HbSus3, 4 and 5 being the predominant isoforms in latex (cytoplasm of rubber-producing laticifers), bark and root, respectively. HbSus1 and 6 were barely expressed in any tissue examined. In mature leaves (source), all HbSus genes were expressed at low levels, but HbSus3 and 4 were abundantly expressed in immature leaves (sink). Low temperature and drought treatments conspicuously induced HbSus5 expression in root and leaf, suggesting a role in stress responses. HbSus2 and 3 transcripts were decreased by ethylene treatment, consistent with the reduced sucrose-synthesizing activity of Sus enzymes in the latex in response to ethylene stimulation. Our results are beneficial to further determination of functions for the Sus genes in Hevea trees, especially roles in regulating latex regeneration. © 2013 FEBS.

  11. Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

    Directory of Open Access Journals (Sweden)

    Toub Omid

    2010-10-01

    Full Text Available Abstract Background Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS were predicted by in silico analysis of the grapevine (Vitis vinifera genome assembly 1. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions. Results We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation. Conclusions The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information

  12. Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

    Science.gov (United States)

    2010-01-01

    Background Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions. Results We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation. Conclusions The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and

  13. Eukaryotic genomes may exhibit up to 10 generic classes of gene promoters

    Directory of Open Access Journals (Sweden)

    Gagniuc Paul

    2012-09-01

    Full Text Available Abstract Background The main function of gene promoters appears to be the integration of different gene products in their biological pathways in order to maintain homeostasis. Generally, promoters have been classified in two major classes, namely TATA and CpG. Nevertheless, many genes using the same combinatorial formation of transcription factors have different gene expression patterns. Accordingly, we tried to ask ourselves some fundamental questions: Why certain genes have an overall predisposition for higher gene expression levels than others? What causes such a predisposition? Is there a structural relationship of these sequences in different tissues? Is there a strong phylogenetic relationship between promoters of closely related species? Results In order to gain valuable insights into different promoter regions, we obtained a series of image-based patterns which allowed us to identify 10 generic classes of promoters. A comprehensive analysis was undertaken for promoter sequences from Arabidopsis thaliana, Drosophila melanogaster, Homo sapiens and Oryza sativa, and a more extensive analysis of tissue-specific promoters in humans. We observed a clear preference for these species to use certain classes of promoters for specific biological processes. Moreover, in humans, we found that different tissues use distinct classes of promoters, reflecting an emerging promoter network. Depending on the tissue type, comparisons made between these classes of promoters reveal a complementarity between their patterns whereas some other classes of promoters have been observed to occur in competition. Furthermore, we also noticed the existence of some transitional states between these classes of promoters that may explain certain evolutionary mechanisms, which suggest a possible predisposition for specific levels of gene expression and perhaps for a different number of factors responsible for triggering gene expression. Our conclusions are based on

  14. Ketide Synthase (KS domain prediction and analysis of iterative type II PKS gene in marine sponge-associated actinobacteria producing biosurfactants and antimicrobial agents

    Directory of Open Access Journals (Sweden)

    George Seghal Kiran

    2016-02-01

    Full Text Available The important biological macromolecules such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS genes was also explored. A unique feature of type II PKS genes is their high amino acid sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of amino acid sequence encoded by PKS domains tailored the chemical structure of polyketide analogues still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species with known PKS and metabolic products to correlate the domain architecture and structural features shared with known PKS proteins. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products.

  15. Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents.

    Science.gov (United States)

    Selvin, Joseph; Sathiyanarayanan, Ganesan; Lipton, Anuj N; Al-Dhabi, Naif Abdullah; Valan Arasu, Mariadhas; Kiran, George S

    2016-01-01

    The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products.

  16. A soluble starch synthase I gene, IbSSI, alters the content, composition, granule size and structure of starch in transgenic sweet potato.

    Science.gov (United States)

    Wang, Yannan; Li, Yan; Zhang, Huan; Zhai, Hong; Liu, Qingchang; He, Shaozhen

    2017-05-24

    Soluble starch synthase I (SSI) is a key enzyme in the biosynthesis of plant amylopectin. In this study, the gene named IbSSI, was cloned from sweet potato, an important starch crop. A high expression level of IbSSI was detected in the leaves and storage roots of the sweet potato. Its overexpression significantly increased the content and granule size of starch and the proportion of amylopectin by up