Science.gov

Sample records for acid invertase gene

  1. Transcripts for genes encoding soluble acid invertase and sucrose synthase accumulate in root tip and cortical cells containing mycorrhizal arbuscules.

    PubMed

    Blee, Kristopher A; Anderson, Anne J

    2002-09-01

    Arbuscule formation by the arbuscular mycorrhizal fungus Glomus intraradices (Schenck & Smith) was limited to cortical cells immediately adjacent to the endodermis. Because these cortical cells are the first to intercept photosynthate exiting the vascular cylinder, transcript levels for sucrose metabolizing-enzymes were compared between mycorrhizal and non-mycorrhizal roots. The probes corresponded to genes encoding a soluble acid invertase with potential vacuolar targeting, which we generated from Phaseolus vulgaris roots, a Rhizobium-responsive sucrose synthase of soybean and a cell wall acid invertase of carrot. Transcripts in non-mycorrhizal roots were developmentally regulated and abundant in the root tips for all three probes but in differentiated roots of P. vulgaris they were predominantly located in phloem tissues for sucrose synthase or the endodermis and phloem for soluble acid invertase. In mycorrhizal roots increased accumulations of transcripts for sucrose synthase and vacuolar invertase were both observed in the same cortical cells bearing arbuscules that fluoresce. There was no effect on the expression of the cell wall invertase gene in fluorescent carrot cells containing arbuscules. Thus, it appears that presence of the fungal hyphae in the fluorescent arbusculated cell stimulates discrete alterations in expression of sucrose metabolizing enzymes to increase the sink potential of the cell. PMID:12175013

  2. Gibberellic acid stimulates acid invertase secretion in pea ovary protoplasts.

    PubMed

    Estruch, J J; Beltrán, J P

    1991-02-25

    Protoplasts purified from mesocarp of nonpollinated pea (Pisum sativum L.) ovaries released acid invertase to the incubation medium. The association of the acid invertase with microsomal fractions, and the sensitivity to energy-metabolism inhibitors and to tunicamycin, indicated the secretory nature of the release process. In the presence of GA3 (10 microM), the protoplasts increased their invertase secretion at about 60 min, this effect being counteracted by tunicamycin but not by cycloheximide. Subcellular fractionation of GA3-treated protoplasts showed that higher invertase secretion was the result of a promotion of invertase transfer from endoplasmic reticulum (ER) to Golgi apparatus. PMID:2001743

  3. Genome-Wide Identification of the Invertase Gene Family in Populus

    PubMed Central

    Su, Xiaoxing; Rao, Pian; An, Xinmin

    2015-01-01

    Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials. PMID:26393355

  4. Genome-Wide Identification of the Invertase Gene Family in Populus.

    PubMed

    Chen, Zhong; Gao, Kai; Su, Xiaoxing; Rao, Pian; An, Xinmin

    2015-01-01

    Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials. PMID:26393355

  5. Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

    PubMed

    McKenzie, Marian J; Chen, Ronan K Y; Harris, John C; Ashworth, Matthew J; Brummell, David A

    2013-01-01

    Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS. PMID:22734927

  6. Expression patterns of Brassica napus genes implicate IPT, CKX, sucrose transporter, cell wall invertase, and amino acid permease gene family members in leaf, flower, silique, and seed development.

    PubMed

    Song, Jiancheng; Jiang, Lijun; Jameson, Paula Elizabeth

    2015-08-01

    Forage brassica (Brassica napus cv. Greenland) is bred for vegetative growth and biomass production, while its seed yield remains to be improved for seed producers without affecting forage yield and quality. Cytokinins affect seed yield by influencing flower, silique and seed number, and seed size. To identify specific cytokinin gene family members as targets for breeding, as well as genes associated with yield and/or quality, a B. napus transcriptome was obtained from a mixed sample including leaves, flower buds and siliques of various stages. Gene families for cytokinin biosynthesis (BnIPT1, 2, 3, 5, 7, 8 and 9), cytokinin degradation (BnCKX1 to BnCKX7), cell wall invertase (BnCWINV1 to BnCWINV6), sugar transporter (BnSUT1 to BnSUT6) and amino acid permease (BnAAP1 to BnAAP8) were identified. As B. napus is tetraploid, homoeologues of each gene family member were sought. Using multiple alignments and phylogenetic analysis, the parental genomes of the two B. napus homoeologues could be differentiated. RT-qPCR was then used to determine the expression of gene family members and their homoeologues in leaves, flowers, siliques and seeds of different developmental stages. The expression analysis showed both temporal and organ-specific expression profiles among members of these multi-gene families. Several pairs of homoeologues showed differential expression, both in terms of level of expression and differences in temporal or organ-specificity. BnCKX2 and 4 were identified as targets for TILLING, EcoTILLING and MAS. PMID:25873685

  7. Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris.

    PubMed

    Veana, Fabiola; Fuentes-Garibay, José Antonio; Aguilar, Cristóbal Noé; Rodríguez-Herrera, Raúl; Guerrero-Olazarán, Martha; Viader-Salvadó, José María

    2014-09-01

    β-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 °C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 °C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry. PMID:25039056

  8. Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE.

    PubMed

    Lin, Yuan; Liu, Tengfei; Liu, Jun; Liu, Xun; Ou, Yongbin; Zhang, Huiling; Li, Meng; Sonnewald, Uwe; Song, Botao; Xie, Conghua

    2015-08-01

    Slowing down cold-induced sweetening (CIS) of potato (Solanum tuberosum) tubers is of economic importance for the potato industry to ensure high-quality products. The conversion of sucrose to reducing sugars by the acid invertase StvacINV1 is thought to be critical for CIS. Identification of the specific StvacINV1 inhibitor StInvInh2B and the α- and β-subunits of the interacting protein SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE from the wild potato species Solanum berthaultii (SbSnRK1) has led to speculation that invertase activity may be regulated via a posttranslational mechanism that remains to be elucidated. Using bimolecular fluorescence complementation assays, this study confirmed the protein complex by pairwise interactions. In vitro kinase assays and protein phosphorylation analysis revealed that phosphorylation of SbSnRK1α is causal for StvacINV1 activity and that its active form blocks the inhibition of StInvInh2B by SbSnRK1β, whereas its inactive form restores the function of SbSnRK1β that prevents StInvInh2B from repressing StvacINV1. Overexpression of SbSnRK1α in CIS-sensitive potato confirmed that SbSnRK1α has significant effects on acid invertase-associated sucrose degradation. A higher level of SbSnRK1α expression was accompanied by elevated SbSnRK1α phosphorylation, reduced acid invertase activity, a higher sucrose-hexose ratio, and improved chip color. Our results lend new insights into a subtle regulatory mode of invertase activity and provide a novel approach for potato CIS improvement. PMID:26134163

  9. Regulation of Invertase Levels in Avena Stem Segments by Gibberellic Acid, Sucrose, Glucose, and Fructose 1

    PubMed Central

    Kaufman, Peter B.; Ghosheh, Najati S.; Lacroix, J. Donald; Soni, Sarvjit L.; Ikuma, Hiroshi

    1973-01-01

    Gibberellic acid and sucrose play significant roles in the increases in invertase and growth in Avena stem segments. About 80% of invertase is readily solubilized, whereas the rest is in the cell wall fraction. The levels of both types of invertase change in a similar manner in the response to gibberellic acid and sucrose treatment. The work described here was carried out with only the soluble enzyme. In response to a treatment, the level of invertase activity typically follows a pattern of increase followed by decrease; the increase in activity is approximately correlated with the active growth phase, whereas the decrease in activity is initiated when growth of the segments slows. A continuous supply of gibberellic acid retards the decline of enzyme activity. When gibberellic acid was pulsed to the segments treated with or without sucrose, the level of invertase activity increased at least twice as high in the presence of sucrose as in its absence, but the lag period is longer with sucrose present. Cycloheximide treatments effectively abolish the gibberellic acid-promoted growth, and the level of enzyme activity drops rapidly. Decay of invertase activity in response to cycloheximide treatment occurs regardless of gibberellic acid or sucrose treatment or both, and it is generally faster when the inhibitor is administered at the peak of enzyme induction than when given at its rising phase. Pulses with sucrose, glucose, fructose, or glucose + fructose elevate the level of invertase significantly with a lag of about 5 to 10 hours. The increase in invertase activity elicited by a sucrose pulse is about one-third that caused by a gibberellic acid pulse given at a comparable time during mid-phase of enzyme induction, and the lag before the enzyme activity increases is nearly twice as long for sucrose as for gibberellic acid. Moreover, the gibberellic acid pulse results in about three times more growth than the sucrose pulse. Our studies support the view that gibberellic

  10. Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

    PubMed

    Yao, Yuan; Wu, Xiao-Hui; Geng, Meng-Ting; Li, Rui-Mei; Liu, Jiao; Hu, Xin-Wen; Guo, Jian-Chun

    2014-01-01

    Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development. PMID:24838076

  11. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

    PubMed

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K; Arias, Cintia L; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E; González, María de la Cruz; Hernández, Jose A; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-02-01

    Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the

  12. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato

    PubMed Central

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K.; Arias, Cintia L.; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E.; González, María de la Cruz; Hernández, Jose A.; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-01-01

    Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of

  13. Gravity-stimulated changes in auxin and invertase gene expression in maize pulvinal cells

    NASA Technical Reports Server (NTRS)

    Long, Joanne C.; Zhao, Wei; Rashotte, Aaron M.; Muday, Gloria K.; Huber, Steven C.; Brown, C. S. (Principal Investigator)

    2002-01-01

    Maize (Zea mays) stem gravitropism involves differential elongation of cells within a highly specialized region, the stem internodal pulvinus. In the present study, we investigated factors that control gravitropic responses in this system. In the graviresponding pulvinus, hexose sugars (D-Glc and D-Fru) accumulated asymmetrically across the pulvinus. This correlated well with an asymmetric increase in acid invertase activity across the pulvinus. Northern analyses revealed asymmetric induction of one maize acid invertase gene, Ivr2, consistent with transcriptional regulation by gravistimulation. Several lines of evidence indicated that auxin redistribution, as a result of polar auxin transport, is necessary for gravity-stimulated Ivr2 transcript accumulation and differential cell elongation across the maize pulvinus. First, the auxin transport inhibitor, N-1-naphthylphthalamic acid, inhibited gravistimulated curvature and Ivr2 transcript accumulation. Second, a transient gradient of free indole-3-acetic acid (IAA) across the pulvinus was apparent shortly after initiation of gravistimulation. This temporarily free IAA gradient appears to be important for differential cell elongation and Ivr2 transcript accumulation. This is based on the observation that N-1-naphthylphthalamic acid will not inhibit gravitropic responses when applied to pulvinus tissue after the free IAA gradient peak has occurred. Third, IAA alone can stimulate Ivr2 transcript accumulation in non-gravistimulated pulvini. The gravity- and IAA-stimulated increase in Ivr2 transcripts was sensitive to the protein synthesis inhibitor, cycloheximide. Based on these results, a two-phase model describing possible relationships between gravitropic curvature, IAA redistribution, and Ivr2 expression is presented.

  14. Gravity-stimulated changes in auxin and invertase gene expression in maize pulvinal cells.

    PubMed

    Long, Joanne C; Zhao, Wei; Rashotte, Aaron M; Muday, Gloria K; Huber, Steven C

    2002-02-01

    Maize (Zea mays) stem gravitropism involves differential elongation of cells within a highly specialized region, the stem internodal pulvinus. In the present study, we investigated factors that control gravitropic responses in this system. In the graviresponding pulvinus, hexose sugars (D-Glc and D-Fru) accumulated asymmetrically across the pulvinus. This correlated well with an asymmetric increase in acid invertase activity across the pulvinus. Northern analyses revealed asymmetric induction of one maize acid invertase gene, Ivr2, consistent with transcriptional regulation by gravistimulation. Several lines of evidence indicated that auxin redistribution, as a result of polar auxin transport, is necessary for gravity-stimulated Ivr2 transcript accumulation and differential cell elongation across the maize pulvinus. First, the auxin transport inhibitor, N-1-naphthylphthalamic acid, inhibited gravistimulated curvature and Ivr2 transcript accumulation. Second, a transient gradient of free indole-3-acetic acid (IAA) across the pulvinus was apparent shortly after initiation of gravistimulation. This temporarily free IAA gradient appears to be important for differential cell elongation and Ivr2 transcript accumulation. This is based on the observation that N-1-naphthylphthalamic acid will not inhibit gravitropic responses when applied to pulvinus tissue after the free IAA gradient peak has occurred. Third, IAA alone can stimulate Ivr2 transcript accumulation in non-gravistimulated pulvini. The gravity- and IAA-stimulated increase in Ivr2 transcripts was sensitive to the protein synthesis inhibitor, cycloheximide. Based on these results, a two-phase model describing possible relationships between gravitropic curvature, IAA redistribution, and Ivr2 expression is presented. PMID:11842162

  15. Gravity-Stimulated Changes in Auxin and Invertase Gene Expression in Maize Pulvinal Cells1

    PubMed Central

    Long, Joanne C.; Zhao, Wei; Rashotte, Aaron M.; Muday, Gloria K.; Huber, Steven C.

    2002-01-01

    Maize (Zea mays) stem gravitropism involves differential elongation of cells within a highly specialized region, the stem internodal pulvinus. In the present study, we investigated factors that control gravitropic responses in this system. In the graviresponding pulvinus, hexose sugars (d-Glc and d-Fru) accumulated asymmetrically across the pulvinus. This correlated well with an asymmetric increase in acid invertase activity across the pulvinus. Northern analyses revealed asymmetric induction of one maize acid invertase gene, Ivr2, consistent with transcriptional regulation by gravistimulation. Several lines of evidence indicated that auxin redistribution, as a result of polar auxin transport, is necessary for gravity-stimulated Ivr2 transcript accumulation and differential cell elongation across the maize pulvinus. First, the auxin transport inhibitor, N-1-naphthylphthalamic acid, inhibited gravistimulated curvature and Ivr2 transcript accumulation. Second, a transient gradient of free indole-3-acetic acid (IAA) across the pulvinus was apparent shortly after initiation of gravistimulation. This temporarily free IAA gradient appears to be important for differential cell elongation and Ivr2 transcript accumulation. This is based on the observation that N-1-naphthylphthalamic acid will not inhibit gravitropic responses when applied to pulvinus tissue after the free IAA gradient peak has occurred. Third, IAA alone can stimulate Ivr2 transcript accumulation in non-gravistimulated pulvini. The gravity- and IAA-stimulated increase in Ivr2 transcripts was sensitive to the protein synthesis inhibitor, cycloheximide. Based on these results, a two-phase model describing possible relationships between gravitropic curvature, IAA redistribution, and Ivr2 expression is presented. PMID:11842162

  16. Functional characterization of an invertase inhibitor gene involved in sucrose metabolism in tomato fruit.

    PubMed

    Zhang, Ning; Jiang, Jing; Yang, Yan-li; Wang, Zhi-he

    2015-10-01

    In this study, we produced tomato plants overexpressing an invertase inhibitor gene (Sly-INH) from tomato, using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction (PCR). Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase (CWIN), which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato. PMID:26465132

  17. Functional characterization of an invertase inhibitor gene involved in sucrose metabolism in tomato fruit*

    PubMed Central

    ZHANG, Ning; JIANG, Jing; YANG, Yan-li; WANG, Zhi-he

    2015-01-01

    In this study, we produced tomato plants overexpressing an invertase inhibitor gene (Sly-INH) from tomato, using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction (PCR). Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase (CWIN), which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato. PMID:26465132

  18. Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects.

    PubMed

    Zhu, Xiaobiao; Richael, Craig; Chamberlain, Patrick; Busse, James S; Bussan, Alvin J; Jiang, Jiming; Bethke, Paul C

    2014-01-01

    Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide. PMID:24695527

  19. Vacuolar invertase gene silencing in potato decreasing the frequency of sugar-end defects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one e...

  20. Characterization of the alkaline/neutral invertase gene in Dendrobium officinale and its relationship with polysaccharide accumulation.

    PubMed

    Gao, F; Cao, X F; Si, J P; Chen, Z Y; Duan, C L

    2016-01-01

    Dendrobium officinale is one of the most well-known traditional Chinese medicines, and polysaccharide is its main active ingredient. Many studies have investigated the synthesis and accumulation mechanisms of polysaccharide, but until recently, little was known about the molecular mechanism of how polysaccharide is synthesized because no related genes have been cloned. In this study, we cloned an alkaline/neutral invertase gene from D. officinale (DoNI) by the rapid amplification of cDNA ends (RACE) method. DoNI was 2231 bp long and contained an open reading frame that predicted a 62.8-kDa polypeptide with 554-amino acid residues. An alkaline/neutral invertase conserved domain was predicted from this deduced amino acid sequence, and DoNI had a similar deduced amino acid sequence to Setaria italica and Oryza brachyantha. We also found that DoNI expression in different tissues was closely related to DoNI activity, and more importantly, polysaccharide level. Our results indicate that DoNI is associated with polysaccharide accumulation in D. officinale. PMID:27173310

  1. Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugar-end defect is a tuber quality disorder that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form da...

  2. Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

    PubMed Central

    Cortés-Romero, Celso; Martínez-Hernández, Aída; Mellado-Mojica, Erika; López, Mercedes G.; Simpson, June

    2012-01-01

    Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants. PMID:22558253

  3. Molecular Basis of the Increase in Invertase Activity Elicited by Gravistimulation of Oat-Shoot Pulvini

    NASA Technical Reports Server (NTRS)

    Wu, Liu-Lai; Song, Il; Kim, Donghern; Kaufman, Peter B.

    1993-01-01

    An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oatshoot pulvini starting within 3h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly(A)(+)RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the Polymerase Chain Reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52 % deduced amino-acid sequence homology to that of carrot beta-fructosi- dase and a 48 % homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

  4. Light/Dark Profiles of Sucrose Phosphate Synthase, Sucrose Synthase, and Acid Invertase in Leaves of Sugar Beets

    PubMed Central

    Vassey, Terry L.

    1989-01-01

    The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves. PMID:16666537

  5. Neurospora Mutant Exhibiting Hyperproduction of Amylase and Invertase

    PubMed Central

    Gratzner, Howard; Sheehan, D. N.

    1969-01-01

    A mutant strain of Neurospora crassa has been isolated which is derepressed for amylase and β-fructofuranosidase (invertase). Large amounts of the two enzymes were secreted into the culture medium upon depletion of exogenous carbon source. The resulting increases of the two extracellular enzymes were prevented by actinomycin D, cycloheximide, and glycerol. The starving cells of the mutant strain produced amylase and invertase de novo, as evidenced by incorporation of radioactive amino acids into the enzymes. Preliminary genetic studies indicate that these elevated enzyme levels described are due to a single gene mutation. PMID:5773010

  6. Involvement of Abscisic Acid in the Coordinated Regulation of a Stress-Inducible Hexose Transporter (VvHT5) and a Cell Wall Invertase in Grapevine in Response to Biotrophic Fungal Infection[W

    PubMed Central

    Hayes, Matthew A.; Feechan, Angela; Dry, Ian B.

    2010-01-01

    Biotrophic fungal and oomycete pathogens alter carbohydrate metabolism in infected host tissues. Symptoms such as elevated soluble carbohydrate concentrations and increased invertase activity suggest that a pathogen-induced carbohydrate sink is established. To identify pathogen-induced regulators of carbohydrate sink strength, quantitative real-time polymerase chain reaction was used to measure transcript levels of invertase and hexose transporter genes in biotrophic pathogen-infected grapevine (Vitis vinifera) leaves. The hexose transporter VvHT5 was highly induced in coordination with the cell wall invertase gene VvcwINV by powdery and downy mildew infection. However, similar responses were also observed in response to wounding, suggesting that this is a generalized response to stress. Analysis of the VvHT5 promoter region indicated the presence of multiple abscisic acid (ABA) response elements, suggesting a role for ABA in the transition from source to sink under stress conditions. ABA treatment of grape leaves was found to reproduce the same gene-specific transcriptional changes as observed under biotic and abiotic stress conditions. Furthermore, the key regulatory ABA biosynthetic gene, VvNCED1, was activated under these same stress conditions. VvHT5 promoter::β-glucuronidase-directed expression in transgenic Arabidopsis (Arabidopsis thaliana) was activated by infection with powdery mildew and by ABA treatment, and the expression was closely associated with vascular tissue adjacent to infected regions. Unlike VvHT1 and VvHT3, which appear to be predominantly involved in hexose transport in developing leaves and berries, VvHT5 appears to have a specific role in enhancing sink strength under stress conditions, and this is controlled through ABA. Our data suggest a central role for ABA in the regulation of VvcwINV and VvHT5 expression during the transition from source to sink in response to infection by biotrophic pathogens. PMID:20348211

  7. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

    PubMed

    Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

    2016-02-01

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. PMID:26079224

  8. Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots

    NASA Technical Reports Server (NTRS)

    Wu, L. L.; Mitchell, J. P.; Cohn, N. S.; Kaufman, P. B.

    1993-01-01

    The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

  9. Soluble Invertase Expression Is an Early Target of Drought Stress during the Critical, Abortion-Sensitive Phase of Young Ovary Development in Maize1

    PubMed Central

    Andersen, Mathias Neumann; Asch, Folkard; Wu, Yong; Jensen, Christian Richardt; Næsted, Henrik; Mogensen, Vagn Overgaard; Koch, Karen Elaine

    2002-01-01

    To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (−6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (−4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at −6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development. PMID:12376627

  10. Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

    NASA Technical Reports Server (NTRS)

    Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

    1993-01-01

    An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

  11. The Miniature-1 (Mn1) gene product, cell wall invertase-2 (INCW2), is associated with wall-in-growths (WIGs) is basal endosperm transfer cells (BETCs) in developing seeds of maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell wall invertases (CWI) are ionically bound to the cell wall in plant cells. A major CWI, INCW2, encoded by the Mn1 gene, provides the gateway to sucrose metabolism in developing seeds as it is entirely and exclusively localized to the BETCs that juxtapose the pedicel. The loss of INCW2 protein ...

  12. Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz).

    PubMed

    Yao, Yuan; Geng, Meng-Ting; Wu, Xiao-Hui; Liu, Jiao; Li, Rui-Mei; Hu, Xin-Wen; Guo, Jian-Chun

    2014-01-01

    The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker. PMID:24786092

  13. Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.

    PubMed

    Weil, M; Rausch, T

    1990-12-01

    The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink. PMID:16667892

  14. Cloning of a CACTA transposon-like insertion in intron I of tomato invertase Lin5 gene and identification of transposase-like sequences of Solanaceae species.

    PubMed

    Proels, Reinhard K; Roitsch, Thomas

    2006-03-01

    Very few CACTA transposon-like sequences have been described in Solanaceae species. Sequence information has been restricted to partial transposase (TPase)-like fragments, and no target gene of CACTA-like transposon insertion has been described in tomato to date. In this manuscript, we report on a CACTA transposon-like insertion in intron I of tomato (Lycopersicon esculentum) invertase gene Lin5 and TPase-like sequences of several Solanaceae species. Consensus primers deduced from the TPase region of the tomato CACTA transposon-like element allowed the amplification of similar sequences from various Solanaceae species of different subfamilies including Solaneae (Solanum tuberosum), Cestreae (Nicotiana tabacum) and Datureae (Datura stramonium). This demonstrates the ubiquitous presence of CACTA-like elements in Solanaceae genomes. The obtained partial sequences are highly conserved, and allow further detection and detailed analysis of CACTA-like transposons throughout Solanaceae species. CACTA-like transposon sequences make possible the evaluation of their use for genome analysis, functional studies of genes and the evolutionary relationships between plant species. PMID:16473661

  15. Auxin and Cell Wall Invertase Related Signaling during Rice Grain Development

    PubMed Central

    Russell French, Sarah; Abu-Zaitoon, Yousef; Uddin, Md. Myn; Bennett, Karina; Nonhebel, Heather M.

    2014-01-01

    Indole-3-acetic acid (IAA) synthesis is required for grain-fill in maize and appears to be regulated by cell-wall invertase (CWIN) activity. OsYUC12 is one of three IAA biosynthesis genes we previously reported as expressed during early rice grain development, correlating with a large increase in IAA content of the grain. This work aimed to investigate further the role of OsYUC12 and its relationship to CWIN activity and invertase inhibitors (INVINH). The analysis shows a brief peak of OsYUC12 expression early in endosperm development. Meta-analysis of microarray data, confirmed by quantitative expression analysis, revealed that OsYUC12 is coexpressed with OsIAA29, which encodes an unusual AUX/IAA transcription factor previously reported as poorly expressed. Maximum expression of OsYUC12 and OsIAA29 coincided with maximum CWIN activity, but also with a peak in INVINH expression. Unlike ZmYUC1, OsYUC12 expression is not reduced in the rice CWIN mutant, gif1. Several reports have investigated CWIN expression in rice grains but none has reported on expression of INVINH in this species. We show that rice has 54 genes encoding putative invertase/pectin methylesterase inhibitors, seven of which are expressed exclusively during grain development. Our results suggest a more complex relationship between IAA, CWIN, and INVINH than previously proposed. PMID:27135493

  16. A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

    PubMed Central

    Lyda, Todd A.; Joshi, Manju B.; Andersen, John F.; Kelada, Andrew Y.; Owings, Joshua P.; Bates, Paul A.; Dwyer, Dennis M.

    2015-01-01

    Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts. PMID:25763714

  17. Simple Practical Investigations Using Invertase.

    ERIC Educational Resources Information Center

    Asare-Brown, Emma; Bullock, Clive

    1988-01-01

    Describes three activities, substrate inhibition, product inhibition by fructose and glucose, and gel immobilization of invertase for use with undergraduate biochemistry classes. Discusses materials, methods, and results. Stresses the advantages of practical exercises in undergraduate classes. (CW)

  18. Molecular and functional characterization of an invertase secreted by Ashbya gossypii.

    PubMed

    Aguiar, Tatiana Q; Dinis, Cláudia; Magalhães, Frederico; Oliveira, Carla; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2014-06-01

    The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii. PMID:24452331

  19. Pleiotropy and its limited dissection through a metabolic gene Miniature1 (Mn1) that encodes a cell wall invertase in developing seeds of maize.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Mn1-encoded endosperm-specific cell wall invertase is a major determinant of sink strength of developing seeds through its control of both sink size, cell number and cell size, and sink activity via sucrose hydrolysis and release of hexoses essential for energy and signaling functions. Consequen...

  20. Improving the Processing Quality of Existing Cultivars by Suppressing the Vacuolar Acid Invertase Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Storing potato tubers at low temperatures is highly advantageous in that it prevents sprouting, minimizes disease losses and increases the marketing window. Unfortunately, cold storage of existing cultivars causes an unacceptable accumulation of reducing sugars, a phenomenon referred to as cold-indu...

  1. Invertase Inhibitor from Potatoes: Purification, Characterization, and Reactivity with Plant Invertases

    PubMed Central

    Pressey, Russell

    1967-01-01

    Invertase inhibitor was extracted from potato tubers and purified nearly 1000-fold. The purification procedure involved precipitation at pH 4.0, fractionation with ammonium sulfate, adsorption on alumina Cγ gel, and gel filtration on Sephadex G-100 and DEAE-Sephadex A-50. The product obtained was homogeneous to electrophoresis on polyacrylamide gel. Exclusion chromatography on Sephadex G-100 indicated a molecular weight of about 17,000. The inhibitor did not inhibit yeast, Neurospora, and several plant invertases. It completely inhibited potato tuber invertase and a number of other plant invertases. Some plant invertases were partially inhibited. Images PMID:16656719

  2. Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

    PubMed Central

    Simpson, C G; Hedley, P E; Watters, J A; Clark, G P; McQuade, C; Machray, G C; Brown, J W

    2000-01-01

    Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon. PMID:10744026

  3. Cell wall invertase-2 (INCW2) encoded by Miniature-1 (Mn1) gene is associated with wall-in-growths (WIGs) in basal endosperm transfer cells (BETCs) in developing seeds of maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell wall invertases (CWI) are ionically bound to the plant cell walls. A major CWI, INCW2, provides the gateway to sucrose metabolism in developing maize seeds as it is entirely localized to the BETCs that juxtapose the pedicel. The loss of INCW2 protein is the causal basis of the mn1 seed phenoty...

  4. Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box.

    PubMed

    Ritsema, Tita; Hernández, Lázaro; Verhaar, Auke; Altenbach, Denise; Boller, Thomas; Wiemken, Andres; Smeekens, Sjef

    2006-10-01

    Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait. PMID:17018033

  5. A higher sink competitiveness of the rooting zone and invertases are involved in dark stimulation of adventitious root formation in Petunia hybrida cuttings.

    PubMed

    Klopotek, Yvonne; Franken, Philipp; Klaering, Hans-Peter; Fischer, Kerstin; Hause, Bettina; Hajirezaei, Mohammad-Reza; Druege, Uwe

    2016-02-01

    The contribution of carbon assimilation and allocation and of invertases to the stimulation of adventitious root formation in response to a dark pre-exposure of petunia cuttings was investigated, considering the rooting zone (stem base) and the shoot apex as competing sinks. Dark exposure had no effect on photosynthesis and dark respiration during the subsequent light period, but promoted dry matter partitioning to the roots. Under darkness, higher activities of cytosolic and vacuolar invertases were maintained in both tissues when compared to cuttings under light. This was partially associated with higher RNA levels of respective genes. However, activity of cell wall invertases and transcript levels of one cell wall invertase isogene increased specifically in the stem base during the first two days after cutting excision under both light and darkness. During five days after excision, RNA accumulation of four invertase genes indicated preferential expression in the stem base compared to the apex. Darkness shifted the balance of expression of one cytosolic and two vacuolar invertase genes towards the stem base. The results indicate that dark exposure before planting enhances the carbon sink competitiveness of the rooting zone and that expression and activity of invertases contribute to the shift in carbon allocation. PMID:26795147

  6. Efficient stabilization of Saccharomyces cerevisiae external invertase by immobilisation on modified beidellite nanoclays.

    PubMed

    Andjelković, Uroš; Milutinović-Nikolić, Aleksandra; Jović-Jovičić, Nataša; Banković, Predrag; Bajt, Teja; Mojović, Zorica; Vujčić, Zoran; Jovanović, Dušan

    2015-02-01

    The external invertase isoform 1 (EINV1) was immobilised on eight differently modified beidellite nanoclays. Modifications were composed of organo-modification with different amounts of surfactant - hexadecyl trimethylammonium cation (HDTMA), pillaring with Al/Fe containing polyhydroxy cations and acid modification of Na-enriched and pillared clays. The modified nanoclays were characterised by XRD, N2-physisorption, SEM and FT-IR spectroscopy. The amount of bound enzyme activity was significantly influenced by the modification of beidellite ranging from 50 to remarkable 2200U/g. Biochemical characterization was performed for five modified nanoclays showing the highest enzyme activity after invertase immobilisation. The investigation demonstrated that after immobilisation the structure and the catalytic properties of invertase were preserved, while Km values were slightly increased from 26 to 37mM. immobilisation significantly improved thermal and storage stability of EINV1. Results indicate that beidellite nanoclays obtained by low cost modifications can be applied as a suitable support for the immobilisation of invertase. The immobilizate can be efficiently engaged in sucrose hydrolysis in batch reactor. PMID:25172709

  7. RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals.

    PubMed

    Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

    2016-05-01

    Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

  8. RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

    PubMed Central

    Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

    2016-01-01

    Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

  9. Sucrose utilization during somatic embryo development in black spruce: involvement of apoplastic invertase in the tissue and of extracellular invertase in the medium.

    PubMed

    Iraqi, Driss; Le, Van Quy; Lamhamedi, Mohammed S; Tremblay, Francine M

    2005-01-01

    The involvement of apoplastic invertase (Ap Inv) and sucrose synthase (SuSy) in the somatic embryo development of black spruce (Picea mariana) was investigated under different maturation conditions. Replacing 6% sucrose with 3% or 1% sucrose in the maturation medium drastically decreased Ap Inv activity and amount in embryogenic tissues. This was accompanied by a decrease in the hexose pool that resulted in a lower starch deposition and protein amount in embryogenic tissues together with a lower embryo production. Conversely, SuSy activity was stable during maturation regardless of the sucrose concentration used in the medium. The presence of an extracellular enzyme responsible for sucrose hydrolysis in the maturation medium was also verified. An immunodetection experiment with anti-acid invertase antibodies revealed the presence of an active 53 kDa polypeptide in the medium, which had a similar molecular mass to that of the Ap Inv polypeptide found in embryogenic tissues. Utilization of sucrose from the medium by the tissues was also studied using labelled 14C-sucrose. Distribution of the radioactivity between tissular sucrose, glucose, and fructose showed that sucrose was diffused into the cell wall of embryogenic tissues and partly hydrolyzed by Ap Inv. These results show that the utilization of sucrose from the medium, the Ap Inv activity in embryogenic tissues, and the release of an active invertase into the medium operate together for the utilization of the carbohydrates during somatic embryo development in black spruce. PMID:15700426

  10. Slow-growth phenotype of transgenic tomato expressing apoplastic invertase

    SciTech Connect

    Dickinson, C.D.; Altabella, T.; Chrispeels, M.J. )

    1991-02-01

    The growth of transgenic tomato (Lycopersicon esculentum) plants that express in their apoplast yeast invertase under the control of the cauliflower mosaic virus 35S promoter is severely inhibited. The higher the level of invertase, the greater the inhibition of growth. A second phenotypic characteristic of these transgenic plants is the development of yellow and necrotic spots on the leaves, and leaf curling. Again the severity of the symptoms is correlated with the level of invertase. These symptoms do not develop in shaded leaves indicating the need for photosynthesis. Keeping the plants in the dark for a prolonged period (24 hours) results in the disappearance of leaf starch from the control plants, but not from the plants with apoplastic invertase. These results are consistent with the interpretation that apoplastic invertase prevents photosynthate export from source leaves and that phloem loading includes an apoplastic step.

  11. Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

    PubMed Central

    Davies, C; Robinson, S P

    1996-01-01

    During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process. PMID:8685267

  12. Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana.

    PubMed

    Park, Jungan; Kim, Soyeon; Choi, Eunseok; Auh, Chung-Kyun; Park, Jong-Bum; Kim, Dong-Giun; Chung, Young-Jae; Lee, Taek-Kyun; Lee, Sukchan

    2013-09-01

    Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation. PMID:23589148

  13. Seed coat-associated invertases of fava bean control both unloading and storage functions: cloning of cDNAs and cell type-specific expression.

    PubMed

    Weber, H; Borisjuk, L; Heim, U; Buchner, P; Wobus, U

    1995-11-01

    We have studied the molecular physiology of photosynthate unloading and partitioning during seed development of fava bean (Vicia faba). During the prestorage phase, high levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat. Three cDNAs were cloned. Sequence comparison revealed genes putatively encoding one soluble and two cell wall-bound isoforms of invertase. Expression was studied in different organs and tissues of developing seeds by RNA gel analysis, in situ hybridization, enzyme assay, and enzyme activity staining. One extracellular invertase gene is expressed during the prestorage phase in the thin-walled parenchyma of the seed coat, a region known to be the site of photoassimilate unloading. We propose a model for an invertase-mediated unloading process during early seed development and the regulation of cotyledonary sucrose metabolism. After unloading from the seed coat, sucrose is hydrolyzed by cell wall-bound invertases. Thus, invertase contributes to establish sink strength in young seeds. The resultant hexoses are loaded into the cotyledons and control carbohydrate partitioning via an influence on the sucrose synthase/sucrose-phosphate synthase pathway. The developmentally regulated degradation of the thin-walled parenchyma expressing the invertase apparently initiates the storage phase. This is characterized by a switch to a low sucrose/hexoses ratio. Feeding hexoses to storage-phase cotyledons in vitro increases the sucrose-phosphate synthase/sucrose synthase ratio and changes carbohydrate partitioning in favor of sucrose. Concomitantly, the transcript level of the major storage product legumin B is downregulated. PMID:8535137

  14. Expression analysis of genes associated with sucrose accumulation in sugarcane (Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthesis/accumulation in sugarcane is a complex process involving many genes and regulatory sequences that control biochemical events in source-sink tissues. Among these, sucrose synthase (SuSy), sucrose-phosphate synthase (SPS), soluble acid (SAI) and cell-wall invertase (CWI) are importan...

  15. Amino acid regulation of gene expression.

    PubMed Central

    Fafournoux, P; Bruhat, A; Jousse, C

    2000-01-01

    The impact of nutrients on gene expression in mammals has become an important area of research. Nevertheless, the current understanding of the amino acid-dependent control of gene expression is limited. Because amino acids have multiple and important functions, their homoeostasis has to be finely maintained. However, amino-acidaemia can be affected by certain nutritional conditions or various forms of stress. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. The aim of the present review is to examine the role of amino acids in regulating mammalian gene expression and protein turnover. It has been reported that some genes involved in the control of growth or amino acid metabolism are regulated by amino acid availability. For instance, limitation of several amino acids greatly increases the expression of the genes encoding insulin-like growth factor binding protein-1, CHOP (C/EBP homologous protein, where C/EBP is CCAAT/enhancer binding protein) and asparagine synthetase. Elevated mRNA levels result from both an increase in the rate of transcription and an increase in mRNA stability. Several observations suggest that the amino acid regulation of gene expression observed in mammalian cells and the general control process described in yeast share common features. Moreover, amino acid response elements have been characterized in the promoters of the CHOP and asparagine synthetase genes. Taken together, the results discussed in the present review demonstrate that amino acids, by themselves, can, in concert with hormones, play an important role in the control of gene expression. PMID:10998343

  16. Differential response of wild and cultivated wheats to water deficits during grain development: changes in soluble carbohydrates and invertases.

    PubMed

    Suneja, Yadhu; Gupta, Anil K; Sharma, Achla; Bains, Navtej S

    2015-04-01

    Wheat, staple food crop of the world, is sensitive to drought, especially during the grain-filling period. Water soluble carbohydrates (WSCs), stem reserve mobilization and higher invertase activity in the developing grains are important biochemical traits for breeding wheat to enhance tolerance to terminal drought. These traits were studied for three accessions of Triticum dicoccoides(a tetraploid wheat progenitor species) - acc 7054 (EC 171812), acc 7079 (EC 171837) and acc 14004 (G-194-3 M-6 M) selected previously on the basis of grain filling characteristics. Check wheat cultivars- PBW-343 (a popular bread wheat cultivar for irrigated environments) and C-306 (widely adapted variety for rain-fed agriculture) were also included in this set. Analysis of variance revealed significant genotypic differences for the content of water soluble carbohydrates, activity of acid invertase and alkaline invertase. Acc 7079 was found to be a very efficient mobilizer of water soluble carbohydrates (236.43 mg g(-1) peduncle DW) when averaged over irrigated and rain-fed conditions. Acid invertase activity revealed marked genotypic differences between wild and cultivated wheats. Alkaline invertase activity was highest in Acc 7079 when pooled across both the environments. On the whole, acc 7079 qualifies as a suitable donor for enhancing tolerance of bread wheat to terminal drought. The association of physio-biochemical differences observed with grain filling attributes on one hand and molecular markers on the other could be of use in improving wheat for water stress conditions. PMID:25964711

  17. Utilization of molasses and sugar cane bagasse for production of fungal invertase in solid state fermentation using Aspergillus niger GH1

    PubMed Central

    Veana, F.; Martínez-Hernández, J.L.; Aguilar, C.N.; Rodríguez-Herrera, R.; Michelena, G.

    2014-01-01

    Agro-industrial wastes have been used as substrate-support in solid state fermentation for enzyme production. Molasses and sugarcane bagasse are by-products of sugar industry and can be employed as substrates for invertase production. Invertase is an important enzyme for sweeteners development. In this study, a xerophilic fungus Aspergillus niger GH1 isolated of the Mexican semi-desert, previously reported as an invertase over-producer strain was used. Molasses from Mexico and Cuba were chemically analyzed (total and reducer sugars, nitrogen and phosphorous contents); the last one was selected based on chemical composition. Fermentations were performed using virgin and hydrolyzate bagasse (treatment with concentrated sulfuric acid). Results indicated that, the enzymatic yield (5231 U/L) is higher than those reported by other A. niger strains under solid state fermentation, using hydrolyzate bagasse. The acid hydrolysis promotes availability of fermentable sugars. In addition, maximum invertase activity was detected at 24 h using low substrate concentration, which may reduce production costs. This study presents an alternative method for invertase production using a xerophilic fungus isolated from Mexican semi-desert and inexpensive substrates (molasses and sugarcane bagasse). PMID:25242918

  18. Utilization of molasses and sugar cane bagasse for production of fungal invertase in solid state fermentation using Aspergillus niger GH1.

    PubMed

    Veana, F; Martínez-Hernández, J L; Aguilar, C N; Rodríguez-Herrera, R; Michelena, G

    2014-01-01

    Agro-industrial wastes have been used as substrate-support in solid state fermentation for enzyme production. Molasses and sugarcane bagasse are by-products of sugar industry and can be employed as substrates for invertase production. Invertase is an important enzyme for sweeteners development. In this study, a xerophilic fungus Aspergillus niger GH1 isolated of the Mexican semi-desert, previously reported as an invertase over-producer strain was used. Molasses from Mexico and Cuba were chemically analyzed (total and reducer sugars, nitrogen and phosphorous contents); the last one was selected based on chemical composition. Fermentations were performed using virgin and hydrolyzate bagasse (treatment with concentrated sulfuric acid). Results indicated that, the enzymatic yield (5231 U/L) is higher than those reported by other A. niger strains under solid state fermentation, using hydrolyzate bagasse. The acid hydrolysis promotes availability of fermentable sugars. In addition, maximum invertase activity was detected at 24 h using low substrate concentration, which may reduce production costs. This study presents an alternative method for invertase production using a xerophilic fungus isolated from Mexican semi-desert and inexpensive substrates (molasses and sugarcane bagasse). PMID:25242918

  19. Polyunsaturated fatty acids and gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose of review. This review focuses on the effect(s) of n-3 polyunsaturated fatty acids (PUFA) on gene transcription as determined from data generated using cDNA microarrays. Introduced within the past decade, this methodology allows detection of the expression of thousands of genes simultaneo...

  20. Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis.

    PubMed

    Privat, Isabelle; Foucrier, Séverine; Prins, Anneke; Epalle, Thibaut; Eychenne, Magali; Kandalaft, Laurianne; Caillet, Victoria; Lin, Chenwei; Tanksley, Steve; Foyer, Christine; McCarthy, James

    2008-01-01

    * Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. * Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). * Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. * It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development - the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re-synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered. PMID:18384509

  1. A new application of humic substances: activation of supports for invertase immobilization.

    PubMed

    Rosa, A H; Vicente, A A; Rocha, J C; Trevisan, H C

    2000-12-01

    Invertase was immobilized on aminopropyl silica (APTS-SiO2) activated with humic substances (APTS-SiO2-HS) and on aminopropyl silica activated with glutaraldehyde (APTS-SiO2-GA). The resulting activity of both systems was compared. Humic substances (HS) used for the activation of the silica were extracted from soil of Cananéia, São Paulo State, Brazil, according to the procedure recommended by the International Humic Substances Society. Activity was determined by measuring the rate of formation of reduced sugars using the reaction with dinitrosalicylic acid (DNS). The amount of HS bound on the APTS-SiO2 was equal to 50 mg. The maximum amount of invertase immobilized on APTS-SiO2-HS was 15,200 U/g while in the system APTS-SiO2-GA it was 13,400 U/g. The experimental enzymatic activity was 3,700 and 3,300 U/g, for the systems APTS-SiO2-HS and APTS-SiO2-GA, respectively. Considering the increased amount and activity of immobilized enzyme compared with the glutaraldehyde method, it was concluded that this technique opens a new perspective in the preparation of supports for enzyme immobilization employing humic substances. PMID:11227556

  2. Critical Roles of Vacuolar Invertase in Floral Organ Development and Male and Female Fertilities Are Revealed through Characterization of GhVIN1-RNAi Cotton Plants1[OPEN

    PubMed Central

    2016-01-01

    Seed number and quality are key traits determining plant fitness and crop yield and rely on combined competence in male and female fertilities. Sucrose metabolism is central to reproductive success. It remains elusive, though, how individual sucrose metabolic enzymes may regulate the complex reproductive processes. Here, by silencing vacuolar invertase (VIN) genes in cotton (Gossypium hirsutum) reproductive organs, we revealed diverse roles that VIN plays in multiple reproductive processes. A set of phenotypic and genetic studies showed significant reductions of viable seeds in GhVIN1-RNAi plants, attributed to pollination failure and impaired male and female fertilities. The former was largely owing to the spatial mismatch between style and stamen and delayed pollen release from the anthers, whereas male defects came from poor pollen viability. The transgenic stamen exhibited altered expression of the genes responsible for starch metabolism and auxin and jasmonic acid signaling. Further analyses identified the reduction of GhVIN expression in the seed coat as the major cause for the reduced female fertility, which appeared to disrupt the expression of some key genes involved in trehalose and auxin metabolism and signaling, leading to programmed cell death or growth repression in the filial tissues. Together, the data provide an unprecedented example of how VIN is required to synchronize style and stamen development and the formation of male and female fertilities for seed development in a crop species, cotton. PMID:26969720

  3. Critical Roles of Vacuolar Invertase in Floral Organ Development and Male and Female Fertilities Are Revealed through Characterization of GhVIN1-RNAi Cotton Plants.

    PubMed

    Wang, Lu; Ruan, Yong-Ling

    2016-05-01

    Seed number and quality are key traits determining plant fitness and crop yield and rely on combined competence in male and female fertilities. Sucrose metabolism is central to reproductive success. It remains elusive, though, how individual sucrose metabolic enzymes may regulate the complex reproductive processes. Here, by silencing vacuolar invertase (VIN) genes in cotton (Gossypium hirsutum) reproductive organs, we revealed diverse roles that VIN plays in multiple reproductive processes. A set of phenotypic and genetic studies showed significant reductions of viable seeds in GhVIN1-RNAi plants, attributed to pollination failure and impaired male and female fertilities. The former was largely owing to the spatial mismatch between style and stamen and delayed pollen release from the anthers, whereas male defects came from poor pollen viability. The transgenic stamen exhibited altered expression of the genes responsible for starch metabolism and auxin and jasmonic acid signaling. Further analyses identified the reduction of GhVIN expression in the seed coat as the major cause for the reduced female fertility, which appeared to disrupt the expression of some key genes involved in trehalose and auxin metabolism and signaling, leading to programmed cell death or growth repression in the filial tissues. Together, the data provide an unprecedented example of how VIN is required to synchronize style and stamen development and the formation of male and female fertilities for seed development in a crop species, cotton. PMID:26969720

  4. Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism.

    PubMed Central

    Godt, D E; Roitsch, T

    1997-01-01

    The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ. PMID:9306701

  5. Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

    PubMed Central

    Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

    2012-01-01

    Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

  6. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase

    PubMed Central

    Zhou, Junpei; He, Limei; Gao, Yajie; Han, Nanyu; Zhang, Rui; Wu, Qian; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Huang, Zunxi

    2016-01-01

    A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0–9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30–32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG* (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol−1, 57.6 kJ mol−1, 62.9 mM and 746.2 s−1, respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged. PMID:27553125

  7. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.

    PubMed

    Zhou, Junpei; He, Limei; Gao, Yajie; Han, Nanyu; Zhang, Rui; Wu, Qian; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Huang, Zunxi

    2016-01-01

    A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0-9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30-32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG(*) (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol(-1), 57.6 kJ mol(-1), 62.9 mM and 746.2 s(-1), respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged. PMID:27553125

  8. Physiological responses of biomass allocation, root architecture, and invertase activity to copper stress in young seedlings from two populations of Kummerowia stipulacea (maxim.) Makino.

    PubMed

    Zhang, Luan; Pan, Yuxue; Lv, Wei; Xiong, Zhi-ting

    2014-06-01

    In the current study, we hypothesize that mine (metallicolous) populations of metallophytes form a trade-off between the roots and shoots when under copper (Cu) stress to adapt themselves to heavy metal contaminated habitats, and thus, differ from normal (non-metallicolous) populations in biomass allocation. To test the hypothesis, two populations of the metallophyte Kummerowia stipulacea, one from an ancient Cu mine (MP) and the other from a non-contaminated site (NMP), were treated with Cu(2+) in hydroponic conditions. The results showed that MP plants had higher root/shoot biomass allocation and more complicated root system architecture compared to those of the NMP plants when under Cu stress. The net photosynthetic capacity was more inhibited in the NMP plants than in the MP plants when under Cu stress. The sugar (sucrose and hexose) contents and acid invertase activities of MP plants were elevated while those in NMP plants were inhibited after Cu treatment. The neutral/alkaline invertase activities and sucrose synthase level showed no significant differences between the two populations when under Cu stress. The results showed that acid invertase played an important role in biomass allocation and that the physiological responses were beneficial for the high root/shoot biomass allocation, which were advantageous during adaptive evolution to Cu-enriched mine soils. PMID:24726940

  9. Inversion of Moraxella lacunata type 4 pilin gene sequences by a Neisseria gonorrhoeae site-specific recombinase.

    PubMed Central

    Rozsa, F W; Meyer, T F; Fussenegger, M

    1997-01-01

    A plasmid library of Neisseria gonorrhoeae sequences was screened for the ability to mediate recombinations on a sequence containing the Moraxella lacunata type 4 pilin gene invertible region in Escherichia coli. A plasmid containing the N. gonorrhoeae sequence encoding the putative recombinase (gcr) was identified and sequenced. Plasmids containing gcr were able to mediate site-specific recombinations despite a weak amino acid homology to Piv, the native M. lacunata pilin gene invertase. The gcr gene is present only in pathogenic strains of Neisseria tested; however, in our assays gene knockouts of gcr did not alter the variation of surface features that play a role in the pathogenesis of N. gonorrhoeae. PMID:9079926

  10. Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae.

    PubMed Central

    Moreno, S; Sanchez, Y; Rodriguez, L

    1990-01-01

    Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2187435

  11. Detection of mercury compounds using invertase-glucose oxidase-based biosensor

    NASA Astrophysics Data System (ADS)

    Amine, A.; Cremisini, C.; Palleschi, G.

    1995-10-01

    Mercury compounds have been determined with an electrochemical biosensor based on invertase inhibition. When invertase is in the presence of mercury its activity decreases; this causes a decrease of glucose production which is monitored by the glucose sensor and correlated to the concentration of mercury in solution. Parameters as pH, enzyme concentration, substrate concentration, and reaction and incubation time were optimized. Mercury compounds determination using soluble or immobilized invertase were reported. Results show that the inhibition was competitive and reversible. Mercury compounds can be detected directly in aqueous solution in the range 2 - 10 ppb.

  12. Invertase immobilized on macroporous p,lystyrene: properties and kinetic characterization

    SciTech Connect

    Mansfeld, J.; Schellenberger, A.

    1987-01-01

    Invertase from baker's yeast (Saccharomyces cerevisiae) was covalently bound via benzoquinone and glutaraldehyde to a macroporous polystyrene anion exchanger. The behavior of the invertase-polystyrene complexes in batch and packed-bed reactors was characterized kinetically. In addition to kinetic studies on sucrose hydrolysis at low initial substrate concentrations, the dependence of conversion degree on flow rate at high, industrially used substrate concentrations was determined. The described invertase-polystyrene complexes are suitable for technical application in the production of glucose-fructose mixtures because of their high specific and relative activities, as well as the good hydrodynamical and mechanical properties of the polystyrene matrix. 25 references.

  13. Fungal invertase as an aid for fermentation of cane molasses into ethanol

    SciTech Connect

    Park, Y.K.; Sato, H.H.

    1982-10-01

    Comparative studies of the fermentation of cane molasses into ethanol by Saccharomyces cerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30 degrees Centigrade and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.4, ethanol production was increased by the addition of invertase. At 40 degrees C, the addition of invertase increased ethanol production by 5.5% at pH 5.0 and by 20.9% at pH 3.5. (Refs. 8).

  14. Age characteristics of changes in invertase activity of the mucous membrane of the small intestine

    NASA Technical Reports Server (NTRS)

    Rakhimov, K. R.; Aleksandrova, N. V.

    1980-01-01

    Rats of varying ages were subjected to stress from heat, cold, and hydrocortisone injection. Invertase activity in homogenates of small intestine mucous membranes was studied following sacrifice. Invertase activity was low in young animals, but increased sharply in 30 day old ones, remaining at a relatively constant level until old age. The study concludes that the stress hormone (corticosteroids, etc.) levels in the blood, which affects the formation of enteric enzyme levels and activities, and that age related peculiarities in invertase activity are a consequence of altered hormone status and epitheliocyte sensitivity.

  15. Detection of mercury compounds using invertase-glucose oxidase-based biosensor

    SciTech Connect

    Amine, A.; Cremisini, C.; Palleschi, G.

    1995-12-31

    Mercury compounds have been determined with an electrochemical biosensor based on invertase inhibition. When invertase is in presence of mercury its activity decreases; this causes a decrease of glucose production which is monitored by the glucose sensor and correlated to the concentration of mercury in solution. Parameters as pH, enzyme concentration, substrate concentration, and reaction and incubation time were optimized. Mercury compounds determination using soluble or immobilized invertase were reported. Results showed that the inhibition was competitive and reversible. Mercury compounds can be detected directly in aqueous solution in the range 2--10 ppb.

  16. Copper-modified gold electrode specific for monosaccharide detection Use in amperometric determination of phenylmercury based on invertase enzyme inhibition.

    PubMed

    Mohammadi, H; Amine, A; El Rhazi, M; Brett, C M A

    2004-04-19

    The electrochemical oxidation of mono- and disaccharides at various copper-modified electrodes is reported: glassy carbon modified at open circuit or by electrochemical deposition of copper, gold modified by electrochemical deposition, and at bulk copper electrodes. A comparative study of these four electrodes was made by linear sweep voltammetry and amperometry. The maximum oxidation peak separation between disaccharides and monosaccharides is about 200mV. After optimization, amperometric determination of monosaccharides was done at +0.30 versus Ag/AgCl in 0.15M NaOH at the copper-modified gold electrode. Using the developed method, the enzymatic activities of invertase and beta-galactosidase were determined through their reaction with sucrose and lactose, respectively. Validation was carried out by a spectrophotometric method based on 3,5-dinitrosalicylic acid, and it was shown that the proposed electrochemical method is more sensitive. The analytical utility of the copper-modified gold electrode was tested for the determination of organic mercury. Addition of phenylmercury standards to the invertase solution caused a decrease in the enzyme activity, and allowed the determination of phenylmercury in pharmaceutical samples. The concentration has been determined in the 10-55ngml(-1) range. PMID:18969385

  17. Comparison of Bacillus monooxygenase genes for unique fatty acid production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reviews Bacillus genes encoding monooxygenase enzymes producing unique fatty acid metabolites. Specifically, it examines standard monooxygenase electron transfer schemes and related domain structures of these fused domain enzymes on route to understanding the observed oxygenase activiti...

  18. Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment.

    PubMed

    Guazzaroni, María-Eugenia; Morgante, Verónica; Mirete, Salvador; González-Pastor, José E

    2013-04-01

    Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizosphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the transcriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowledge about the different strategies used by microorganisms to survive under extremely acid conditions. PMID:23145860

  19. Purification and characterization of neutral invertase from chickpea nodules.

    PubMed

    Asthir, B; Singh, R

    1997-12-01

    Neutral invertase from nodules of chickpea (Cicer arietinum L.) was isolated and purified by ammonium sulphate fractionation, gel filtration and DEAE-cellulose column chromatography. The purified enzyme was stable between 0 to 40 degrees C beyond which it was irreversibly denatured. Optimum temperature and pH of the enzyme were 37 degrees C and 7.0, respectively. K(m) for sucrose was 14.2 mM and Vmax was 4.8 mumole hr-1. The enzyme was inhibited by several metal ions. From the temperature effect on K(m) and Vmax values, the energy of activation (Ea), enthalpy change (delta H) and entropy change (delta S) of the enzyme were calculated to be 147 kJmol-1, -4.10 kJmol-1 and -2.33 JK-1mol-1, respectively. By employing photo-oxidation and chemical modification and by studying the effect of pH on K(m) and Vmax, the involvement of sulphydryl-, imidazole- and alpha-amino groups in the active site of the enzyme has been indicated. PMID:9594435

  20. Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death.

    PubMed

    Liu, Yong-Hua; Offler, Christina E; Ruan, Yong-Ling

    2016-09-01

    Reduced cell wall invertase (CWIN) activity has been shown to be associated with poor seed and fruit set under abiotic stress. Here, we examined whether genetically increasing native CWIN activity would sustain fruit set under long-term moderate heat stress (LMHS), an important factor limiting crop production, by using transgenic tomato (Solanum lycopersicum) with its CWIN inhibitor gene silenced and focusing on ovaries and fruits at 2 d before and after pollination, respectively. We found that the increase of CWIN activity suppressed LMHS-induced programmed cell death in fruits. Surprisingly, measurement of the contents of H2O2 and malondialdehyde and the activities of a cohort of antioxidant enzymes revealed that the CWIN-mediated inhibition on programmed cell death is exerted in a reactive oxygen species-independent manner. Elevation of CWIN activity sustained Suc import into fruits and increased activities of hexokinase and fructokinase in the ovaries in response to LMHS Compared to the wild type, the CWIN-elevated transgenic plants exhibited higher transcript levels of heat shock protein genes Hsp90 and Hsp100 in ovaries and HspII17.6 in fruits under LMHS, which corresponded to a lower transcript level of a negative auxin responsive factor IAA9 but a higher expression of the auxin biosynthesis gene ToFZY6 in fruits at 2 d after pollination. Collectively, the data indicate that CWIN enhances fruit set under LMHS through suppression of programmed cell death in a reactive oxygen species-independent manner that could involve enhanced Suc import and catabolism, HSP expression, and auxin response and biosynthesis. PMID:27462084

  1. Contributions of Sucrose Synthase and Invertase to the Metabolism of Sucrose in Developing Leaves 1

    PubMed Central

    Schmalstig, J. Gougler; Hitz, William D.

    1987-01-01

    The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1′-deoxy-1′-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [14C]Sucrose and [14C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [14C]sucrose was supplied than when [14C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%. PMID:16665711

  2. Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

    NASA Astrophysics Data System (ADS)

    Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

    2015-11-01

    Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

  3. Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

    NASA Astrophysics Data System (ADS)

    Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

    2016-07-01

    Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

  4. Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin.

    PubMed

    Gómez, Leissy; Ramírez, Hector L; Neira-Carrillo, Andrónico; Villalonga, Reynaldo

    2006-05-01

    Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 degrees C and its thermostability was enhanced by about 10 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively. PMID:16775742

  5. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

  6. Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

    PubMed

    Arez, Bruno F; Alves, Luís; Paixão, Susana M

    2014-11-01

    The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition. PMID:25163885

  7. Role of invertase activity in processing quality of potatoes: Effect of storage temperature and duration.

    PubMed

    Bandana; Sharma, Vineet; Singh, Brajesh; Raigond, Pinky; Kaushik, S K

    2016-03-01

    Invertase activity and processing attributes of three potato cultivars were studied to find the reason for deterioration of processing quality during their prolonged storage in commercial cold stores (4°C) as compared to elevated temperature storage (12 ± 0.5°C), with CIPC {Isopropyl-N-(3-Cholorophenyl) carbamate}. Lower storage temperature (4°C) tended to be more effective in increasing invertase activity of potato tubers than elevated temperature. Non-processing cultivar viz., Kufri Pukhraj resulted in accumulation of more invertase activity than relatively two processing cultivars. Kufri Chipsona-1 and Kufri Chipsona-3 at 12 ± 0.5°C possessed basal invertase activity ranging from 39.3 to 79.8 and 54.1 to 93.8 (pmoles hexose h⁻¹ g⁻¹ f.wt.) respectively, during two years. Total invertase activity at 4°C increased abruptly and remained high from 30 to 60 days of storage. The activity progressively reached 90.6 to 106.6 and 81.4 to 101.3 during both the years respectively, after 60 days of storage to that observed initially. Reducing sugar content increased from 23.3 to 105.7 and 389.0 to 1138.2 (mg 100g⁻¹ f.wt.) after 90 days of storage at 12 ± 0.5°C and 4°C, respectively. Studies concluded that basal and total invertase, were responsible for cold-induced sweetening and resulted in deterioration of processing quality of potatoes during storage at 4°C. Since this activity is low at 12 ± 0.5°C, the processing traits remained acceptable to industry and consumers. PMID:27097443

  8. Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

    PubMed Central

    Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

    2014-01-01

    Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community. PMID:24498417

  9. Cadmium induces retinoic acid signaling by regulating retinoic acid metabolic gene expression.

    PubMed

    Cui, Yuxia; Freedman, Jonathan H

    2009-09-11

    The transition metal cadmium is an environmental teratogen. In addition, cadmium and retinoic acid can act synergistically to induce forelimb malformations. The molecular mechanism underlying the teratogenicity of cadmium and the synergistic effect with retinoic acid has not been addressed. An evolutionarily conserved gene, beta,beta-carotene 15,15'-monooxygenase (BCMO), which is involved in retinoic acid biosynthesis, was studied in both Caenorhabditis elegans and murine Hepa 1-6 cells. In C. elegans, bcmo-1 was expressed in the intestine and was cadmium inducible. Similarly, in Hepa 1-6 cells, Bcmo1 was induced by cadmium. Retinoic acid-mediated signaling increased after 24-h exposures to 5 and 10 microm cadmium in Hepa 1-6 cells. Examination of gene expression demonstrated that the induction of retinoic acid signaling by cadmium may be mediated by overexpression of Bcmo1. Furthermore, cadmium inhibited the expression of Cyp26a1 and Cyp26b1, which are involved in retinoic acid degradation. These results indicate that cadmium-induced teratogenicity may be due to the ability of the metal to increase the levels of retinoic acid by disrupting the expression of retinoic acid-metabolizing genes. PMID:19556237

  10. Cadmium Induces Retinoic Acid Signaling by Regulating Retinoic Acid Metabolic Gene Expression*

    PubMed Central

    Cui, Yuxia; Freedman, Jonathan H.

    2009-01-01

    The transition metal cadmium is an environmental teratogen. In addition, cadmium and retinoic acid can act synergistically to induce forelimb malformations. The molecular mechanism underlying the teratogenicity of cadmium and the synergistic effect with retinoic acid has not been addressed. An evolutionarily conserved gene, β,β-carotene 15,15′-monooxygenase (BCMO), which is involved in retinoic acid biosynthesis, was studied in both Caenorhabditis elegans and murine Hepa 1–6 cells. In C. elegans, bcmo-1 was expressed in the intestine and was cadmium inducible. Similarly, in Hepa 1–6 cells, Bcmo1 was induced by cadmium. Retinoic acid-mediated signaling increased after 24-h exposures to 5 and 10 μm cadmium in Hepa 1–6 cells. Examination of gene expression demonstrated that the induction of retinoic acid signaling by cadmium may be mediated by overexpression of Bcmo1. Furthermore, cadmium inhibited the expression of Cyp26a1 and Cyp26b1, which are involved in retinoic acid degradation. These results indicate that cadmium-induced teratogenicity may be due to the ability of the metal to increase the levels of retinoic acid by disrupting the expression of retinoic acid-metabolizing genes. PMID:19556237

  11. Reassessment of an Arabidopsis cell wall invertase inhibitor AtCIF1 reveals its role in seed germination and early seedling growth.

    PubMed

    Su, Tao; Wolf, Sebastian; Han, Mei; Zhao, Hongbo; Wei, Hongbin; Greiner, Steffen; Rausch, Thomas

    2016-01-01

    In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) are recognized as essential players in sugar metabolism and sugar signaling, thereby affecting source-sink interactions, plant development and responses to environmental cues. CWI and VI expression levels are transcriptionally controlled; however, both enzymes are also subject to posttranslational control by invertase inhibitor proteins. The physiological significances of inhibitor proteins during seed germination and early seedling development are not yet fully understood. Here, we demonstrate that the inhibitor isoform AtCIF1 impacted on seed germination and early seedling growth in Arabidopsis. The primary target of AtCIF1 was shown to be localized to the apoplast after expressing an AtCIF1 YFP-fusion construct in tobacco epidermis and transgenic Arabidopsis root. The analysis of expression patterns showed that AtCWI1 was co-expressed spatiotemporally with AtCIF1 within the early germinating seeds. Seed germination was observed to be accelerated independently of exogenous abscisic acid (ABA) in the AtCIF1 loss-of-function mutant cif1-1. This effect coincided with a drastic increase of CWI activity in cif1-1 mutant seeds by 24 h after the onset of germination, both in vitro and in planta. Accordingly, quantification of sugar content showed that hexose levels were significantly boosted in germinating seeds of the cif1-1 mutant. Further investigation of AtCIF1 overexpressors in Arabidopsis revealed a markedly suppressed CWI activity as well as delayed seed germination. Thus, we conclude that the posttranslational modulation of CWI activity by AtCIF1 helps to orchestrate seed germination and early seedling growth via fine-tuning sucrose hydrolysis and, possibly, sugar signaling. PMID:26546341

  12. Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family.

    PubMed

    Körbes, Ana Paula; Kulcheski, Franceli Rodrigues; Margis, Rogério; Margis-Pinheiro, Márcia; Turchetto-Zolet, Andreia Carina

    2016-03-01

    Lysophosphatidic acid acyltransferases (LPAATs) perform an essential cellular function by controlling the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane, signaling and storage lipids. Although LPAATs have been extensively explored by functional and biotechnological studies, little is known about their molecular evolution and diversification. We performed a genome-wide analysis using data from several plants and animals, as well as other eukaryotic and prokaryotic species, to identify LPAAT genes and analyze their evolutionary history. We used phylogenetic and molecular evolution analysis to test the hypothesis of distinct origins for these genes. The reconstructed phylogeny supported the ancient origin of some isoforms (plant LPAAT1 and LPAATB; animal AGPAAT1/2), while others emerged more recently (plant LPAAT2/3/4/5; AGPAAT3/4/5/8). Additionally, the hypothesis of endosymbiotic origin of the plastidic isoform LPAAT1 was confirmed. LPAAT genes from plants and animals mainly experienced strong purifying selection pressures with limited functional divergence after the species-specific duplications. Gene expression analyses of LPAAT isoforms in model plants demonstrated distinct LPAAT expression patterns in these organisms. The results showed that distinct origins followed by diversification of the LPAAT genes shaped the evolution of TAG biosynthesis. The expression pattern of individual genes may be responsible for adaptation into multiple ecological niches. PMID:26721558

  13. Tomato ABSCISIC ACID STRESS RIPENING (ASR) Gene Family Revisited

    PubMed Central

    Golan, Ido; Dominguez, Pia Guadalupe; Konrad, Zvia; Shkolnik-Inbar, Doron; Carrari, Fernando; Bar-Zvi, Dudy

    2014-01-01

    Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding. PMID:25310287

  14. Tomato ABSCISIC ACID STRESS RIPENING (ASR) gene family revisited.

    PubMed

    Golan, Ido; Dominguez, Pia Guadalupe; Konrad, Zvia; Shkolnik-Inbar, Doron; Carrari, Fernando; Bar-Zvi, Dudy

    2014-01-01

    Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding. PMID:25310287

  15. VARIED GROWTH, BIOMASS AND CELLULOSE CONTENT IN TOBACCO EXPRESSING YEAST-DERIVED INVERTASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of the expression of yeast-derived apoplastic (AI) and cytosolic (CI) invertases on biomass and structural carbohydrate accumulation in tobacco (Nicotiana tabacum cv. Xanthi) were evaluated. Transgenic tobacco plants expressing AI or CI under the control of either a tandem repeat of the...

  16. Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by-product.

    PubMed

    Mouelhi, Refka; Abidi, Ferid; Galai, Said; Marzouki, M Nejib

    2014-03-01

    The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K(m) and V(max) for sucrose were estimated to be respectively 5.8 mM and 0.11 μmol/min. The invertase was activated by β-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month's storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %. PMID:24142426

  17. Abscisic acid represses the transcription of chloroplast genes*

    PubMed Central

    Yamburenko, Maria V.; Zubo, Yan O.; Börner, Thomas

    2013-01-01

    Numerous studies have shown effects of abscisic acid (ABA) on nuclear genes encoding chloroplast-localized proteins. ABA effects on the transcription of chloroplast genes, however, have not been investigated yet thoroughly. This work, therefore, studied the effects of ABA (75 μM) on transcription and steady-state levels of transcripts in chloroplasts of basal and apical segments of primary leaves of barley (Hordeum vulgare L.). Basal segments consist of young cells with developing chloroplasts, while apical segments contain the oldest cells with mature chloroplasts. Exogenous ABA reduced the chlorophyll content and caused changes of the endogenous concentrations not only of ABA but also of cytokinins to different extents in the basal and apical segments. It repressed transcription by the chloroplast phage-type and bacteria-type RNA polymerases and lowered transcript levels of most investigated chloroplast genes drastically. ABA did not repress the transcription of psbD and a few other genes and even increased psbD mRNA levels under certain conditions. The ABA effects on chloroplast transcription were more pronounced in basal vs. apical leaf segments and enhanced by light. Simultaneous application of cytokinin (22 μM 6-benzyladenine) minimized the ABA effects on chloroplast gene expression. These data demonstrate that ABA affects the expression of chloroplast genes differentially and points to a role of ABA in the regulation and coordination of the activities of nuclear and chloroplast genes coding for proteins with functions in photosynthesis. PMID:24078671

  18. Gene therapy for aromatic L-amino acid decarboxylase deficiency.

    PubMed

    Hwu, Wuh-Liang; Muramatsu, Shin-ichi; Tseng, Sheng-Hong; Tzen, Kai-Yuan; Lee, Ni-Chung; Chien, Yin-Hsiu; Snyder, Richard O; Byrne, Barry J; Tai, Chun-Hwei; Wu, Ruey-Meei

    2012-05-16

    Aromatic L-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector-mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[(18)F]fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function. PMID:22593174

  19. Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High oleic acid soybeans were produced by combining a mutant FAD2-1A and a mutant FAD2-1B gene. Despite having a high oleic acid content, the linolenic acid content of these soybeans was in the range of 4-6%. Therefore, a study was conducted to incorporate one or two mutant FAD3 genes into the high ...

  20. Benzoic Acid-Inducible Gene Expression in Mycobacteria

    PubMed Central

    Dragset, Marte S.; Barczak, Amy K.; Kannan, Nisha; Mærk, Mali; Flo, Trude H.; Valla, Svein; Rubin, Eric J.; Steigedal, Magnus

    2015-01-01

    Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance. PMID:26348349

  1. Identification of genes regulated by UV/salicylic acid.

    SciTech Connect

    Paunesku, T.; Chang-Liu, C.-M.; Shearin-Jones, P.; Watson, C.; Milton, J.; Oryhon, J.; Salbego, D.; Milosavljevic, A.; Woloschak, G. E.; CuraGen Corp.

    2000-02-01

    Purpose : Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells. The presumed effector molecule responsible for this modulation is NF-kappaB. In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid. Materials and methods : Differential-display RT-PCR was used to identify differentially expressed genes. Results : Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156). Conclusions : Sequences of four of these genes contained NF-kappaB DNA binding sites of the type that may attract transrepressor p55/p55 NF-kappaB homodimers. Down-regulation of these genes upon UV irradiation may contribute to increased cell survival via suppression of p53 independent apoptosis.

  2. Comparison of gene expression methods to identify genes responsive to perfluorooctane sulfonic acid.

    PubMed

    Hu, Wenyue; Jones, Paul D; Decoen, Wim; Newsted, John L; Giesy, John P

    2005-01-01

    Genome-wide expression techniques are being increasingly used to assess the effects of environmental contaminants. Oligonucleotide or cDNA microarray methods make possible the screening of large numbers of known sequences for a given model species, while differential display analysis makes possible analysis of the expression of all the genes from any species. We report a comparison of two currently popular methods for genome-wide expression analysis in rat hepatoma cells treated with perfluorooctane sulfonic acid. The two analyses provided 'complimentary' information. Approximately 5% of the 8000 genes analyzed by the GeneChip array, were altered by a factor of three or greater. Differential display results were more difficult to interpret, since multiple gene products were present in most gel bands so a probabilistic approach was used to determine which pathways were affected. The mechanistic interpretation derived from these two methods was in agreement, both showing similar alterations in a specific set of genes. PMID:21783471

  3. Immobilization of invertase on chitosan coated γ-Fe2O3 magnetic nanoparticles to facilitate magnetic separation.

    PubMed

    Waifalkar, P P; Parit, S B; Chougale, A D; Sahoo, Subasa C; Patil, P S; Patil, P B

    2016-11-15

    Industrially important invertase enzyme was immobilized on chitosan coated sol gel derived γ-Fe2O3 magnetic nanoparticles (MNPs) to enable it for repetitive use by magnetic separation. MNPs were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), field emission scanning electron microscope (FE-SEM), Fourier transform infrared (FTIR) spectrometer and magnetic measurements. FTIR studies confirmed successful immobilization of invertase on MNPs. The ability to convert sucrose into invert syrup was enhanced in immobilized invertase compared to that of free enzyme. Further it was found that invertase immobilized on MNPs (IIMNPs) were more stable at varying pH and temperature conditions. Magnetic separation technique was successfully employed for reuse of the IIMNPs for 20 times without significant loss of activity. PMID:27501039

  4. Molecular cloning, expression and characterization of a novel apoplastic invertase inhibitor from tomato (Solanum lycopersicum) and its use to purify a vacuolar invertase.

    PubMed

    Reca, Ida Barbara; Brutus, Alexandre; D'Avino, Rossana; Villard, Claude; Bellincampi, Daniela; Giardina, Thierry

    2008-01-01

    Protein inhibitors are molecules secreted by many plants. In a functional genomics approach, an invertase inhibitor (SolyCIF) of Solanum lycopersicum was identified at the Solanaceae Cornell University data bank (www.sgn.cornell.edu). It was established that this inhibitor is expressed mainly in the leaves, flowers and green fruit of the plant and localized in the cell wall compartment. The SolyCIF cDNA was cloned by performing RT-PCR, fully sequenced and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by performing ion-exchange chromatography and gel filtration was further biochemically characterized and used to perform affinity chromatography. The latter step made it possible to purify natural vacuolar invertase (TIV-1), which showed high rates of catalytic activity (438.3 U mg(-1)) and efficiently degraded saccharose (K(m)=6.4mM, V(max)=2.9 micromol saccharosemin(-1) and k(c)(at)=7.25 x 10(3)s(-1) at pH 4.9 and 37 degrees C). The invertase activity was strongly inhibited in a dose-dependent manner by SolyCIF produced in P. pastoris. In addition, Gel-SDS-PAGE analysis strongly suggests that TIV-1 was proteolyzed in planta and it was established that the fragments produced have to be tightly associated for its enzymatic activity to occur. We further investigated the location of the proteolytic sites by performing NH(2)-terminal Edman degradation on the fragments. The molecular model for TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two halves, which confirms that the enzymatic activity is possible only when the fragments are tightly associated. PMID:18573306

  5. Nucleic Acid Modifications in Regulation of Gene Expression.

    PubMed

    Chen, Kai; Zhao, Boxuan Simen; He, Chuan

    2016-01-21

    Nucleic acids carry a wide range of different chemical modifications. In contrast to previous views that these modifications are static and only play fine-tuning functions, recent research advances paint a much more dynamic picture. Nucleic acids carry diverse modifications and employ these chemical marks to exert essential or critical influences in a variety of cellular processes in eukaryotic organisms. This review covers several nucleic acid modifications that play important regulatory roles in biological systems, especially in regulation of gene expression: 5-methylcytosine (5mC) and its oxidative derivatives, and N(6)-methyladenine (6mA) in DNA; N(6)-methyladenosine (m(6)A), pseudouridine (Ψ), and 5-methylcytidine (m(5)C) in mRNA and long non-coding RNA. Modifications in other non-coding RNAs, such as tRNA, miRNA, and snRNA, are also briefly summarized. We provide brief historical perspective of the field, and highlight recent progress in identifying diverse nucleic acid modifications and exploring their functions in different organisms. Overall, we believe that work in this field will yield additional layers of both chemical and biological complexity as we continue to uncover functional consequences of known nucleic acid modifications and discover new ones. PMID:26933737

  6. Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue

    NASA Technical Reports Server (NTRS)

    Karuppiah, N.; Vadlamudi, B.; Kaufman, P. B.

    1989-01-01

    Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (beta-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organo-mercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, Km of 12 millimolar, and a Vmax of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, Km of millimolar, and a Vmax of 9 micromole per minute per milligram of protein.

  7. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  8. Sucrose and invertases, a part of the plant defense response to the biotic stresses

    PubMed Central

    Tauzin, Alexandra S.; Giardina, Thierry

    2014-01-01

    Sucrose is the main form of assimilated carbon which is produced during photosynthesis and then transported from source to sink tissues via the phloem. This disaccharide is known to have important roles as signaling molecule and it is involved in many metabolic processes in plants. Essential for plant growth and development, sucrose is engaged in plant defense by activating plant immune responses against pathogens. During infection, pathogens reallocate the plant sugars for their own needs forcing the plants to modify their sugar content and triggering their defense responses. Among enzymes that hydrolyze sucrose and alter carbohydrate partitioning, invertases have been reported to be affected during plant-pathogen interactions. Recent highlights on the role of invertases in the establishment of plant defense responses suggest a more complex regulation of sugar signaling in plant-pathogen interaction. PMID:25002866

  9. One-pot production of fructooligosaccharides by a Saccharomyces cerevisiae strain expressing an engineered invertase.

    PubMed

    Marín-Navarro, Julia; Talens-Perales, David; Polaina, Julio

    2015-03-01

    We describe a simple, efficient process for the production of 6-kestose, a trisaccharide with well-documented prebiotic properties. A key factor is the use of a yeast transformant expressing an engineered version of Saccharomyces invertase with enhanced transfructosylating activity. When the yeast transformant was grown with 30 % sucrose as the carbon source, 6-kestose accumulated up to ca. 100 g/L in the culture medium. The 6-kestose yield was significantly enhanced (up to 200 g/L) using a two-stage process carried out in the same flask. In the first stage, the culture was grown in 30 % sucrose at physiological temperature (30 °C) to allow overexpression of the invertase. In the second stage, sucrose was added to the culture at high concentration (60 %) and the temperature shifted to 50 °C. In both cases, 6-kestose was synthesized with high specificity, representing more than 95 % of total FOS. PMID:25547837

  10. Associations between a fatty acid desaturase gene polymorphism and blood arachidonic acid compositions in Japanese elderly.

    PubMed

    Horiguchi, Sayaka; Nakayama, Kazuhiro; Iwamoto, Sadahiko; Ishijima, Akiko; Minezaki, Takayuki; Baba, Mamiko; Kontai, Yoshiko; Horikawa, Chika; Kawashima, Hiroshi; Shibata, Hiroshi; Kagawa, Yasuo; Kawabata, Terue

    2016-02-01

    We investigated whether the single nucleotide polymorphism rs174547 (T/C) of the fatty acid desaturase-1 gene, FADS1, is associated with changes in erythrocyte membrane and plasma phospholipid (PL) long-chain polyunsaturated fatty acid (LCPUFA) composition in elderly Japanese participants (n=124; 65 years or older; self-feeding and oral intake). The rs174547 C-allele carriers had significantly lower arachidonic acid (ARA; n-6 PUFA) and higher linoleic acid (LA, n-6 PUFA precursor) levels in erythrocyte membrane and plasma PL (15% and 6% ARA reduction, respectively, per C-allele), suggesting a low LA to ARA conversion rate in erythrocyte membrane and plasma PL of C-allele carriers. α-linolenic acid (n-3 PUFA precursor) levels were higher in the plasma PL of C-allele carriers, whereas levels of the n-3 LCPUFAs eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were unchanged in erythrocyte membrane and plasma PL. Thus, rs174547 genotypes were significantly associated with different ARA compositions of the blood of elderly Japanese. PMID:26869086

  11. Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

    PubMed Central

    Ewert, Kai K.; Majzoub, Ramsey N.; Leal, Cecília

    2014-01-01

    Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications. PMID:25587216

  12. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

    PubMed

    Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

    2015-12-01

    In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. PMID:26041210

  13. A gene network engineering platform for lactic acid bacteria

    PubMed Central

    Kong, Wentao; Kapuganti, Venkata S.; Lu, Ting

    2016-01-01

    Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas. PMID:26503255

  14. A gene network engineering platform for lactic acid bacteria.

    PubMed

    Kong, Wentao; Kapuganti, Venkata S; Lu, Ting

    2016-02-29

    Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas. PMID:26503255

  15. Formulation and drying of alginate beads for controlled release and stabilization of invertase.

    PubMed

    Santagapita, Patricio R; Mazzobre, M Florencia; Buera, M Pilar

    2011-09-12

    Several alternatives to the conventional alginate beads formulation were studied for encapsulation of invertase. Pectin was added to the alginate/enzyme solution while trehalose and β-cyclodextrin were added to the calcium gelation media. The effect of composition changes, freezing, drying methods (freeze, vacuum, or air drying), and thermal treatment were evaluated on invertase stability and its release kinetics from beads. The enzyme release mechanism from wet beads depended on pH. The addition of trehalose, pectin, and β-cyclodextrin modified the bead structure, leading in some cases to a release mechanism that included the relaxation of the polymer chains, besides Fickian diffusion. Enzyme release from vacuum-dried beads was much faster than from freeze-dried beads, probably due to their higher pore size. The inclusion of β-cyclodextrin and especially of pectin prevented enzyme activity losses during bead generation, and trehalose addition was fundamental for achieving adequate invertase protection during freezing, drying, and thermal treatment. Present results showed that several alternatives such as drying method, composition, as well as pH of the relese medium can be managed to control enzyme release. PMID:21809830

  16. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  17. Light-induced expression of fatty acid desaturase genes

    PubMed Central

    Kis, Mihály; Zsiros, Otto; Farkas, Tibor; Wada, Hajime; Nagy, Ferenc; Gombos, Zoltán

    1998-01-01

    In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for ω3 desaturase. However, the induction of both desA and desD mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases. PMID:9539715

  18. Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-07-28

    Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'. PMID:27298431

  19. [Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca].

    PubMed

    Yu, Shu-lin; Huang, Lu-qi; Yuan, Yuan; Qi, Lin-jie; Liu, Da-hui

    2015-03-01

    To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients. PMID:26087546

  20. Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves : estimation by alternate substrate utilization.

    PubMed

    Schmalstig, J G; Hitz, W D

    1987-10-01

    The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1'-deoxy-1'-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [(14)C]Sucrose and [(14)C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [(14)C]sucrose was supplied than when [(14)C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%. PMID:16665711

  1. Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves: estimation by alternate substrate utilization

    SciTech Connect

    Schmalstig, J.G.; Hitz, W.D.

    1987-10-01

    The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1'-deoxy-1'-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS.(/sup 14/C)Sucrose and (/sup 14/C)FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when (/sup 14/C)sucrose was supplied than when (/sup 14/C)FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.

  2. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    SciTech Connect

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  3. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

  4. Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

    PubMed Central

    2012-01-01

    Background Biofuel has been the focus of intensive global research over the past few years. The development of 4th generation biofuel production (algae-to-biofuels) based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA) biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation. Results We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP), 3-ketoacyl-ACP-synthase (KAS), and acyl-ACP thioesterase (FATA) gene expression had significant correlations with monounsaturated FA (MUFA) synthesis and polyunsaturated FA (PUFA) synthesis. Conclusions We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production. PMID:22448811

  5. Hybrubins: Bipyrrole Tetramic Acids Obtained by Crosstalk between a Truncated Undecylprodigiosin Pathway and Heterologous Tetramic Acid Biosynthetic Genes.

    PubMed

    Zhao, Zhilong; Shi, Ting; Xu, Min; Brock, Nelson L; Zhao, Yi-Lei; Wang, Yemin; Deng, Zixin; Pang, Xiuhua; Tao, Meifeng

    2016-02-01

    Heterologous expression of bacterial artificial chromosome (BAC) clones from the genomic library of Streptomyces variabilis Snt24 in Streptomyces lividans SBT5 which carried a truncated undecylprodigiosin biosynthetic gene cluster led to the identification of hybrubins A-C. The hybrubins represent a new carbon skeleton in which a tetramic acid moiety is fused to a 2,2'-dipyrrole building block. Gene knockout experiments confirmed that hybrubins are derived from two convergent biosynthetic pathways including the remaining genomic red genes of S. lividans SBT5 as well as the BAC encoded hbn genes for the production of 5-ethylidenetetramic acid. A possible biosynthetic pathway was also proposed. PMID:26800378

  6. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  7. The PH gene determines fruit acidity and contributes to the evolution of sweet melons.

    PubMed

    Cohen, Shahar; Itkin, Maxim; Yeselson, Yelena; Tzuri, Galil; Portnoy, Vitaly; Harel-Baja, Rotem; Lev, Shery; Sa'ar, Uzi; Davidovitz-Rikanati, Rachel; Baranes, Nadine; Bar, Einat; Wolf, Dalia; Petreikov, Marina; Shen, Shmuel; Ben-Dor, Shifra; Rogachev, Ilana; Aharoni, Asaph; Ast, Tslil; Schuldiner, Maya; Belausov, Eduard; Eshed, Ravit; Ophir, Ron; Sherman, Amir; Frei, Benedikt; Neuhaus, H Ekkehard; Xu, Yimin; Fei, Zhangjun; Giovannoni, Jim; Lewinsohn, Efraim; Tadmor, Yaakov; Paris, Harry S; Katzir, Nurit; Burger, Yosef; Schaffer, Arthur A

    2014-01-01

    Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago. PMID:24898284

  8. Effect of hypokinesia on invertase activity of the mucosa of the small intestine

    NASA Technical Reports Server (NTRS)

    Abdusattarov, A.

    1980-01-01

    The effect of prolonged hypokinesia on the enzyme activity of the middle portion of the small intestine was investigated. Eighty-four mongrel white male rats weighing 170-180 g were divided into two equal groups. The experimental group were maintained in single cages under 30 days of hypokinetic conditions and the control animals were maintained under ordinary laboratory conditions. It is concluded that rates of invertase formation and its inclusion in the composition if the cellular membrane, if judged by the enzyme activity studied in sections of the small intestine, are subject to phase changes in the course of prolonged hypokinesia.

  9. Preparation and properties of co-reticulated invertase supported by an ultrafiltration membrane.

    PubMed Central

    Cantarella, M; Gianfreda, L; Palescandolo, R; Scardi, V

    1977-01-01

    Yeast invertase was co-reticulated with glutaraldehyde to bovine serum albumin to give a soluble bound enzyme that was immobilized as a tightly adhering layer on the active surface of an ultrafiltration membrane. The Michaelis constant and stability of this immobilized-enzyme system are compared with those of the enzyme either in the native form or immobilized as a dynamically formed gel layer on an ultrafiltration membrane, as previously described by us [Drioli, Gianfreda, Palescandolo & Scardi (1975) Biotechnol, Bioeng, 17, 1365-1367]. PMID:588266

  10. Preparation and properties of co-reticulated invertase supported by an ultrafiltration membrane.

    PubMed

    Cantarella, M; Gianfreda, L; Palescandolo, R; Scardi, V

    1977-10-01

    Yeast invertase was co-reticulated with glutaraldehyde to bovine serum albumin to give a soluble bound enzyme that was immobilized as a tightly adhering layer on the active surface of an ultrafiltration membrane. The Michaelis constant and stability of this immobilized-enzyme system are compared with those of the enzyme either in the native form or immobilized as a dynamically formed gel layer on an ultrafiltration membrane, as previously described by us [Drioli, Gianfreda, Palescandolo & Scardi (1975) Biotechnol, Bioeng, 17, 1365-1367]. PMID:588266

  11. The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases.

    PubMed Central

    Ullrich, M; Bender, C L

    1994-01-01

    Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an intermediate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription. The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including nonribosomal peptide synthetases. Furthermore, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(II) enzymes which catalyze oxidative cyclizations. The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins. These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesis were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature. Images PMID:8002582

  12. Mutation and gene transfer of neutral amino acid transport System L genes in mammalian cells

    SciTech Connect

    El-Gewely, M.R.; Collarini, E.J.; Campbell, G.S.; Oxender, D.L.

    1987-05-01

    The authors are attempting to clone the genes coding for amino acid transport System L. Chinese hamster ovary (CHO) cell mutants that are temperature sensitive in their leucyl-tRNA synthetase show temperature-dependent regulation of System L. Temperature resistant mutants isolated from these cells have constitutively derepressed System L activity. Somatic cell fusion studies using these mutants have suggested that a trans-acting element controls regulation of System L. Mutants with reduced transport activity were isolated by a TH-suicide selection. The growth of these mutant cells is limited by the transport defect. CHO mutants were transformed with a human cosmid library, followed by selection at high temperatures and low leucine concentrations. Some transformants have increased levels of System L activity, suggesting that human genes coding for leucine transport have been incorporated into the CHO genome. Human sequences were rescued by a lambda in vitro packaging system. These sequences hybridize to vector and total human DNA. Experiments are being done to confirm that these sequences indeed code for transport System L. They are also attempting to label membrane components of amino acid transporters by group-specific modifying reagents.

  13. The rice OsLpa1 gene encodse a novel protein involved in phytic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rice low phytic acid 1 (OsLpa1) gene was originally identified using a forward genetics approach. Mutation of this gene resulted in a 45% reduction in rice seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, the rice lpa1 mutant does not appear to differ significa...

  14. Identifying and assessing the impact of wine acid-related genes in yeast.

    PubMed

    Chidi, Boredi S; Rossouw, Debra; Bauer, Florian F

    2016-02-01

    Saccharomyces cerevisiae strains used for winemaking show a wide range of fermentation phenotypes, and the genetic background of individual strains contributes significantly to the organoleptic properties of wine. This strain-dependent impact extends to the organic acid composition of the wine, an important quality parameter. However, little is known about the genes which may impact on organic acids during grape must fermentation. To generate novel insights into the genetic regulation of this metabolic network, a subset of genes was identified based on a comparative analysis of the transcriptomes and organic acid profiles of different yeast strains showing different production levels of organic acids. These genes showed significant inter-strain differences in their transcription levels at one or more stages of fermentation and were also considered likely to influence organic acid metabolism based on existing functional annotations. Genes selected in this manner were ADH3, AAD6, SER33, ICL1, GLY1, SFC1, SER1, KGD1, AGX1, OSM1 and GPD2. Yeast strains carrying deletions for these genes were used to conduct fermentations and determine organic acid levels at various stages of alcoholic fermentation in synthetic grape must. The impact of these deletions on organic acid profiles was quantified, leading to novel insights and hypothesis generation regarding the role/s of these genes in wine yeast acid metabolism under fermentative conditions. Overall, the data contribute to our understanding of the roles of selected genes in yeast metabolism in general and of organic acid metabolism in particular. PMID:26040556

  15. Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress).

    PubMed Central

    Dumas, R; Curien, G; DeRose, R T; Douce, R

    1993-01-01

    Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene. Images Figure 3 Figure 5 PMID:8379936

  16. Identification of a 12-gene fusaric acid biosynthetic gene cluster in Fusarium species through comparative and functional genomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a ...

  17. Glassy state and thermal inactivation of invertase and lactase in dried amorphous matrices.

    PubMed

    Schebor, C; Burin, L; Buera, M P; Aguilera, J M; Chirife, J

    1997-01-01

    The thermal stability of enzymes lactase and invertase in dried, amorphous matrices of sugars (trehalose, maltose, lactose, sucrose, raffinose) and some other selected systems (casein, PVP, milk) was studied. The glass transition temperature (Tg) was limited as a threshold parameter for predicting enzyme inactivation because (a) enzyme inactivation was observed in glassy matrices, (b) a specific effect of enzyme stabilization by certain matrices particularly trehalose was observed, and (c) enzyme stability appeared to depend on heating temperature (T) "per se" rather than (T-Tg). For these reasons, a protective mechanism by sugars related to the maintenance of the tertiary structure of the enzyme was favored. A rapid loss of enzyme (lactase) activity was observed in heated sucrose systems at T > Tg, and this was attributed to sucrose crystallization since it is known that upon crystallization the protective effect of sugars is lost. Thus, the stabilizing effect could be indirectly affected by the Tg of the matrix, since crystallization of sugars only occurs above Tg. Trehalose model systems (with added invertase) showed an exceptional stability toward "darkening" (e.g., non-enzymatic browning) when heated in the dried state to elevated temperatures and for long periods of time. PMID:9413144

  18. Microsomal Omega-3 Fatty Acid Desaturase Genes in Low Linolenic Acid Soybean Line RG10 and Validation of Major Linolenic Acid QTL

    PubMed Central

    Reinprecht, Yarmilla; Pauls, K. Peter

    2016-01-01

    High levels of linolenic acid (80 g kg−1) are associated with the development of off-flavors and poor stability in soybean oil. The development of low linolenic acid lines such as RG10 (20 g kg−1 linolenic acid) can reduce these problems. The level of linolenic acid in seed oil is determined by the activities of microsomal omega-3 fatty acid desaturases (FAD3). A major linolenic acid QTL (>70% of variation) on linkage group B2 (chromosome Gm14) was previously detected in a recombinant inbred line population from the RG10 × OX948 cross. The objectives of this study were to validate the major linolenic acid QTL in an independent population and characterize all the soybean FAD3 genes. Four FAD3 genes were sequenced and localized in RG10 and OX948 and compared to the genes in the reference Williams 82 genome. The FAD3A gene sequences mapped to the locus Glyma.14g194300 [on the chromosome Gm14 (B2)], which is syntenic to the FAD3B gene (locus Glyma.02g227200) on the chromosome Gm02 (D1b). The location of the FAD3A gene is the same as was previously determined for the fan allele, that conditions low linolenic acid content and several linolenic acid QTL, including Linolen 3-3, mapped previously with the RG10 × OX948 population and confirmed in the PI 361088B × OX948 population as Linolen-PO (FAD3A). The FAD3B gene-based marker, developed previously, was mapped to the chromosome Gm02 (D1b) in a region containing a newly detected linolenic acid QTL [Linolen-RO(FAD3B)] in the RG10 × OX948 genetic map and corresponds well with the in silico position of the FAD3B gene sequences. FAD3C and FAD3D gene sequences, mapped to syntenic regions on chromosomes Gm18 (locus Glyma.18g062000) and Gm11 (locus Glyma.11g227200), respectively. Association of linolenic acid QTL with the desaturase genes FAD3A and FAD3B, their validation in an independent population, and development of FAD3 gene-specific markers should simplify and accelerate breeding for low linolenic acid soybean

  19. The PH gene determines fruit acidity and contributes to the evolution of sweet melons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acids are one of the three major components of fleshy fruit taste, together with sugars and volatile flavor compounds. However, the molecular-genetic control of acid accumulation in fruit is poorly understood and, to date, no genes responsible for acid accumulation in fleshy fruit have been function...

  20. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene.

    PubMed

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆⁶desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T₀ and T₁ generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%-0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T₁ generation as well as in immature and mature grains of the T₂ generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%-1.40% (v/v) and 0%-1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  1. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    PubMed Central

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  2. Student Collaboration in a Series of Integrated Experiments to Study Enzyme Reactor Modeling with Immobilized Cell-Based Invertase

    ERIC Educational Resources Information Center

    Taipa, M. A^ngela; Azevedo, Ana M.; Grilo, Anto´nio L.; Couto, Pedro T.; Ferreira, Filipe A. G.; Fortuna, Ana R. M.; Pinto, Ine^s F.; Santos, Rafael M.; Santos, Susana B.

    2015-01-01

    An integrative laboratory study addressing fundamentals of enzyme catalysis and their application to reactors operation and modeling is presented. Invertase, a ß-fructofuranosidase that catalyses the hydrolysis of sucrose, is used as the model enzyme at optimal conditions (pH 4.5 and 45 °C). The experimental work involves 3 h of laboratory time…

  3. The development of invertase activity in slices of the root of Beta vulgaris L. washed under aseptic conditions

    PubMed Central

    Bacon, J. S. D.; MacDonald, I. R.; Knight, A. H.

    1965-01-01

    1. When disks of root tissue from sugar or red beet (Beta vulgaris L.) are washed in running aerated tap water the sucrose contained in them disappears and glucose and fructose are formed. 2. Invertase activity in the disks has been measured by a polarimetric method. Freshly cut tissue has a very low activity, but a considerable increase occurs during the first 3–4 days of washing, the final activity being sufficient to hydrolyse the sucrose contained in the disk within a few hours. 3. Disks of red beet have been cut and shaken in water under aseptic conditions. Sucrose breakdown and invertase development still took place. Microbial contamination is therefore not responsible. 4. Trisaccharides that appear in sugar-beet disks during the washing process have been isolated and identified; their formation also suggests that a higher-plant invertase is acting. 5. The significance of these results is discussed in relation to protein synthesis in washed storage-tissue slices, and the occurrence of high invertase activity in growing plant cells. PMID:14342226

  4. The Isolation of Invertase from Baker's Yeast: A Four-Part Exercise in Protein Purification and Characterization

    ERIC Educational Resources Information Center

    Timerman, Anthony P.; Fenrick, Angela M.; Zamis, Thomas M.

    2009-01-01

    A sequence of exercises for the isolation and characterization of invertase (E.C. 3.1.2.26) from baker's yeast obtained from a local grocery store is outlined. Because the enzyme is colorless, the use of colored markers and the sequence of purification steps are designed to "visualize" the process by which a colorless protein is selectively…

  5. Gene Activation in Eukaryotes: Are Nuclear Acidic Proteins the Cause or the Effect?

    PubMed Central

    Pederson, Thoru

    1974-01-01

    Nuclear acidic proteins have been implicated in the positive control of gene transcription in eukaryotes. This hypothesis was examined in greater detail by analysis of these proteins during experimental gene activation by a technique for fractionating nuclei into chromatin and the ribonucleoprotein particles that contain heterogeneous nuclear RNA. When synthesis of rat-liver heterogeneous nuclear RNA was stimulated by administration of hydrocortisone, there was a parallel increase in the labeling of acidic proteins in ribonucleoprotein particles. However, there was no detectable effect on the labeling of either acidic chromatin proteins or histones. Thus, the nuclear acidic proteins that respond to the hormone are concerned with a post-transcriptional event, namely the assembly and processing of ribonucleoprotein particles that contain heterogeneous RNA, rather than with direct gene activation. Increases in synthesis of “chromatin” acidic proteins during gene activation observed by others may reflect the presence of these ribonucleoprotein particles in crude chromatin preparations. Images PMID:4522777

  6. Induction of nodD Gene in a Betarhizobium Isolate, Cupriavidus sp. of Mimosa pudica, by Root Nodule Phenolic Acids.

    PubMed

    Mandal, Santi M; Chakraborty, Dipjyoti; Dutta, Suhrid R; Ghosh, Ananta K; Pati, Bikas R; Korpole, Suresh; Paul, Debarati

    2016-06-01

    A range of phenolic acids, viz., p-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, ferulic acid, and cinnamic acid have been isolated and identified by LC-MS analysis in the roots and root nodules of Mimosa pudica. The effects of identified phenolic acids on the regulation of nodulation (nod) genes have been evaluated in a betarhizobium isolate of M. pudica root nodule. Protocatechuic acid and p-hydroxybenzoic acid were most effective in inducing nod gene, whereas caffeic acid had no significant effect. Phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activities were estimated, indicating regulation and metabolism of phenolic acids in root nodules. These results showed that nodD gene expression of betarhizobium is regulated by simple phenolic acids such as protocatechuic acid and p-hydroxybenzoic acid present in host root nodule and sustains nodule organogenesis. PMID:26897126

  7. Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

    PubMed

    O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

    2015-08-01

    The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms. PMID:25912312

  8. HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

    PubMed

    Liu, Shujin; Lan, Jixian; Zhou, Binhui; Qin, Yunxia; Zhou, Yihua; Xiao, Xiaohu; Yang, Jianghua; Gou, Jiqing; Qi, Jiyan; Huang, Yacheng; Tang, Chaorong

    2015-04-01

    In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular. PMID:25581169

  9. Functional expression of a Δ12 fatty acid desaturase gene from spinach in transgenic pigs

    PubMed Central

    Saeki, Kazuhiro; Matsumoto, Kazuya; Kinoshita, Mikio; Suzuki, Iwane; Tasaka, Yasushi; Kano, Koichiro; Taguchi, Yoshitomo; Mikami, Koji; Hirabayashi, Masumi; Kashiwazaki, Naomi; Hosoi, Yoshihiko; Murata, Norio; Iritani, Akira

    2004-01-01

    Linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Δ12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Δ12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were ≈10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained ≈20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases. PMID:15067141

  10. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  11. Assessment of Suitable Reference Genes for Quantitative Gene Expression Studies in Melon Fruits

    PubMed Central

    Kong, Qiusheng; Gao, Lingyun; Cao, Lei; Liu, Yue; Saba, Hameed; Huang, Yuan; Bie, Zhilong

    2016-01-01

    Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated. This study aims to assess the suitability of 20 genes for their potential use as reference genes in melon fruits. Expression variations of these genes were measured in 24 samples that represented different developmental stages of fertilized and parthenocarpic melon fruits by qRT-PCR analysis. GeNorm identified ribosomal protein L (CmRPL) and cytosolic ribosomal protein S15 (CmRPS15) as the best pair of reference genes, and as many as five genes including CmRPL, CmRPS15, TIP41-like family protein (CmTIP41), cyclophilin ROC7 (CmCYP7), and ADP ribosylation factor 1 (CmADP) were required for more reliable normalization. NormFinder ranked CmRPS15 as the best single reference gene, and RAN GTPase gene family (CmRAN) and TATA-box binding protein (CmTBP2) as the best combination of reference genes in melon fruits. Their effectiveness was further validated by parallel analyses on the activities of soluble acid invertase and sucrose phosphate synthase, and expression profiles of their respective encoding genes CmAIN2 and CmSPS1, as well as sucrose contents during melon fruit ripening. The validated reference genes will help to improve the accuracy of gene expression studies in melon fruits. PMID:27536316

  12. Assessment of Suitable Reference Genes for Quantitative Gene Expression Studies in Melon Fruits.

    PubMed

    Kong, Qiusheng; Gao, Lingyun; Cao, Lei; Liu, Yue; Saba, Hameed; Huang, Yuan; Bie, Zhilong

    2016-01-01

    Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated. This study aims to assess the suitability of 20 genes for their potential use as reference genes in melon fruits. Expression variations of these genes were measured in 24 samples that represented different developmental stages of fertilized and parthenocarpic melon fruits by qRT-PCR analysis. GeNorm identified ribosomal protein L (CmRPL) and cytosolic ribosomal protein S15 (CmRPS15) as the best pair of reference genes, and as many as five genes including CmRPL, CmRPS15, TIP41-like family protein (CmTIP41), cyclophilin ROC7 (CmCYP7), and ADP ribosylation factor 1 (CmADP) were required for more reliable normalization. NormFinder ranked CmRPS15 as the best single reference gene, and RAN GTPase gene family (CmRAN) and TATA-box binding protein (CmTBP2) as the best combination of reference genes in melon fruits. Their effectiveness was further validated by parallel analyses on the activities of soluble acid invertase and sucrose phosphate synthase, and expression profiles of their respective encoding genes CmAIN2 and CmSPS1, as well as sucrose contents during melon fruit ripening. The validated reference genes will help to improve the accuracy of gene expression studies in melon fruits. PMID:27536316

  13. Effects of Long Chain Fatty Acid Synthesis and Associated Gene Expression in Microalga Tetraselmis sp

    PubMed Central

    Adarme-Vega, T. Catalina; Thomas-Hall, Skye R.; Lim, David K. Y.; Schenk, Peer M.

    2014-01-01

    With the depletion of global fish stocks, caused by high demand and effective fishing techniques, alternative sources for long chain omega-3 fatty acids are required for human nutrition and aquaculture feeds. Recent research has focused on land-based cultivation of microalgae, the primary producers of omega-3 fatty acids in the marine food web. The effect of salinity on fatty acids and related gene expression was studied in the model marine microalga, Tetraselmis sp. M8. Correlations were found for specific fatty acid biosynthesis and gene expression according to salinity and the growth phase. Low salinity was found to increase the conversion of C18:4 stearidonic acid (SDA) to C20:4 eicosatetraenoic acid (ETA), correlating with increased transcript abundance of the Δ-6-elongase-encoding gene in salinities of 5 and 10 ppt compared to higher salinity levels. The expression of the gene encoding β-ketoacyl-coenzyme was also found to increase at lower salinities during the nutrient deprivation phase (Day 4), but decreased with further nutrient stress. Nutrient deprivation also triggered fatty acids synthesis at all salinities, and C20:5 eicosapentaenoic acid (EPA) increased relative to total fatty acids, with nutrient starvation achieving a maximum of 7% EPA at Day 6 at a salinity of 40 ppt. PMID:24901700

  14. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  15. Impact of Docosahexaenoic Acid on Gene Expression during Osteoclastogenesis in Vitro—A Comprehensive Analysis

    PubMed Central

    Akiyama, Masako; Nakahama, Ken-ichi; Morita, Ikuo

    2013-01-01

    Polyunsaturated fatty acids (PUFAs), especially n-3 polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are known to protect against inflammation-induced bone loss in chronic inflammatory diseases, such as rheumatoid arthritis, periodontitis and osteoporosis. We previously reported that DHA, not EPA, inhibited osteoclastogenesis induced by the receptor activator of nuclear factor-κB ligand (sRANKL) in vitro. In this study, we performed gene expression analysis using microarrays to identify genes affected by the DHA treatment during osteoclastogenesis. DHA strongly inhibited osteoclastogenesis at the late stage. Among the genes upregulated by the sRANKL treatment, 4779 genes were downregulated by DHA and upregulated by the EPA treatment. Gene ontology analysis identified sets of genes related to cell motility, cell adhesion, cell-cell signaling and cell morphogenesis. Quantitative PCR analysis confirmed that DC-STAMP, an essential gene for the cell fusion process in osteoclastogenesis, and other osteoclast-related genes, such as Siglec-15, Tspan7 and Mst1r, were inhibited by DHA. PMID:23945674

  16. Regulation of the expression of key genes involved in HDL metabolism by unsaturated fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine the effects, and possible mechanisms of action, of unsaturated fatty acids on the expression of genes involved in HDL metabolism in HepG2 cells. The mRNA concentration of target genes was assessed by real time PCR. Protein concentrations were determined by wes...

  17. MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    EPA Science Inventory


    MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

  18. Gene expression profiles of soybeans with mid-oleic acid seed phenotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seeds of the mid-oleic acid soybean mutant M23 accumulate higher levels of oleic acid (50-60% oleate) by virtue of a deletion of GmFAD2-1A, an isoform of the microsomal omega-6 oleate desaturase gene. In other less well characterized natural soybean varieties that are phenotypically mid-oleic, litt...

  19. EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHLOROACETIC ACID

    EPA Science Inventory

    EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHLOROACETIC ACID

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have shown that DCA induces liver tumors in rodents when administered in drinking wate...

  20. EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHOLORACETC ACID

    EPA Science Inventory

    EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHLOROACETIC ACID

    Dichloroacetic acid COCA) is a major by-product ofwater disinfection by cWorination. Several
    studies have shown that DCA induces liver tumors in rodents when administered in drinkmg wate...

  1. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    PubMed

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective. PMID:25055961

  2. Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

    PubMed Central

    Bihlmaier, C.; Welle, E.; Hofmann, C.; Welzel, K.; Vente, A.; Breitling, E.; Müller, M.; Glaser, S.; Bechthold, A.

    2006-01-01

    The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic α-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that α-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway. PMID:16723573

  3. Developing cold-chipping potato varieties by silencing the vacuolar invertase gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accumulation of reducing sugars during cold storage is a persistent and costly problem for the potato processing industry. High temperature processing of potato tubers with elevated amounts of reducing sugars results in potato chips, fries and other products that are unacceptable to consumers becaus...

  4. Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Storing potato (Solanum tuberosum) tubers at cold temperatures prevents sprouting and minimizes losses due to disease. Unfortunately, cold storage triggers an accumulation of reducing sugars, a phenomenon referred to as cold-induced sweetening (CIS). High-temperature processing of potato tubers wit...

  5. Impact of supramolecular interactions of dextran-β-cyclodextrin polymers on invertase activity in freeze-dried systems.

    PubMed

    Santagapita, Patricio R; Mazzobre, M Florencia; Buera, M Pilar; Ramirez, Héctor L; Brizuela, Leissy Gómez; Corti, Horacio R; Villalonga, Reynaldo

    2015-01-01

    β-Cyclodextrin (β-CD)-grafted dextrans with spacer arms of different length were employed to evaluate the impact of supramolecular interactions on invertase activity. The modified dextrans were used as single additives or combined with trehalose in freeze-dried formulations containing invertase. Enzyme activity conservation was analyzed after freeze-drying and thermal treatment. The change of glass transition temperature (Tg ) was also evaluated and related to effective interactions. Outstanding differences on enzyme stability were mainly related to the effect of the spacer arm length on polymer-enzyme interactions, since both the degree of substitution and the molecular weight were similar for the two polymers. This change of effective interactions was also manifested in the pronounced reduction of Tg values, and were related to the chemical modification of the backbone during oxidation, and to the attachment of the β-CD units with spacer arms of different length on dextran. PMID:25736897

  6. Metarhizium robertsii Produces an Extracellular Invertase (MrINV) That Plays a Pivotal Role in Rhizospheric Interactions and Root Colonization

    PubMed Central

    Liao, Xinggang; Fang, Weiguo; Lin, Liangcai; Lu, Hsiao-Ling; Leger, Raymond J. St.

    2013-01-01

    As well as killing pest insects, the rhizosphere competent insect-pathogenic fungus Metarhizium robertsii also boosts plant growth by providing nitrogenous nutrients and increasing resistance to plant pathogens. Plant roots secrete abundant nutrients but little is known about their utilization by Metarhizium spp. and the mechanistic basis of Metarhizium-plant associations. We report here that M. robertsii produces an extracellular invertase (MrInv) on plant roots. Deletion of MrInv (⊿MrInv) reduced M. robertsii growth on sucrose and rhizospheric exudates but increased colonization of Panicum virgatum and Arabidopsis thaliana roots. This could be accounted for by a reduction in carbon catabolite repression in ⊿MrInv increasing production of plant cell wall-degrading depolymerases. A non-rhizosphere competent scarab beetle specialist Metarhizium majus lacks invertase which suggests that rhizospheric competence may be related to the sugar metabolism of different Metarhizium species. PMID:24205119

  7. Gene Expression Levels Are Correlated with Synonymous Codon Usage, Amino Acid Composition, and Gene Architecture in the Red Flour Beetle, Tribolium castaneum

    PubMed Central

    Williford, Anna; Demuth, Jeffery P.

    2012-01-01

    Gene expression levels correlate with multiple aspects of gene sequence and gene structure in phylogenetically diverse taxa, suggesting an important role of gene expression levels in the evolution of protein-coding genes. Here we present results of a genome-wide study of the influence of gene expression on synonymous codon usage, amino acid composition, and gene structure in the red flour beetle, Tribolium castaneum. Consistent with the action of translational selection, we find that synonymous codon usage bias increases with gene expression. However, the correspondence between tRNA gene copy number and optimal codons is weak. At the amino acid level, translational selection is suggested by the positive correlation between tRNA gene numbers and amino acid usage, which is stronger for highly expressed genes. In addition, there is a clear trend for increased use of metabolically cheaper, less complex amino acids as gene expression increases. tRNA gene numbers also correlate negatively with amino acid size/complexity (S/C) score indicating the coupling between translational selection and selection to minimize the use of large/complex amino acids. Interestingly, the analysis of 10 additional genomes suggests that the correlation between tRNA gene numbers and amino acid S/C score is widespread and might be explained by selection against negative consequences of protein misfolding. At the level of gene structure, three major trends are detected: 1) complete coding region length increases across low and intermediate expression levels but decreases in highly expressed genes; 2) the average intron size shows the opposite trend, first decreasing with expression, followed by a slight increase in highly expressed genes; and 3) intron density remains nearly constant across all expression levels. These changes in gene architecture are only in partial agreement with selection favoring reduced cost of biosynthesis. PMID:22826459

  8. Identification of a 12-gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics.

    PubMed

    Brown, Daren W; Lee, Seung-Ho; Kim, Lee-Han; Ryu, Jae-Gee; Lee, Soohyung; Seo, Yunhee; Kim, Young Ho; Busman, Mark; Yun, Sung-Hwan; Proctor, Robert H; Lee, Theresa

    2015-03-01

    In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production. PMID:25372119

  9. Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylic acid synthase genes in Hevea brasiliensis.

    PubMed

    Zhu, Jia-Hong; Xu, Jing; Chang, Wen-Jun; Zhang, Zhi-Li

    2015-01-01

    Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1-7) of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment. These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production. PMID:25690030

  10. Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties.

    PubMed

    Pérez de los Santos, Ara Itzel; Cayetano-Cruz, Maribel; Gutiérrez-Antón, Marina; Santiago-Hernández, Alejandro; Plascencia-Espinosa, Miguel; Farrés, Amelia; Hidalgo-Lara, María Eugenia

    2016-02-01

    Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses. PMID:26777250

  11. In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches.

    PubMed

    Hovasse, Agnès; Alayi, Tchilabalo Dilezitoko; Van Dorsselaer, Alain; Marchal, Richard; Jégou, Sandrine; Schaeffer-Reiss, Christine

    2016-06-01

    Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins. PMID:26830584

  12. Stimulation of extracellular invertase production from spent yeast when sugarcane pressmud used as substrate through solid state fermentation.

    PubMed

    Kumar, Rahul; Kesavapillai, Balakrishnan

    2012-12-01

    Efforts were made to utilize the waste/by-product of two agro-process industries namely pressmud from sugar processing industries and spent yeast from distilleries manufacturing ethanol from cane molasses, for the production of microbial invertase. Our experimentation indicated that these two sources could be ideally utilized for the production of invertase through solid substrate fermentation (SSF). SSF with spent yeast had given highest specific activity of 430 U/mg in 72 h of fermentation. Inoculum percentage of yeast cells on pressmud was optimized as 50% (w/w) with a combination inoculum of spent yeast and fresh cultured yeast at a ratio of 7:3. Crude enzyme was characterized for optimum pH and temperature and maximum activity was recorded at pH 5.0 and at a temperature of 40°C. Impacts of metal ions and detergents on invertase action were studied in which Mn(2+), Fe(3+), Al(3+) and detergents had enhanced the activity of the enzyme whereas Cu(2+) and Zn(2+) inhibited the enzyme activity. Purification of 9.8 folds was obtained by using three phase partition method. PMID:23420549

  13. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

    PubMed

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-05-01

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production. PMID:23610393

  14. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

    PubMed Central

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L.; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-01-01

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production. PMID:23610393

  15. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells

    PubMed Central

    Park, Hui Gyu; Park, Woo Jung; Kothapalli, Kumar S. D.; Brenna, J. Thomas

    2015-01-01

    Docosahexaenoic acid (DHA) is a Δ4-desaturated C22 fatty acid and the limiting highly unsaturated fatty acid (HUFA) in neural tissue. The biosynthesis of Δ4-desaturated docosanoid fatty acids 22:6n-3 and 22:5n-6 are believed to proceed via a circuitous biochemical pathway requiring repeated use of a fatty acid desaturase 2 (FADS2) protein to perform Δ6 desaturation on C24 fatty acids in the endoplasmic reticulum followed by 1 round of β-oxidation in the peroxisomes. We demonstrate here that the FADS2 gene product can directly Δ4-desaturate 22:5n-3→22:6n-3 (DHA) and 22:4n-6→22:5n-6. Human MCF-7 cells lacking functional FADS2-mediated Δ6-desaturase were stably transformed with FADS2, FADS1, or empty vector. When incubated with 22:5n-3 or 22:4n-6, FADS2 stable cells produce 22:6n-3 or 22:5n-6, respectively. Similarly, FADS2 stable cells when incubated with d5-18:3n-3 show synthesis of d5-22:6n-3 with no labeling of 24:5n-3 or 24:6n-3 at 24 h. Further, both C24 fatty acids are shown to be products of the respective C22 fatty acids via elongation. Our results demonstrate that the FADS2 classical transcript mediates direct Δ4 desaturation to yield 22:6n-3 and 22:5n-6 in human cells, as has been widely shown previously for desaturation by fish and many other organisms.—Park, H. G., Park, W. J., Kothapalli, K. S. D., Brenna, J. T. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells. PMID:26065859

  16. Identification of the Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase Gene

    PubMed Central

    Mehl, Ryan A.; Kinsland, Cynthia; Begley, Tadhg P.

    2000-01-01

    The gene (ybeN) coding for nicotinate mononucleotide adenylyltransferase, an NAD(P) biosynthetic enzyme, has been identified and overexpressed in Escherichia coli. This enzyme catalyzes the reversible adenylation of nicotinate mononucleotide and shows product inhibition. The rate of adenylation of nicotinate mononucleotide is at least 20 times faster than the rate of adenylation of nicotinamide mononucleotide. PMID:10894752

  17. ALTERED GENE EXPRESSION IN MOUSE LIVERS AFTER DICHLOROACETIC ACID EXPOSURE

    EPA Science Inventory

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated that DCA exhibits hepatocarcinogenic effects in rodents when administered in drinking water. The mechanism(s) involved in DCA induction of cancer are not clear...

  18. Gene expression analysis of Corynebacterium glutamicum subjected to long-term lactic acid adaptation.

    PubMed

    Jakob, Kinga; Satorhelyi, Peter; Lange, Christian; Wendisch, Volker F; Silakowski, Barbara; Scherer, Siegfried; Neuhaus, Klaus

    2007-08-01

    Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced >or=2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low p

  19. Gene Expression Analysis of Corynebacterium glutamicum Subjected to Long-Term Lactic Acid Adaptation▿ ¶

    PubMed Central

    Jakob, Kinga; Satorhelyi, Peter; Lange, Christian; Wendisch, Volker F.; Silakowski, Barbara; Scherer, Siegfried; Neuhaus, Klaus

    2007-01-01

    Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of Corynebacterium glutamicum adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a sigB knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A sigE mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the sigB mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the nrd (ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced ≥2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low p

  20. [Expression of Mortierella isabellina delta6-fatty acid desaturase gene in gamma-linolenic acid production in transgenic tobacco].

    PubMed

    Li, Ming-Chun; Liu, Li; Hu, Guo-Wu; Xing, Lai-Jun

    2003-03-01

    Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were

  1. A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis.

    PubMed

    Martín, Mariana L; Lechner, Leandra; Zabaleta, Eduardo J; Salerno, Graciela L

    2013-03-01

    Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle. PMID:23135328

  2. Multiplexed analysis of genes using nucleic acid-stabilized silver-nanocluster quantum dots.

    PubMed

    Enkin, Natalie; Wang, Fuan; Sharon, Etery; Albada, H Bauke; Willner, Itamar

    2014-11-25

    Luminescent nucleic acid-stabilized Ag nanoclusters (Ag NCs) are applied for the optical detection of DNA and for the multiplexed analysis of genes. Two different sensing modules including Ag NCs as luminescence labels are described. One sensing module involves the assembly of a three-component sensing module composed of a nucleic acid-stabilized Ag NC and a quencher-modified nucleic acid hybridized with a nucleic acid scaffold that is complementary to the target DNA. The luminescence of the Ag NCs is quenched in the sensing module nanostructure. The strand displacement of the scaffold by the target DNA separates the nucleic acid-functionalized Ag NCs, leading to the turned-on luminescence of the NCs and to the optical readout of the sensing process. By implementing two different-sized Ag NC-modified sensing modules, the parallel multiplexed analysis of two genes (the Werner Syndrome gene and the HIV, human immunodeficiency, gene), using 615 and 560 nm luminescent Ag NCs, is demonstrated. The second sensing module includes the nucleic acid functionalized Ag NCs and the quencher-modified nucleic acid hybridized with a hairpin DNA scaffold. The luminescence of the Ag NCs is quenched in the sensing module. Opening of the hairpin by the target DNA triggers the luminescence of the Ag NCs, due to the spatial separation of the Ag NCs/quencher units. The system is applied for the optical detection of the BRAC1 gene. In addition, by implementing two-sized Ag NCs, the multiplexed analysis of two genes by the hairpin sensing module approach is demonstrated. PMID:25327411

  3. The expansion of amino-acid repeats is not associated to adaptive evolution in mammalian genes

    PubMed Central

    2009-01-01

    Background The expansion of amino acid repeats is determined by a high mutation rate and can be increased or limited by selection. It has been suggested that recent expansions could be associated with the potential of adaptation to new environments. In this work, we quantify the strength of this association, as well as the contribution of potential confounding factors. Results Mammalian positively selected genes have accumulated more recent amino acid repeats than other mammalian genes. However, we found little support for an accelerated evolutionary rate as the main driver for the expansion of amino acid repeats. The most significant predictors of amino acid repeats are gene function and GC content. There is no correlation with expression level. Conclusions Our analyses show that amino acid repeat expansions are causally independent from protein adaptive evolution in mammalian genomes. Relaxed purifying selection or positive selection do not associate with more or more recent amino acid repeats. Their occurrence is slightly favoured by the sequence context but mainly determined by the molecular function of the gene. PMID:20021652

  4. MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

    PubMed

    Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

    2009-07-01

    Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions. PMID:19445975

  5. The Genes for Cytoplasmic Ribosomal Ribonucleic Acid in Higher Plants

    PubMed Central

    Scott, N. Steele; Ingle, J.

    1973-01-01

    The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 × 106 molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction. PMID:16658392

  6. Foreign gene recruitment to the fatty acid biosynthesis pathway in diatoms.

    PubMed

    Chan, Cheong Xin; Baglivi, Francesca L; Jenkins, Christina E; Bhattacharya, Debashish

    2013-09-01

    Diatoms are highly successful marine and freshwater algae that contribute up to 20% of global carbon fixation. These species are leading candidates for biofuel production owing to ease of culturing and high fatty acid content. To assist in strain improvement and downstream applications for potential use as a biofuel, it is important to understand the evolution of lipid biosynthesis in diatoms. The evolutionary history of diatoms is however complicated by likely multiple endosymbioses involving the capture of foreign cells and horizontal gene transfer into the host genome. Using a phylogenomic approach, we assessed the evolutionary history of 12 diatom genes putatively encoding functions related to lipid biosynthesis. We found evidence of gene transfer likely from a green algal source for seven of these genes, with the remaining showing either vertical inheritance or evolutionary histories too complicated to interpret given current genome data. The functions of horizontally transferred genes encompass all aspects of lipid biosynthesis (initiation, biosynthesis, and desaturation of fatty acids) as well as fatty acid elongation, and are not restricted to plastid-targeted proteins. Our findings demonstrate that the transfer, duplication, and subfunctionalization of genes were key steps in the evolution of lipid biosynthesis in diatoms and other photosynthetic eukaryotes. This target pathway for biofuel research is highly chimeric and surprisingly, our results suggest that research done on related genes in green algae may have application to diatom models. PMID:24404416

  7. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

    PubMed

    Haun, William; Coffman, Andrew; Clasen, Benjamin M; Demorest, Zachary L; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre; Cedrone, Frederic; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

    2014-09-01

    Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement. PMID:24851712

  8. Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

    PubMed Central

    Fukushima, J; Yamamoto, S; Morihara, K; Atsumi, Y; Takeuchi, H; Kawamoto, S; Okuda, K

    1989-01-01

    The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin. PMID:2493453

  9. Comparative genomics of lactic acid bacteria reveals a niche-specific gene set

    PubMed Central

    2009-01-01

    Background The recently sequenced genome of Lactobacillus helveticus DPC4571 [1] revealed a dairy organism with significant homology (75% of genes are homologous) to a probiotic bacteria Lb. acidophilus NCFM [2]. This led us to hypothesise that a group of genes could be determined which could define an organism's niche. Results Taking 11 fully sequenced lactic acid bacteria (LAB) as our target, (3 dairy LAB, 5 gut LAB and 3 multi-niche LAB), we demonstrated that the presence or absence of certain genes involved in sugar metabolism, the proteolytic system, and restriction modification enzymes were pivotal in suggesting the niche of a strain. We identified 9 niche specific genes, of which 6 are dairy specific and 3 are gut specific. The dairy specific genes identified in Lactobacillus helveticus DPC4571 were lhv_1161 and lhv_1171, encoding components of the proteolytic system, lhv_1031 lhv_1152, lhv_1978 and lhv_0028 encoding restriction endonuclease genes, while bile salt hydrolase genes lba_0892 and lba_1078, and the sugar metabolism gene lba_1689 from Lb. acidophilus NCFM were identified as gut specific genes. Conclusion Comparative analysis revealed that if an organism had homologs to the dairy specific geneset, it probably came from a dairy environment, whilst if it had homologs to gut specific genes, it was highly likely to be of intestinal origin. We propose that this "barcode" of 9 genes will be a useful initial guide to researchers in the LAB field to indicate an organism's ability to occupy a specific niche. PMID:19265535

  10. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68

    PubMed Central

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase. PMID:25242926

  11. Gene-related strain variation of Staphylococcus aureus for homologous resistance response to acid stress.

    PubMed

    Lee, Soomin; Ahn, Sooyeon; Lee, Heeyoung; Kim, Won-Il; Kim, Hwang-Yong; Ryu, Jae-Gee; Kim, Se-Ri; Choi, Kyoung-Hee; Yoon, Yohan

    2014-10-01

    This study investigated the effect of adaptation of Staphylococcus aureus strains to the acidic condition of tomato in response to environmental stresses, such as heat and acid. S. aureus ATCC 13565, ATCC 14458, ATCC 23235, ATCC 27664, and NCCP10826 habituated in tomato extract at 35°C for 24 h were inoculated in tryptic soy broth. The culture suspensions were then subjected to heat challenge or acid challenge at 60°C and pH 3.0, respectively, for 60 min. In addition, transcriptional analysis using quantitative real-time PCR was performed to evaluate the expression level of acid-shock genes, such as clpB, zwf, nuoF, and gnd, from five S. aureus strains after the acid habituation of strains in tomato at 35°C for 15 min and 60 min in comparison with that of the nonhabituated strains. In comparison with the nonhabituated strains, the five tomato-habituated S. aureus strains did not show cross protection to heat, but tomato-habituated S. aureus ATCC 23235 showed acid resistance. In quantitative real-time-PCR analysis, the relative expression levels of acid-shock genes (clpB, zwf, nuoF, and gnd) were increased the most in S. aureus ATCC 23235 after 60 min of tomato habituation, but there was little difference in the expression levels among the five S. aureus strains after 15 min of tomato habituation. These results indicate that the variation of acid resistance of S. aureus is related to the expression of acid-shock genes during acid habituation. PMID:25285500

  12. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells.

    PubMed

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  13. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    PubMed Central

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  14. Cloning and phylogenetic analysis of a fatty acid elongase gene from Nannochloropsis oculata CS179

    NASA Astrophysics Data System (ADS)

    Pan, Kehou; Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Yang, Guanpin

    2009-12-01

    Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5' and 3' RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the NJ-tree revealed that the N. oculata CS179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.

  15. Engineering Clostridium beijerinckii with the Cbei_4693 gene knockout for enhanced ferulic acid tolerance.

    PubMed

    Liu, Jun; Guo, Ting; Shen, Xiaoning; Xu, Jiahui; Wang, Junzhi; Wang, Yanyan; Liu, Dong; Niu, Huanqing; Liang, Lei; Ying, Hanjie

    2016-07-10

    A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23g/L of butanol was produced in P2 medium containing 0.5g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii. PMID:27164255

  16. Serum homocysteine, vitamin B12, folic acid levels and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in vitiligo.

    PubMed

    Yasar, Ali; Gunduz, Kamer; Onur, Ece; Calkan, Mehmet

    2012-01-01

    The aim of this study was to determine serum vitamin B12, folic acid and homocysteine (Hcy) levels as well as MTHFR (C677, A1298C) gene polymorphisms in patients with vitiligo, and to compare the results with healthy controls. Forty patients with vitiligo and 40 age and sex matched healthy subjects were studied. Serum vitamin B12 and folate levels were determined by enzyme-linked immunosorbent assay. Plasma Hcy levels and MTHFR polymorphisms were determined by chemiluminescence and real time PCR methods, respectively. Mean serum vitamin B12 and Hcy levels were not significantly different while folic acid levels were significantly lower in the control group. There was no significant relationship between disease activity and vitamin B12, folic acid and homocystein levels. No significant difference in C677T gene polymorphism was detected. Heterozygote A1298C gene polymorphism in the patient group was statistically higher than the control group. There was no significant relationship between MTHFR gene polymorphisms and vitamin B12, folic acid and homocysteine levels. In conclusion, vitamin B12, folate and Hcy levels are not altered in vitiligo and MTHFR gene mutations (C677T and A1298C) do not seem to create susceptibility for vitiligo. PMID:22846211

  17. Archaeal Lipid Genes: Clues to Life in Acid and the Evolution of Membranes

    NASA Astrophysics Data System (ADS)

    Macalady, J. L.; Croft, L.; Vestling, M. M.; Harms, A. C.; Zheng, L.; Baumler, D. J.; Kaspar, C. W.; Banfield, J. F.

    2002-12-01

    Microorganisms living in acid mine drainage environments face extraordinary challenges. Acid-loving archaea such as Ferroplasma acidarmanus maintain pH gradients of 4 to 5 pH units across their membranes and thrive in hot, extremely low pH (0-1), metal-rich, solutions. New lipid analyses for two extremely acidophilic archaea, F. acidarmanus and F. acidiphilum, reveal that all known archaeal acidophiles have cell membranes composed primarily of tetraether-linked lipids. Because tetraether lipids assemble in rigid monolayers that exclude protons and metals, we suggest that tetraether synthesis genes are essential for archaeal survival in acid. Fusion of two diether-linked lipids to form a tetraether-linked lipid is a distinctive biochemical reaction with no analogy in bacteria and eukaryotes. In addition to archaeal acidophiles, tetraethers are present in members of every archaeal lineage except halophiles. Genes responsible for tetraether synthesis and subsequent biochemical steps which "tune" membrane lipid properties in response to environmental changes have not been identified to date. Comparative genomic analyses using the newly completed genome of F. acidarmanus and available genomes from Bacteria, Archaea and Eukarya have generated candidate tetraether synthase genes found only in archaea. Because tetraether-linked lipids are advantageous for acid-loving and possibly also for heat-loving archaea, the phylogeny of these genes has the potential to shed new light on role of hot, acid environments in early evolution.

  18. The prostatic acid phosphatase (ACPP) gene is localized to human chromosome 3q21-q23

    SciTech Connect

    Li, S.S.L.; Sharief, F.S. )

    1993-09-01

    Human prostatic acid phosphatase (ACPP) has been used as a diagnostic marker for prostate cancer. It is synthesized under androgen regulation and secreted by the epithelial cells of the prostate gland. The authors have confirmed the previous assignment of the ACPP gene to chromosome 3 by probing a panel of 25 human-Chinese hamster somatic cell hybrids, and they have further localized the ACPP gene to chromosome 3q21-q23 by fluorescence in situ hybridization. 10 refs., 1 fig.

  19. Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

    PubMed Central

    Gopal-Srivastava, R; Mallonee, D H; White, W B; Hylemon, P B

    1990-01-01

    Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium. Images PMID:2376563

  20. Antitumor Molecular Mechanism of Chlorogenic Acid on Inducting Genes GSK-3 β and APC and Inhibiting Gene β -Catenin.

    PubMed

    Xu, Ruoshi; Kang, Qiumei; Ren, Jie; Li, Zukun; Xu, Xiaoping

    2013-01-01

    Objective. Inhibiting gene β -catenin and inducting genes GSK-3 β and APC, promoting the tumor cell apoptosis in Wnt pathway, by chlorogenic acid were discussed (CGA). Method. The different genes were scanned by the 4∗44K mouse microarray chips. The effect of the three genes was confirmed by RT-PCR technique with CGA dosage of 5, 10, and 20 mg/kg. Result. The expression of GSK-3 β and APC was upregulated in group of 20 mg/kg dosage (P < 0.05) and the expression of β -catenin was downregulated in the same dosage (P < 0.05). Conclusion. The results infer that the multimeric protein complex of β -catenin could be increased by CGA upregulated genes GSK-3 β and APC, which could inhibit the free β -catenin into the nucleus to connect with TCF. So the transcriptional expression of the target genes will be cut to abnormal cell proliferation. It is probably one of the ways that can stop the tumor increase by CGA. PMID:23844319

  1. Regulation of invertase activity in different root zones of wheat (Triticum aestivum L.) seedlings in the course of osmotic adjustment under water deficit conditions.

    PubMed

    Königshofer, Helga; Löppert, Hans-Georg

    2015-07-01

    Osmotic adjustment of roots is an essential adaptive mechanism to sustain water uptake and root growth under water deficit. In this paper, the role of invertases (β-fructofuranosidase, EC 3.2.1.26) in osmotic adjustment was investigated in the root tips (cell division and elongation zone) and the root maturation zone of wheat (Triticum aestivum L. cv. Josef) in the course of osmotic stress imposed by 20% polyethylene glycol (PEG) 6000. The two root zones investigated differed distinctly in the response of invertases to water deprivation. In the root tips, the activity of the vacuolar and cell wall-bound invertases increased markedly under water stress resulting in the accumulation of hexoses (glucose and fructose) that contributed significantly to osmotic adjustment. A transient rise in hydrogen peroxide (H2O2) preceded the enhancement of invertases upon exposure to osmotic stress. Treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished the stress induced H2O2 production and suppressed the stimulation of the vacuolar invertase activity, whereas the activity of the cell wall-bound invertase was not influenced by DPI. As a consequence of the inhibitory effect of DPI on the vacuolar invertase, hexose levels and osmotic adjustment were also markedly decreased in the root tips under water deficit in the presence of DPI. These data suggest that H2O2 probably generated by a NADPH oxidase is required as a signalling molecule for the up-regulation of the vacuolar invertase activity in the root tips under osmotic stress, thereby enhancing the capacity for osmotic adjustment. In the root maturation zone, an early H2O2 signal could not be detected in response to PEG application. Only an increase in the glucose level that was not paralleled by fructose and a slight stimulation of the activity of the vacuolar invertase occurred in the maturation zone after water deprivation. The stress induced accumulation of glucose in the maturation zone was not

  2. Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

    PubMed

    Thambugala, Dinushika; Cloutier, Sylvie

    2014-11-01

    Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax. PMID:24871199

  3. Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers.

    PubMed

    Szarka, András; Horemans, Nele; Passarella, Salvatore; Tarcsay, Akos; Orsi, Ferenc; Salgó, András; Bánhegyi, Gábor

    2008-10-01

    Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism. PMID:18600345

  4. Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major.

    PubMed

    Belaz, Sorya; Rattier, Thibault; Lafite, Pierre; Moreau, Philippe; Routier, Françoise H; Robert-Gangneux, Florence; Gangneux, Jean-Pierre; Daniellou, Richard

    2015-10-13

    The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism. PMID:26279524

  5. Effect of Mg²⁺ and Al²⁺ Ions on Thermodynamic and Physiochemical Properties of Aspergillus niger Invertases.

    PubMed

    Nadeem, Habibullah; Rashid, Muhammad H; Siddique, Muhammad H

    2015-01-01

    In the present study, we reported for the first time the effect of various concentrations (0.5- 3.0 mM) of Mg(2+) and Al(3+) ions on the kinetics and thermodynamics of Aspergillus niger invertases for sucrose hydrolysis. We found that both metal ions enhanced the affinity of invertase for sucrose by decreasing the Km. In the presence of 0.5 mM Al(3+) ions invertase have maximum affinity for sucrose (Km = 0.00914 M sucrose). Invertase was activated by Mg(2+) ions at low concentrations (0.5-2.0 mM) and 341% increase in turnover (Kcat) and maximum decrease in ΔG* was observed in the presence of 0.5 mM Mg(2+) ions. The entropy change for activation of substrate hydrolysis (ΔS*) was increased by all concentrations of Mg(2+) ions and was highest (-94 J mol(-1) K(-1)) for invertases bound with 1.5 mM Mg(2+) ions. PMID:26021385

  6. Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

    PubMed Central

    Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

    2014-01-01

    Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p < 0.05) the α-linolenic acid (C18:3n-3) concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05) decreased the arachidonic acid (C20:4n-6) and conjugated linolenic acid (CLA) c-9 t-11 content in the ST muscle. There was a significant (p < 0.05) upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD) gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression. PMID:25255382

  7. Gene expression profiles of murine fatty liver induced by the administration of valproic acid

    SciTech Connect

    Lee, Min-Ho; Hong, Il; Kim, Mingoo; Lee, Byung Hoon; Kim, Ju-Han; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-Il; Chung, Heekyoung; Kong, Gu; Lee, Mi-Ock . E-mail: molee@snu.ac.kr

    2007-04-01

    Valproic acid (VPA) has been used as anticonvulsants, however, it induces hepatotoxicity such as microvesicular steatosis and necrosis in the liver. To explore the mechanisms of VPA-induced steatosis, we profiled the gene expression patterns of the mouse liver that were altered by treatment with VPA using microarray analysis. VPA was orally administered as a single dose of 100 mg/kg (low-dose) or 1000 mg/kg (high-dose) to ICR mice and the animals were killed at 6, 24, or 72 h after treatment. Serum alanine aminotransferase and aspartate aminotransferase levels were not significantly altered in the experimental animals. However, symptoms of steatosis were observed at 72 h with low-dose and at 24 h and 72 h with high-dose. After microarray data analysis, 1910 genes were selected by two-way ANOVA (P < 0.05) as VPA-responsive genes. Hierarchical clustering revealed that gene expression changes depended on the time rather than the dose of VPA treatment. Gene profiling data showed striking changes in the expression of genes associated with lipid, fatty acid, and steroid metabolism, oncogenesis, signal transduction, and development. Functional categorization of 1156 characteristically up- and down-regulated genes (cutoff > 1.5-fold) revealed that 60 genes were involved in lipid metabolism that was interconnected with biological pathways for biosynthesis of triglyceride and cholesterol, catabolism of fatty acid, and lipid transport. This gene expression profile may be associated with the known steatogenic hepatotoxicity of VPA and it may provide useful information for prediction of hepatotoxicity of unknown chemicals or new drug candidates through pattern recognition.

  8. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  9. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  10. Isolation and partial characterization of the gene for goose fatty acid synthase.

    PubMed

    Kameda, K; Goodridge, A G

    1991-01-01

    Fatty acid synthase is regulated by diet and hormones, with regulation being primarily transcriptional. In chick embryo hepatocytes in culture, triiodothyronine stimulates accumulation of enzyme and transcription of the gene. Since the 5'-flanking region of this gene is likely involved in hormonal regulation of its expression, we have isolated and partially characterized an avian fatty acid synthase gene. A genomic DNA library was constructed in a cosmid vector and screened with cDNA clones that contained sequence complementary to the 3' end of goose fatty acid synthase mRNA. A genomic clone (approximately 35 kilobase pairs (kb] was isolated, and a 6.5-kb EcoRI fragment thereof contained DNA complementary to the 3' noncoding region of fatty acid synthase mRNA. Additional cosmid libraries were screened with 5' fragments of previously isolated genomic clones, resulting in the isolation of five overlapping cosmid DNAs. The entire region of cloned DNA spans approximately 105 kb. Exon-containing fragments were identified by hybridization with end-labeled poly(A)+ RNA and by hybridization of labeled exon-containing genomic DNA fragments to fatty acid synthase mRNA. A new set of cDNA clones spanning approximately 3.2 kb was isolated from a lambda-ZAP goose liver cDNA library using the 5'-most exon-containing fragment of the 5'-most genomic DNA clone. This region of mRNA contains a 5'-untranslated sequence and a continuous open reading frame which includes a region that codes for the essential cysteine of the beta-ketoacyl synthase domain. The entire fatty acid synthase gene spans about 50 kb. The 5' 15 kb of the gene contain 7 exons. S1 nuclease and primer extension analyses were used to identify a single site for initiation of transcription, 174 nucleotides upstream from the putative translation initiation codon. Putative "TATA" and "CCAAT" boxes are located 28 and 60 base pairs (bp), respectively, upstream of the site of initiation of transcription. The 5'-flanking 597

  11. A Systems Genetics Approach Identifies Gene Regulatory Networks Associated with Fatty Acid Composition in Brassica rapa Seed.

    PubMed

    Basnet, Ram Kumar; Del Carpio, Dunia Pino; Xiao, Dong; Bucher, Johan; Jin, Mina; Boyle, Kerry; Fobert, Pierre; Visser, Richard G F; Maliepaard, Chris; Bonnema, Guusje

    2016-01-01

    Fatty acids in seeds affect seed germination and seedling vigor, and fatty acid composition determines the quality of seed oil. In this study, quantitative trait locus (QTL) mapping of fatty acid and transcript abundance was integrated with gene network analysis to unravel the genetic regulation of seed fatty acid composition in a Brassica rapa doubled haploid population from a cross between a yellow sarson oil type and a black-seeded pak choi. The distribution of major QTLs for fatty acids showed a relationship with the fatty acid types: linkage group A03 for monounsaturated fatty acids, A04 for saturated fatty acids, and A05 for polyunsaturated fatty acids. Using a genetical genomics approach, expression quantitative trait locus (eQTL) hotspots were found at major fatty acid QTLs on linkage groups A03, A04, A05, and A09. An eQTL-guided gene coexpression network of lipid metabolism-related genes showed major hubs at the genes BrPLA2-ALPHA, BrWD-40, a number of seed storage protein genes, and the transcription factor BrMD-2, suggesting essential roles for these genes in lipid metabolism. Three subnetworks were extracted for the economically important and most abundant fatty acids erucic, oleic, linoleic, and linolenic acids. Network analysis, combined with comparison of the genome positions of cis- or trans-eQTLs with fatty acid QTLs, allowed the identification of candidate genes for genetic regulation of these fatty acids. The generated insights in the genetic architecture of fatty acid composition and the underlying complex gene regulatory networks in B. rapa seeds are discussed. PMID:26518343

  12. Comparative Analysis of Human, Mouse, and Pig Glial Fibrillary Acidic Protein Gene Structures.

    PubMed

    Eun, Kiyoung; Hwang, Seon-Ung; Jeon, Hye-Min; Hyun, Sang-Hwan; Kim, Hyunggee

    2016-01-01

    Comparing the coding and regulatory sequences of genes in different species provides information on whether proteins translated from genes have conserved functions or gene expressions are regulated by analogical mechanisms. Herein, we compared the coding and regulatory sequences of glial fibrillary acidic protein (GFAP) from humans, mice, and pigs. The GFAP gene encodes a class III intermediate filament protein expressed specifically in astrocytes of the central nervous system. On comparing the mRNA, regulatory region (promoter), and protein sequences of GFAP gene in silico, we found that GFAP mRNA 3'-untranslated region (3'-UTR), promoter, and amino acid sequences showed higher similarities between humans and pigs than between humans and mice. In addition, the promoter-luciferase reporter gene assay revealed that the pig GFAP promoter functioned in human astrocytes. Notably, the 1.8-kb promoter fragment upstream from transcription initiation site showed strongest transcriptional activity compared to 5.2-kb DNA fragment or other regions of GFAP promoter. We also found that pig GFAP mRNA and promoter activity increased in pig fibroblasts by human IL-1β treatment. Taken together, these results suggest that the regulatory mechanisms and functions of pig genes might be more similar to those of humans than mice, indicating that pigs, particularly miniature pigs, are a useful model for studying human biological and pathological events. PMID:26913554

  13. Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

    PubMed Central

    Zhang, Ling; Yang, Xiang-dong; Zhang, Yuan-yu; Yang, Jing; Qi, Guang-xun; Guo, Dong-quan; Xing, Guo-jie; Yao, Yao; Xu, Wen-jing; Li, Hai-yun; Li, Qi-yun; Dong, Ying-shan

    2014-01-01

    The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts. PMID:25197629

  14. The rolC gene increases caffeoylquinic acid production in transformed artichoke cells.

    PubMed

    Vereshchagina, Y V; Bulgakov, V P; Grigorchuk, V P; Rybin, V G; Veremeichik, G N; Tchernoded, G K; Gorpenchenko, T Y; Koren, O G; Phan, N H T; Minh, N T; Chau, L T; Zhuravlev, Y N

    2014-09-01

    Caffeoylquinic acids are found in artichokes, and they are currently considered important therapeutic or preventive agents for treating Alzheimer's disease and diabetes. We transformed artichoke [the cultivated cardoon or Cynara cardunculus var. altilis DC (Asteraceae)] with the rolC gene, which is a known inducer of secondary metabolism. High-performance liquid chromatography with UV and high-resolution mass spectrometry (HPLC-UV-HRMS) revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and chlorogenic acid. The rolC-transformed calli contained 1.5% caffeoylquinic acids by dry weight. The overall production of these metabolites was three times higher than that of the corresponding control calli. The enhancing effect of rolC remained stable over long-term cultivation. PMID:24938208

  15. Bugs, genes, fatty acids, and serotonin: Unraveling inflammatory bowel disease?

    PubMed Central

    Kaunitz, Jonathan; Nayyar, Piyush

    2015-01-01

    The annual incidence of the inflammatory bowel diseases (IBDs) ulcerative colitis and Crohn’s disease has increased at an alarming rate. Although the specific pathophysiology underlying IBD continues to be elusive, it is hypothesized that IBD results from an aberrant and persistent immune response directed against microbes or their products in the gut, facilitated by the genetic susceptibility of the host and intrinsic alterations in mucosal barrier function. In this review, we will describe advances in the understanding of how the interaction of host genetics and the intestinal microbiome contribute to the pathogenesis of IBD, with a focus on bacterial metabolites such as short chain fatty acids (SCFAs) as possible key signaling molecules.  In particular, we will describe alterations of the intestinal microbiota in IBD, focusing on how genetic loci affect the gut microbial phylogenetic distribution and the production of their major microbial metabolic product, SCFAs. We then describe how enteroendocrine cells and myenteric nerves express SCFA receptors that integrate networks such as the cholinergic and serotonergic neural systems and the glucagon-like peptide hormonal pathway, to modulate gut inflammation, permeability, and growth as part of an integrated model of IBD pathogenesis.  Through this integrative approach, we hope that novel hypotheses will emerge that will be tested in reductionist, hypothesis-driven studies in order to examine the interrelationship of these systems in the hope of better understanding IBD pathogenesis and to inform novel therapies.

  16. Monitoring Gene Expression In Vivo with Nucleic Acid Molecular Switches

    SciTech Connect

    David C. Ward; Patricia Bray-Ward

    2005-01-26

    The overall objectives of this project were (1) to develop allosteric ribozymes capable of acting as molecular switches for monitoring the levels of both wild-type and mutant mRNA species in living cells and whole animals and (2) to develop highly efficient reagents to deliver nucleic acid molecular switches into living cells, tissues and animals with the ultimate goal of expression profiling specific mRNAs of diagnostic or prognostic value within tumors in animals. During the past year, we have moved our laboratory to Nevada and in the moving process we have lost electronic and paper copies of prior progress reports concerning the construction and biological properties of the molecular switches. Since there was minimal progress during the last year on molecular switches, we are relying on past project reports to provide a summary of our data on this facet of the grant. Here we are summarizing the work done on the delivery reagents and their application to inducing mutations in living cells, which will include work done during the no cost extension.

  17. Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furfural (2-furaldehyde) is a furan formed by dehydration of pentose sugars. Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid and Coenzyme A intermediates. To identify genes involved in furan metabolism, two P. putida transposo...

  18. Efflux Pump Gene Expression in Erwinia Chrysanthemi is Induced by Exposure to Phenolic Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salicylic acid (SA) is an important signaling molecule in local and systemic plant resistance. Following infection by microbial pathogens and the initial oxidative burst in plants, SA accumulation functions in the amplification of defense gene expression. Production of pathogenesisrelated proteins a...

  19. A Low Phytic Acid Barley Mutation Alters Gene Expression in Early Seed Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley (Hordeum vulgare L.) low phytic acid (lpa) mutants have reduced levels of seed phytate, the most abundant form of phosphorus in seeds, and increases in seed inorganic phosphorus. To understand how lpa mutations affect metabolic and developmental processes during seed growth, gene expression ...

  20. Differential influence of distinct fatty acids on cardiomyocyte metabolic gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diabetes mellitus increases risk for cardiovascular disease, and exposes the heart to high plasma fatty acid (FA) levels, which induce genes promoting FA oxidation (e.g., malonyl-CoA decarboxylase; mcd), as well as those suppressing carbohydrate oxidation (e.g., pyruvate dehydrogenase kinase 4; pdk4...

  1. DIFFERENTIAL INFLUENCE OF DISTINCT FATTY ACIDS ON CARDIOMYOCYTE METABOLIC GENE EXPRESSION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diabetes mellitus is a major risk factor for development of cardiovascular disease. Metabolic adaptation of the heart to increased fatty acids (FAs) in the diabetic milieu is mediated by induction of genes promoting FA oxidation (e.g. malonyl-CoA decarboxylase; mcd), as well as those suppressing car...

  2. GENE EXPRESSION PATTERNS OF CD-1 DAY-8 EMBRYO CULTURES EXPOSED TO BROMOCHLORO ACETIC ACID

    EPA Science Inventory

    Gene expression patterns of CD-1 day-8 embryo cultures exposed to bromochloro acetic acid

    Edward D. Karoly?*, Judith E. Schmid* and E. Sidney Hunter III*
    ?Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina and *Reproductiv...

  3. Metatranscriptomic analysis of lactic acid bacterial gene expression during kimchi fermentation.

    PubMed

    Jung, Ji Young; Lee, Se Hee; Jin, Hyun Mi; Hahn, Yoonsoo; Madsen, Eugene L; Jeon, Che Ok

    2013-05-15

    Barcode-based 16S rRNA gene pyrosequencing showed that the kimchi microbiome was dominated by six lactic acid bacteria (LAB), Leuconostoc (Lc.) mesenteroides, Lactobacillus (Lb.) sakei, Weissella (W.) koreensis, Lc. gelidum, Lc. carnosum, and Lc. gasicomitatum. Therefore, we used completed genome sequences of representatives of these bacteria to investigate metatranscriptomic gene-expression profiles during kimchi fermentation. Total mRNA was extracted from kimchi samples taken at five time points during a 29 day-fermentation. Nearly all (97.7%) of the metagenome sequences that were recruited on all LAB genomes of GenBank mapped onto the six LAB strains; this high coverage rate indicated that this approach for assessing processes carried out by the kimchi microbiome was valid. Expressed mRNA sequences (as cDNA) were determined using Illumina GA IIx. Assignment of mRNA sequences to metabolic genes using MG-RAST revealed the prevalence of carbohydrate metabolism and lactic acid fermentation. The mRNA sequencing reads were mapped onto genomes of the six LAB strains, which showed that Lc. mesenteroides was most active during the early-stage fermentation, whereas gene expression by Lb. sakei and W. koreensis was high during later stages. However, gene expression by Lb. sakei decreased rapidly at 25 days of fermentation, which was possibly caused by bacteriophage infection of the Lactobacillus species. Many genes related to carbohydrate transport and hydrolysis and lactate fermentation were actively expressed, which indicated typical heterolactic acid fermentation. Mannitol dehydrogenase-encoding genes (mdh) were identified from all Leuconostoc species and especially Lc. mesenteroides, which harbored three copies (two copies on chromosome and one copy on plasmid) of mdh with different expression patterns. These results contribute to knowledge of the active populations and gene expression in the LAB community responsible for an important fermentation process. PMID

  4. Characterization of the Fatty Acid Desaturase Genes in Cucumber: Structure, Phylogeny, and Expression Patterns

    PubMed Central

    Dong, Chun-Juan; Cao, Ning; Zhang, Zhi-Gang; Shang, Qing-Mao

    2016-01-01

    Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber. PMID:26938877

  5. Characterization of the Fatty Acid Desaturase Genes in Cucumber: Structure, Phylogeny, and Expression Patterns.

    PubMed

    Dong, Chun-Juan; Cao, Ning; Zhang, Zhi-Gang; Shang, Qing-Mao

    2016-01-01

    Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber. PMID:26938877

  6. Regulation of inflammatory and lipid metabolism genes by eicosapentaenoic acid-rich oil[S

    PubMed Central

    Gillies, Peter J.; Bhatia, Sujata K.; Belcher, Leigh A; Hannon, Daniel B.; Thompson, Jerry T.; Vanden Heuvel, John P.

    2012-01-01

    Omega-3-PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are associated with prevention of various aspects of metabolic syndrome. In the present studies, the effects of oil rich in EPA on gene expression and activation of nuclear receptors was examined and compared with other ω3-PUFAs. The EPA-rich oil (EO) altered the expression of FA metabolism genes in THP-1 cells, including stearoyl CoA desaturase (SCD) and FA desaturase-1 and -2 (FASDS1 and -2). Other ω3-PUFAs resulted in a similar gene expression response for a subset of genes involved in lipid metabolism and inflammation. In reporter assays, EO activated human peroxisome proliferator-activated receptor α (PPARα) and PPARβ/γ with minimal effects on PPARγ, liver X receptor, retinoid X receptor, farnesoid X receptor, and retinoid acid receptor γ (RARγ); these effects were similar to that observed for purified EPA. When serum from a 6 week clinical intervention with dietary supplements containing olive oil (control), DHA, or two levels of EPA were applied to THP-1 cells, the expression of SCD and FADS2 decreased in the cells treated with serum from the ω3-PUFA-supplemented individuals. Taken together, these studies indicate regulation of gene expression by EO that is consistent with treating aspects of dyslipidemia and inflammation. PMID:22556214

  7. Exploring the diversity of arsenic resistance genes from acid mine drainage microorganisms.

    PubMed

    Morgante, Verónica; Mirete, Salvador; de Figueras, Carolina G; Postigo Cacho, Marina; González-Pastor, José E

    2015-06-01

    The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance. PMID:24801164

  8. Polyamidoamine dendrimer and oleic acid-functionalized graphene as biocompatible and efficient gene delivery vectors.

    PubMed

    Liu, Xiahui; Ma, Dongmei; Tang, Hao; Tan, Liang; Xie, Qingji; Zhang, Youyu; Ma, Ming; Yao, Shouzhuo

    2014-06-11

    Functionalized graphene has good potential in biomedical applications. To address a better and multiplex design of graphene-based gene vectors, the graphene-oleate-polyamidoamine (PAMAM) dendrimer hybrids were synthesized by the oleic acid adsorption and covalent linkage of PAMAM dendrimers. The micromorphology, electrical charge property, and amount of free amine groups of the graphene-oleate-PAMAM hybrids were characterized, and the peripheral functional groups were identified. The PAMAM dendrimers could be tethered onto graphene surface in high density. The graphene-oleate-PAMAM hybrids exhibit relatively good dispersity and stability in aqueous solutions. To evaluate the potential application of the hybrids in gene delivery vectors, cytotoxicity to HeLa and MG-63 cells and gene (plasmid DNA of enhanced green fluorescent protein) transfection capacity of the hybrids were investigated in detail. The graphene-oleate-PAMAM hybrids show mammalian cell type- and dose-dependent in vitro cytotoxicity. Under the optimal condition, the hybrids possess good biocompatibility and gene transfection capacity. The surface modification of graphene with oleic acid and PAMAM improves the gene transfection efficiency 13 times in contrast to the ultrasonicated graphene. Moreover, the hybrids show better transfection efficiency than the graphene oxide-PAMAM without the oleic acid modification. PMID:24836601

  9. Skin Commensal Staphylococci May Act as Reservoir for Fusidic Acid Resistance Genes

    PubMed Central

    Hung, Wei-Chun; Chen, Hsiao-Jan; Lin, Yu-Tzu; Tsai, Jui-Chang; Chen, Chiao-Wei; Lu, Hsiao-Hung; Tseng, Sung-Pin; Jheng, Yao-Yu; Leong, Kin Hong; Teng, Lee-Jene

    2015-01-01

    We analyzed the occurrence and mechanisms of fusidic acid resistance present in staphylococci isolated from 59 healthy volunteers. The fingers of the volunteers were screened for the presence of staphylococci, and the collected isolates were tested for resistance to fusidic acid. A total of 34 fusidic acid resistant staphylococcal strains (all were coagulase-negative) were isolated from 22 individuals (22/59, 37.3%). Examination of the resistance genes revealed that acquired fusB or fusC was present in Staphylococcus epidermidis, Staphylococcus capitis subsp. urealyticus, Staphylococcus hominis subsp. hominis, Staphylococcus warneri and Staphylococcus haemolyticus. Resistance islands (RIs) carrying fusB were found in S. epidermidis and S. capitis subsp. urealyticus, while staphylococcal chromosome cassette (SCC)-related structures harboring fusC were found in S. hominis subsp. hominis. Genotypic analysis of S. epidermidis and S. hominis subsp. hominis indicated that the fus elements were disseminated in diverse genetic strain backgrounds. The fusC elements in S. hominis subsp. hominis strains were highly homologous to SCCfusC in the epidemic sequence type (ST) 239/SCCmecIII methicillin-resistant S. aureus (MRSA) or the pseudo SCCmec in ST779 MRSA. The presence of acquired fusidic acid resistance genes and their genetic environment in commensal staphylococci suggested that the skin commensal staphylococci may act as reservoir for fusidic acid resistance genes. PMID:26581090

  10. MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria

    PubMed Central

    Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S.; Bhatt, Apoorva

    2012-01-01

    Summary Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. PMID:22520756

  11. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    PubMed

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  12. The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production.

    PubMed

    Ishida, Nobuhiro; Saitoh, Satoshi; Onishi, Toru; Tokuhiro, Kenro; Nagamori, Eiji; Kitamoto, Katsuhiko; Takahashi, Haruo

    2006-05-01

    A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported. PMID:16717415

  13. Expression of genes associated with fatty acid metabolism during maturation in diploid and triploid female rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To study effects of sexual maturation on fatty acid metabolism in fish on a high nutritional plane, expression of thirty-five genes involved in fatty acid metabolism was determined in sexually maturing diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout. Gene expression was assesse...

  14. Candidate gene expression affects intramuscular fat content and fatty acid composition in pigs.

    PubMed

    Wang, Wei; Xue, Wenda; Jin, Bangquan; Zhang, Xixia; Ma, Fei; Xu, Xiaofeng

    2013-02-01

    The objective of this study was to correlate the expression pattern of candidate genes with the intramuscular fat (IMF) content and fatty acid composition of the Longissimus dorsi muscle of Duroc × Shanzhu commercial crossbred pigs. Animals of both sexes were slaughtered at a body weight of about 90 kg. The IMF content and fatty acid composition of the Longissimus dorsi muscle were measured and correlated with candidate genes mRNA expression (AdPLA, ADRB3, LEPR, MC4R, PPARγ, PPARα, LPL, PEPCK, and SCD). Females presented higher IMF content (p < 0.05) than males. The total saturated fatty acid (SFA) in males was greater (p < 0.01), whereas the total monounsaturated fatty acid (MUFA) (p < 0.01) and polyunsaturated fatty acid (PUFA) (p < 0.05) were lower than in females. The expressions of AdPLA, MC4R, PEPCK, and SCD correlated with the IMF content (p < 0.05). AdPLA showed a positive association with MUFA and a negative association with SFA (p < 0.05). LEPR and MC4R were both positively and significantly associated with C18:3 and C20:0 (p < 0.05). PPARα and PPARγ were negatively correlated with SFA, and PPARγ was positively associated with MUFA (p < 0.05). LPL was positively associated with MUFA and negatively associated with SFA (p < 0.05). PEPCK was negatively correlated with PUFA (p < 0.05). SCD was positively associated with MUFA (p < 0.05). The revealed correlations may confirm that these candidate genes are important for fat deposition and fatty acid composition in pigs, and the evaluation and use of these genes may be useful for improving porcine meat quality. PMID:23275256

  15. Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils.

    PubMed

    Jung, Jaejoon; Yeom, Jinki; Han, Jiwon; Kim, Jisun; Park, Woojun

    2012-06-01

    The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change. PMID:22752898

  16. Subchronic effects of valproic acid on gene expression profiles for lipid metabolism in mouse liver

    SciTech Connect

    Lee, Min-Ho |; Kim, Mingoo |; Lee, Byung-Hoon |; Kim, Ju-Han |; Kang, Kyung-Sun |; Kim, Hyung-Lae |; Yoon, Byung-Il |; Chung, Heekyoung; Kong, Gu |; Lee, Mi-Ock ||

    2008-02-01

    Valproic acid (VPA) is used clinically to treat epilepsy, however it induces hepatotoxicity such as microvesicular steatosis. Acute hepatotoxicity of VPA has been well documented by biochemical studies and microarray analysis, but little is known about the chronic effects of VPA in the liver. In the present investigation, we profiled gene expression patterns in the mouse liver after subchronic treatment with VPA. VPA was administered orally at a dose of 100 mg/kg/day or 500 mg/kg/day to ICR mice, and the livers were obtained after 1, 2, or 4 weeks. The activities of serum liver enzymes did not change, whereas triglyceride concentration increased significantly. Microarray analysis revealed that 1325 genes of a set of 32,996 individual genes were VPA responsive when examined by two-way ANOVA (P < 0.05) and fold change (> 1.5). Consistent with our previous results obtained using an acute VPA exposure model (Lee et al., Toxicol Appl Pharmacol. 220:45-59, 2007), the most significantly over-represented biological terms for these genes included lipid, fatty acid, and steroid metabolism. Biological pathway analysis suggests that the genes responsible for increased biosynthesis of cholesterol and triglyceride, and for decreased fatty acid {beta}-oxidation contribute to the abnormalities in lipid metabolism induced by subchronic VPA treatment. A comparison of the VPA-responsive genes in the acute and subchronic models extracted 15 commonly altered genes, such as Cyp4a14 and Adpn, which may have predictive power to distinguish the mode of action of hepatotoxicants. Our data provide a better understanding of the molecular mechanisms of VPA-induced hepatotoxicity and useful information to predict steatogenic hepatotoxicity.

  17. Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome

    PubMed Central

    Meslin, Camille; Desert, Colette; Callebaut, Isabelle; Djari, Anis; Klopp, Christophe; Pitel, Frédérique; Leroux, Sophie; Martin, Pascal; Froment, Pascal; Guilbert, Edith; Gondret, Florence; Lagarrigue, Sandrine; Monget, Philippe

    2015-01-01

    Free fatty acid receptors (FFAR) belong to a family of five G-protein coupled receptors that are involved in the regulation of lipid metabolism, so that their loss of function increases the risk of obesity. The aim of this study was to determine the expansion of genes encoding paralogs of FFAR2 in the chicken, considered as a model organism for developmental biology and biomedical research. By estimating the gene copy number using quantitative polymerase chain reaction, genomic DNA resequencing, and RNA sequencing data, we showed the existence of 23 ± 1.5 genes encoding FFAR2 paralogs in the chicken genome. The FFAR2 paralogs shared an identity from 87.2% up to 99%. Extensive gene conversion was responsible for this high degree of sequence similarities between these genes, and this concerned especially the four amino acids known to be critical for ligand binding. Moreover, elevated nonsynonymous/synonymous substitution ratios on some amino acids within or in close-vicinity of the ligand-binding groove suggest that positive selection may have reduced the effective rate of gene conversion in this region, thus contributing to diversify the function of some FFAR2 paralogs. All the FFAR2 paralogs were located on a microchromosome in a same linkage group. FFAR2 genes were expressed in different tissues and cells such as spleen, peripheral blood mononuclear cells, abdominal adipose tissue, intestine, and lung, with the highest rate of expression in testis. Further investigations are needed to determine whether these chicken-specific events along evolution are the consequence of domestication and may play a role in regulating lipid metabolism in this species. PMID:25912043

  18. Fatty acid regulates gene expression and growth of human prostate cancer PC-3 cells

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Chen, Y.; Tjandrawinata, R. R.

    2001-01-01

    It has been proposed that the omega-6 fatty acids increase the rate of tumor growth. Here we test that hypothesis in the PC-3 human prostate tumor. We found that the essential fatty acids, linoleic acid (LA) and arachidonic acid (AA), and the AA metabolite PGE(2) stimulate tumor growth while oleic acid (OA) and the omega-3 fatty acid, eicosapentaenoic acid (EPA) inhibited growth. In examining the role of AA in growth response, we extended our studies to analyze changes in early gene expression induced by AA. We demonstrate that c-fos expression is increased within minutes of addition in a dose-dependent manner. Moreover, the immediate early gene cox-2 is also increased in the presence of AA in a dose-dependent manner, while the constitutive cox-1 message was not increased. Three hours after exposure to AA, the synthesis of PGE(2) via COX-2 was also increased. Previous studies have demonstrated that AA was primarily delivered by low density lipoprotein (LDL) via its receptor (LDLr). Since it is known that hepatomas, acute myelogenous leukemia and colorectal tumors lack normal cholesterol feedback, we examined the role of the LDLr in growth regulation of the PC-3 prostate cancer cells. Analysis of ldlr mRNA expression and LDLr function demonstrated that human PC-3 prostate cancer cells lack normal feedback regulation. While exogenous LDL caused a significant stimulation of cell growth and PGE(2) synthesis, no change was seen in regulation of the LDLr by LDL. Taken together, these data show that normal cholesterol feedback of ldlr message and protein is lost in prostate cancer. These data suggest that unregulated over-expression of LDLr in tumor cells would permit increased availability of AA, which induces immediate early genes c-fos and cox-2 within minutes of uptake.

  19. Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids.

    PubMed

    Liu, Weitian; Jiao, Hongmei; Murray, Nancy C; O'Connor, Marion; Roelofs, Wendell L

    2002-01-22

    Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, that makes E11 isomers of monounsaturated (E11-16 and E11-14) fatty acids and a diunsaturated (E9,E11-14) fatty acid. In the pheromone gland, the monoene precursor is made available by beta oxidation of E11-16 acid with a subsequent two-carbon loss to E9-14 acid. A functional assay using a baculovirus expression system required addition of myristic acid and E9-14 acid precursors to demonstrate the unusual regiospecificity and stereospecificity of this desaturase. The amino acid sequence of this desaturase has approximately 61% identity to that of Z11-desaturases from two other insect species, and only approximately 48% identity to the metabolic Z9-desaturases in those species. A pheromone-gland Z9-desaturase gene also was found with the light brown apple moth that differed in its deduced amino acid sequence (66% identity) with the metabolic Z9-desaturase from fat body in this species. PMID:11805319

  20. Incorporation of D-alanine into lipoteichoic acid and wall teichoic acid in Bacillus subtilis. Identification of genes and regulation.

    PubMed

    Perego, M; Glaser, P; Minutello, A; Strauch, M A; Leopold, K; Fischer, W

    1995-06-30

    The Bacillus subtilis dlt operon (D-alanyl-lipoteichoic acid) is responsible for D-alanine esterification of both lipoteichoic acid (LTA) and wall teichoic acid (WTA). The dlt operon contains five genes, dltA-dltE. Insertional inactivation of dltA-dltD results in complete absence of D-alanine from both LTA and WTA. Based on protein sequence similarity with the Lactobacillus casei dlt gene products (Heaton, M. P., and Neuhaus, F. C. (1992) J. Bacteriol. 174, 4707-4717), we propose that dltA encodes the D-alanine-D-alanyl carrier protein ligase (Dcl) and dltC the D-alanyl carrier protein (Dcp). We further hypothesize that the products of dltB and dltD are concerned with the transport of activated D-alanine through the membrane and the final incorporation of D-alanine into LTA. The hydropathy profiles of the dltB and dltD gene products suggest a transmembrane location for the former and an amino-terminal signal peptide for the latter. The incorporation of D-alanine into LTA and WTA did not separate in any of the mutants studied which indicates that either one and the same enzyme is responsible for D-alanine incorporation into both polymers or a separate enzyme, encoded outside the dlt operon, transfers the D-alanyl residues from LTA to WTA (Haas, R., Koch, H.-U., and Fischer, W. (1984) FEMS Microbiol. Lett. 21, 27-31). Inactivation of dltE has no effect on D-alanine ester content of both LTA and WTA, and at present we cannot propose any function for its gene product. Transcription analysis shows that the dlt operon is transcribed from a sigma D-dependent promoter and follows the pattern of transcription of genes belonging to the sigma D regulon. However, the turn off of transcription observed before sporulation starts seems to be dependent on the Spo0A and AbrB sporulation proteins and results in a D-alanine-free purely anionic LTA in the spore membrane. The dlt operon is dispensable for cell growth; its inactivation does not affect cell growth or morphology as

  1. The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2010-10-15

    An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes. PMID:20674726

  2. Controllably local gene delivery mediated by polyelectrolyte multilayer films assembled from gene-loaded nanopolymersomes and hyaluronic acid

    PubMed Central

    Teng, Wei; Wang, Qinmei; Chen, Ying; Huang, Hongzhang

    2014-01-01

    To explore a spatiotemporally controllable gene delivery system with high efficiency and safety, polyelectrolyte multilayer (PEM) films were constructed on titanium or quartz substrates via layer-by-layer self-assembly technique by using plasmid deoxyribonucleic acid-loaded lipopolysaccharide–amine nanopolymersomes (pNPs) as polycations and hyaluronic acid (HA) as polyanions. pNPs were chosen because they have high transfection efficiency (>95%) in mesenchymal stem cells (MSCs) and induce significant angiogenesis in zebrafish in conventional bolus transfection. The assembly process of PEM films was confirmed by analyses of quartz crystal microbalance with dissipation, X-ray photoelectron spectroscopy, infrared, contact angle, and zeta potential along with atomic force microscopy observation. Quartz crystal microbalance with dissipation analysis reveals that this film grows in an exponential mode, pNPs are the main contributor to the film mass, and the film mass can be modulated in a relatively wide range (1.0–29 μg/cm2) by adjusting the deposition layer number. Atomic force microscopy observation shows that the assembly leads to the formation of a patterned film with three-dimensional tree-like nanostructure, where the branches are composed of beaded chains (pNP beads are strung on HA molecular chains), and the incorporated pNPs keep structure intact. In vitro release experiment shows that plasmid deoxyribonucleic acid can be gradually released from films over 14 days, and the released plasmid deoxyribonucleic acid exists in a complex form. In vitro cell experiments demonstrate that PEM films can enhance the adhesion and proliferation of MSCs and efficiently transfect MSCs in situ in vitro for at least 4 days. Our results suggest that a (pNPs/HA)n system can mediate efficient transfection in stem cells in a spatially and temporally controllable pattern, highlighting its huge potential in local gene therapy. PMID:25378927

  3. Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes.

    PubMed

    Nagano, Naoki; Sakaguchi, Keishi; Taoka, Yousuke; Okita, Yuji; Honda, Daiske; Ito, Makoto; Hayashi, Masahiro

    2011-01-01

    Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms. PMID:21852747

  4. A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides.

    PubMed

    Ment, Dana; Alkan, Noam; Luria, Neta; Bi, Fang-Cheng; Reuveni, Eli; Fluhr, Robert; Prusky, Dov

    2015-02-01

    Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity. PMID:25317668

  5. Transcriptome analysis and identification of genes associated with ¿-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Perilla (Perilla frutescens (L.) var frutescens) produces high levels of a-linolenic acid (ALA), a '-3 fatty acid important to health and development. To uncover key genes involved in fatty acid (FA) and triacylglycerol (TAG) synthesis in perilla, we conducted deep sequencing of cDNAs f...

  6. [Succinic acid production from sucrose and sugarcane molasses by metabolically engineered Escherichia coli].

    PubMed

    Li, Feng; Ma, Jiangfeng; Wu, Mingke; Ji, Yaliang; Chen, Wufang; Ren, Xinyi; Jiang, Min

    2015-04-01

    Sugarcane molasses containing large amounts of sucrose is an economical substrate for succinic acid production. However, Escherichia coli AFP111 cannot metabolize sucrose although it is a promising candidate for succinic acid production. To achieve sucrose utilizing ability, we cloned and expressed cscBKA genes encoding sucrose permease, fructokinase and invertase of non-PTS sucrose-utilization system from E. coli W in E. coli AFP111 to generate a recombinant strain AFP111/pMD19T-cscBKA. After 72 h of anaerobic fermentation of the recombinant in serum bottles, 20 g/L sucrose was consumed and 12 g/L succinic acid was produced. During dual-phase fermentation comprised of initial aerobic growth phase followed by anaerobic fermentation phase, the concentration of succinic acid from sucrose and sugarcane molasses was 34 g/L and 30 g/L, respectively, at 30 h of anaerobic phase in a 3 L fermentor. The results show that the introduction of non-PTS sucrose-utilization system has sucrose-metabolizing capability for cell growth and succinic acid production, and can use cheap sugarcane molasses to produce succinic acid. PMID:26380410

  7. Increased Missense Mutation Burden of Fatty Acid Metabolism Related Genes in Nunavik Inuit Population

    PubMed Central

    Zhou, Sirui; Xiong, Lan; Xie, Pingxing; Ambalavanan, Amirthagowri; Bourassa, Cynthia V.; Dionne-Laporte, Alexandre; Spiegelman, Dan; Turcotte Gauthier, Maude; Henrion, Edouard; Diallo, Ousmane; Dion, Patrick A.; Rouleau, Guy A.

    2015-01-01

    Background Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three carnitine acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism. Methods Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the carnitine acyltransferases in 113 Nunavik Inuit individuals. Results Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on carnitine acyltransferase gene variations between Nunavik Inuit and Asians. Conclusion The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit. PMID:26010953

  8. Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei

    SciTech Connect

    Clayton, C.E.; Fueri, J.P.; Itzhaki, J.E.; Bellofatto, V.; Sherman, D.R.; Wisdom, G.S.; Vijayasarathy, S.; Mowatt, M.R. )

    1990-06-01

    The procyclic acidic repetitive protein (parp) genes of Trypanosoma brucei encode a small family of abundant surface proteins whose expression is restricted to the procyclic form of the parasite. They are found at two unlinked loci, parpA and parpB; transcription of both loci is developmentally regulated. The region of homology upstream of the A and B parp genes is only 640 base pairs long and may contain sequences responsible for transcriptional initiation and regulation. Transcription upstream of this putative promoter region is not developmentally regulated and is much less active than that of the parp genes; the polymerase responsible is inhibited by alpha-amanitin, whereas that transcribing the parp genes is not. Transcription of the parp genes is strongly stimulated by low levels of UV irradiation. The putative parp promoter, when placed upstream of the chloramphenicol acetyltransferase gene, is sufficient to cause production of chloramphenicol acetyltransferase in a T. brucei DNA transformation assay. Taken together, these results suggest that a promoter for an alpha-amanitin-resistant RNA polymerase lies less than 600 nucleotides upstream of the parp genes.

  9. Identification and Functional Analysis of the Mycophenolic Acid Gene Cluster of Penicillium roqueforti.

    PubMed

    Del-Cid, Abdiel; Gil-Durán, Carlos; Vaca, Inmaculada; Rojas-Aedo, Juan F; García-Rico, Ramón O; Levicán, Gloria; Chávez, Renato

    2016-01-01

    The filamentous fungus Penicillium roqueforti is widely known as the ripening agent of blue-veined cheeses. Additionally, this fungus is able to produce several secondary metabolites, including the meroterpenoid compound mycophenolic acid (MPA). Cheeses ripened with P. roqueforti are usually contaminated with MPA. On the other hand, MPA is a commercially valuable immunosuppressant. However, to date the molecular basis of the production of MPA by P. roqueforti is still unknown. Using a bioinformatic approach, we have identified a genomic region of approximately 24.4 kbp containing a seven-gene cluster that may be involved in the MPA biosynthesis in P. roqueforti. Gene silencing of each of these seven genes (named mpaA, mpaB, mpaC, mpaDE, mpaF, mpaG and mpaH) resulted in dramatic reductions in MPA production, confirming that all of these genes are involved in the biosynthesis of the compound. Interestingly, the mpaF gene, originally described in P. brevicompactum as a MPA self-resistance gene, also exerts the same function in P. roqueforti, suggesting that this gene has a dual function in MPA metabolism. The knowledge of the biosynthetic pathway of MPA in P. roqueforti will be important for the future control of MPA contamination in cheeses and the improvement of MPA production for commercial purposes. PMID:26751579

  10. Structure and expression of the Drosophila ubiquitin-80-amino-acid fusion-protein gene.

    PubMed Central

    Barrio, R; del Arco, A; Cabrera, H L; Arribas, C

    1994-01-01

    In the fruitfly Drosophila, as in all eukaryotes examined so far, some ubiquitin-coding sequences appear fused to unrelated open reading frames. Two of these fusion genes have been previously described (the homologues of UBI1-UBI2 and UBI4 in yeast), and we report here the organization and expression of a third one, the DUb80 gene (the homologue of UBI3 in yeast). This gene encodes a ubiquitin monomer fused to an 80-amino-acid extension which is homologous with the ribosomal protein encoded by the UB13 gene. The 5' regulatory region of DUb80 shares common features with another ubiquitin fusion gene, DUb52, and with the ribosomal protein genes of Drosophila, Xenopus and mouse. We also find helix-loop-helix protein-binding sequences (E-boxes). The DUb80 gene is transcribed to a 0.9 kb mRNA which is particularly abundant under conditions of high protein synthesis, such as in ovaries and exponentially growing cells. Images Figure 3 Figure 4 PMID:8068011

  11. Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat

    PubMed Central

    Goszczynski, Daniel Estanislao; Mazzucco, Juliana Papaleo; Ripoli, María Verónica; Villarreal, Edgardo Leopoldo; Rogberg-Muñoz, Andrés; Mezzadra, Carlos Alberto; Melucci, Lilia Magdalena; Giovambattista, Guillermo

    2014-01-01

    LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01), and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01), may be helpful to improve the quality of meat and improve health. PMID:25606458

  12. Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification.

    PubMed Central

    Kranz, R G; Gabbert, K K; Madigan, M T

    1997-01-01

    The photosynthetic bacterium Rhodobacter capsulatus can grow with short- to long-chain fatty acids as the sole carbon source (R. G. Kranz, K. K. Gabbert, T. A. Locke, and M. T. Madigan, Appl. Environ. Microbiol. 63:3003-3009, 1997). Concomitant with growth on fatty acids is the production to high levels of the polyester storage compounds called polyhydroxyalkanoates (PHAs). Here, we describe colony screening and selection systems to analyze the production of PHAs in R. capsulatus. A screen with Nile red dissolved in acetone distinguishes between PHA producers and nonproducers. Unlike the wild type, an R. capsulatus PhaC- strain with the gene encoding PHA synthase deleted is unable to grow on solid media containing high concentrations of certain fatty acids. It is proposed that this deficiency is due to the inability of the PhaC- strain to detoxify the surrounding medium by consumption of fatty acids and their incorporation into PHAs. This fatty acid toxicity phenotype is used in selection for the cloning and characterization of heterologous phaC genes. PMID:9251190

  13. Identification and heterologous expression of a Δ4-fatty acid desaturase gene from Isochrysis sphaerica.

    PubMed

    Guo, Bing; Jiang, Mulan; Wan, Xia; Gong, Yangmin; Liang, Zhuo; Hu, Chuanjiong

    2013-10-28

    The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a Δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops. PMID:23851273

  14. Genetic engineering to contain the Vitreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia

    SciTech Connect

    Liu, S.C.; Webster, D.A.; Wei, M.L.; Stark, B.C.

    1996-01-05

    Xanthomonas maltophilia was transformed with the gene encoding Vitreoscilla (bacterial) hemoglobin, vgb, and the growth of the engineered strain was compared with that of the untransformed strain using benzoic acid as the sole carbon source. In general, growth of the engineered strain was greater than that of the untransformed strain; this was true for experiments using both overnight cultures and log phase cells as inocula, but particularly for the latter. In both cases the engineered strain was also more efficiency than the untransformed strain in converting benzoic acid into biomass.

  15. Production of thermostable invertases by Aspergillus caespitosus under submerged or solid state fermentation using agroindustrial residues as carbon source

    PubMed Central

    Alegre, Ana Cláudia Paiva; de Lourdes Teixeira de Moraes Polizeli, Maria; Terenzi, Héctor Francisco; Jorge, João Atílio; Guimarães, Luis Henrique Souza

    2009-01-01

    The filamentous fungus Aspergillus caespitosus was a good producer of intracellular and extracellular invertases under submerged (SbmF) or solid-state fermentation (SSF), using agroindustrial residues, such as wheat bran, as carbon source. The production of extracellular enzyme under SSF at 30°C, for 72h, was enhanced using SR salt solution (1:1, w/v) to humidify the substrate. The extracellular activity under SSF using wheat bran was around 5.5-fold higher than that obtained in SbmF (Khanna medium) with the same carbon source. However, the production of enzyme with wheat bran plus oat meal was 2.2-fold higher than wheat bran isolated. The enzymatic production was affected by supplementation with nitrogen and phosphate sources. The addition of glucose in SbmF and SSF promoted the decreasing of extracellular activity, but the intracellular form obtained in SbmF was enhanced 3-5-fold. The invertase produced in SSF exhibited optimum temperature at 50°C while the extra- and intracellular enzymes produced in SbmF exhibited maximal activities at 60°C. All enzymatic forms exhibited maximal activities at pH 4.0-6.0 and were stable up to 1 hour at 50°C. PMID:24031406

  16. Kinetic and Energetic Parameters of Carob Wastes Fermentation by Saccharomyces cerevisiae: Crabtree Effect, Ethanol Toxicity, and Invertase Repression.

    PubMed

    Rodrigues, B; Peinado, J M; Raposo, S; Constantino, A; Quintas, C; Lima-Costa, M E

    2015-06-01

    Carob waste is a useful raw material for the second-generation ethanol because 50% of its dry weight is sucrose, glucose, and fructose. To optimize the process, we have studied the influence of the initial concentration of sugars on the fermentation performance of Saccharomyces cerevisiae. With initial sugar concentrations (S0) of 20 g/l, the yeasts were derepressed and the ethanol produced during the exponential phase was consumed in a diauxic phase. The rate of ethanol consumption decreased with increasing S0 and disappeared at 250 g/l when the Crabtree effect was complete and almost all the sugar consumed was transformed into ethanol with a yield factor of 0.42 g/g. Sucrose hydrolysis was delayed at high S0 because of glucose repression of invertase synthesis, which was triggered at concentrations above 40 g/l. At S0 higher than 250 g/l, even when glucose had been exhausted, sucrose was hydrolyzed very slowly, probably due to an inhibition at this low water activity. Although with lower metabolic rates and longer times of fermentation, 250 g/l is considered the optimal initial concentration because it avoids the diauxic consumption of ethanol and maintains enough invertase activity to consume all the sucrose, and also avoids the inhibitions due to lower water activities at higher S0. PMID:25588557

  17. Changes in Gene Expression Profiling of Apoptotic Genes in Neuroblastoma Cell Lines upon Retinoic Acid Treatment

    PubMed Central

    Celay, Jon; Blanco, Idoia; Lázcoz, Paula; Rotinen, Mirja; Castresana, Javier S.; Encío, Ignacio

    2013-01-01

    To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used. PMID:23650528

  18. Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

    DOE PAGESBeta

    Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.; Inmon, Jay J.; Millhollon, Jon C.; Liechty, Zach; Page, Justin T.; Jenks, Matthew A.; Chapman, Kent D.; Udall, Joshua A.; et al

    2014-11-18

    The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior

  19. Effects of candidate gene polymorphisms on the detailed fatty acids profile determined by gas chromatography in bovine milk.

    PubMed

    Pegolo, S; Cecchinato, A; Mele, M; Conte, G; Schiavon, S; Bittante, G

    2016-06-01

    Association analyses between candidate genes and bovine milk fatty acids can improve our understanding of genetic variation in milk fatty acid profiles and reveal potential opportunities to tailor milk fat composition through selection strategies. In this work, we investigated the association of 51 single nucleotide polymorphisms (SNP) selected from 37 candidate genes using a functional and positional approach, with 47 fatty acids, 9 fatty acid groups, and 5 Δ(9)-desaturation indices in milk samples from Brown Swiss cows. Individual milk samples were collected from 1,158 Italian Brown Swiss cows, and gas chromatography was used to obtain detailed milk fatty acid compositions. A GoldenGate assay system (Illumina, San Diego, CA) was used to perform genotype 96 selected SNP located in 54 genes across 22 chromosomes. In total, 51 polymorphic SNP in 37 candidate genes were retained for the association analysis. A Bayesian linear animal model was used to estimate the contribution of each SNP. A total of 129 tests indicated relevant additive effects between a given SNP and a single fatty acid trait; 38 SNP belonging to 30 genes were relevant for a total of 57 fatty acid traits. Most of the studied fatty acid traits (~81%) were relevantly associated with multiple SNP. Relevantly associated SNP were mainly found in genes related to fat metabolism, linked to or contained in previously identified quantitative trait loci for fat yield or content, or associated with genes previously identified in association analyses with milk fatty acid profiles in other cow breeds. The most representative candidate genes were LEP, PRL, STAT5A, CCL3, ACACA, GHR, ADRB2, LPIN1, STAT1, FABP4, and CSN2. In particular, relevant associations with SNP located on bovine chromosome 19 (BTA19) were found. Two candidate genes on BTA19 (CCL3 and ACACA) were relevantly associated with de novo short- and medium-chain fatty acids, likely explaining the high heritability values found for these fatty acids

  20. Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

    PubMed

    Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

    2016-01-01

    Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. PMID:26514651

  1. Analysis of ldh genes in Lactobacillus casei BL23: role on lactic acid production.

    PubMed

    Rico, Juan; Yebra, María Jesús; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

    2008-06-01

    Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid. PMID:18231816

  2. Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells.

    PubMed

    Young, Kaylie L; Scott, Alexander W; Hao, Liangliang; Mirkin, Sarah E; Liu, Guoliang; Mirkin, Chad A

    2012-07-11

    Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach. PMID:22725653

  3. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    PubMed Central

    Birkedal, Henrik

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies. PMID:21686249

  4. The ionotropic γ-aminobutyric acid receptor gene family of the silkworm, Bombyx mori.

    PubMed

    Yu, Lin-Lin; Cui, Ying-Jun; Lang, Guo-Jun; Zhang, Ming-Yan; Zhang, Chuan-Xi

    2010-09-01

    γ-Aminobutyric acid (GABA) is a very important inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. GABA receptors (GABARs) are known to be the molecular targets of a class of insecticides. Members of the GABAR gene family of the silkworm, Bombyx mori, a model insect of Lepidoptera, have been identified and characterized in this study. All putative silkworm GABAR cDNAs were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Bombyx mori appears to have the largest insect GABAR gene family known to date, including three RDL, one LCCH3, and one GRD subunit. The silkworm RDL1 gene has RNA-editing sites, and the RDL1 and RDL3 genes possess alternative splicing. These mRNA modifications enhance the diversity of the silkworm's GABAR gene family. In addition, truncated transcripts were found for the RDL1 and LCCH3 genes. In particular, the three RDL subunits may have arisen from two duplication events. PMID:20924418

  5. Miniature1-encoded cell wall invertase is essential for assembly and function of wall-in-growth in the maize endosperm transfer cell

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The miniature1 (mn1) seed phenotype in maize is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase, INCW2, which localizes exclusively to the basal endosperm transfer cells (BETC) of developing seeds. A common feature of all transfer cells is the labyrinth-like wa...

  6. Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes.

    PubMed Central

    Doerner, K C; Takamine, F; LaVoie, C P; Mallonee, D H; Hylemon, P B

    1997-01-01

    Eubacterium sp. strain VPI 12708 has several bile acid-inducible (bai) genes which encode enzymes in the bile acid 7 alpha-dehydroxylation (7 alpha DeOH) pathway. Twelve 7 alpha DeOH-positive intestinal bacterial strains were assayed for 7 alpha DeOH activity, and 13 strains were tested for hybridization with bai genes. Cholic acid 7 alpha DeOH activity varied greatly (> 100-fold) among these strains. Southern blot experiments showed that DNA prepared from 7 of 13 strains hybridized with at least one of the bai genes from Eubacterium sp. strain VPI 12708. PMID:9055436

  7. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering.

    PubMed

    Chen, Yingying; Stabryla, Lisa; Wei, Na

    2016-01-01

    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production. PMID:26826231

  8. Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

    SciTech Connect

    Jiang, S.-Y.; Wu, M.-S.; Chen, L.-M.; Hung, M.-W.; Lin, H.-E.; Chang, G.-G.; Chang, T.-C. . E-mail: tcchang@ndmctsgh.edu.tw

    2005-06-03

    The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the at

  9. Isolation and characterization of all-trans-retinoic acid-responsive genes in the rat testis.

    PubMed

    Gaemers, I C; Van Pelt, A M; Themmen, A P; De Rooij, D G

    1998-05-01

    By way of differential screening of testis cDNA libraries from vitamin A-deficient (VAD) rats before and after administration of all-trans retinoic acid (ATRA), genes, the transcription of which was influenced by ATRA, were isolated. Most clones with an increased transcription encoded different subunits of the same mitochondrial protein complex, cytochrome c oxidase (COX). The mRNA expression of COX increased by a factor 3.9 +/- 1.5 (mean +/- SD, n = 4). This increased expression seems to reflect an increased energy demand in the ATRA-supplemented VAD testis. Also, one gene was isolated, the transcription of which was reduced to about 70% by ATRA. This gene, sulfated glycoprotein 2 (Sgp-2), is a major secretion product of Sertoli cells, the function of which is still unknown. The effect of ATRA on Sgp-2 expression may be direct, since the promoter of Sgp-2 contains a putative ATRA-responsive element (RARE). PMID:9547504

  10. Role of a liver fatty acid-binding protein gene in lipid metabolism in chicken hepatocytes.

    PubMed

    Gao, G L; Na, W; Wang, Y X; Zhang, H F; Li, H; Wang, Q G

    2015-01-01

    This study investigated the role of the chicken liver fatty acid-binding protein (L-FABP) gene in lipid metabolism in hepatocytes, and the regulatory relationships between L-FABP and genes related to lipid metabolism. The short hairpin RNA (shRNA) interference vector with L-FABP and an eukaryotic expression vector were used. Chicken hepatocytes were subjected to shRNA-mediated knockdown or L-FABP cDNA overexpression. Expression levels of lipid metabolism-related genes and biochemical parameters were detected 24, 36, 48, 60, and 72 h after transfection with the interference or overexpression plasmids for L-FABP, PPARα and L-BABP expression levels, and the total amount of cholesterol, were significantly affected by L-FABP expression. L-FABP may affect lipid metabolism by regulating PPARα and L-BABP in chicken hepatocytes. PMID:25966259

  11. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    SciTech Connect

    Ding, J.H.; Yang, B.Z.; Liu, H.M.

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  12. Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors.

    PubMed

    Junquera, Elena; Aicart, Emilio

    2016-07-01

    Due to the potential use as transfecting agents of nucleic acids (DNA or RNA), multivalent cationic non-viral vectors have received special attention in the last decade. Much effort has been addressed to synthesize more efficient and biocompatible gene vectors able to transport nucleic acids into the cells without provoking an immune response. Among them, the mostly explored to compact and transfect nucleic acids are: (a) gemini and multivalent cationic lipids, mixed with a helper lipid, by forming lipoplexes; and (b) cationic polymers, polycations, and polyrotaxanes, by forming polyplexes. This review is focused on the progress and recent advances experimented in this area, mainly during the present decade, devoting special attention to the lipoplexes and polyplexes, as follows: (a) to its biophysical characterization (mainly electrostatics, structure, size and morphology) using a wide variety of experimental methods; and (b) to its biological activity (transfection efficacy and cytotoxicity) addressed to confirm the optimum formulations and viability of these complexes as very promising gene vectors of nucleic acids in nanomedicine. PMID:26265376

  13. Transcriptome Profiling of Shewanella oneidensis Gene Expressionfollowing Exposure to Acidic and Alkaline pH

    SciTech Connect

    Leaphart, Adam B.; Thompson, Dorothea K.; Huang, Katherine; Alm,Eric; Wan, Xiu-Feng; Arkin, Adam P.; Brown, Steven D.; Wu, Liyou; Yan,Tingfen; Liu, Xueduan; Wickham, Gene S.; Zhou, Jizhong

    2007-04-02

    The molecular response of Shewanella oneidensis MR-1 tovariations in extracellular pH was investigated based on genomewide geneexpression profiling. Microarray analysis revealed that cells elicitedboth general and specific transcriptome responses when challenged withenvironmental acid (pH 4) or base (pH 10) conditions over a 60-minperiod. Global responses included the differential expression of genesfunctionally linked to amino acid metabolism, transcriptional regulationand signal transduction, transport, cell membrane structure, andoxidative stress protection. Response to acid stress included theelevated expression of genes encoding glycogen biosynthetic enzymes,phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS),whereas the molecular response to alkaline pH was characterized byupregulation of nhaA and nhaR, which are predicted to encode an Na+/H+antiporter and transcriptional activator, respectively, as well assulfate transport and sulfur metabolism genes. Collectively, theseresults suggest that S. oneidensis modulates multiple transporters, cellenvelope components, and pathways of amino acid consumption and centralintermediary metabolism as part of its transcriptome response to changingexternal pH conditions.

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

    SciTech Connect

    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.

  15. Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds.

    PubMed

    Dehghan Nayeri, Fatemeh; Yarizade, Kazem

    2014-08-01

    Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In plants, seed-specific delta-12 fatty acid desaturase 2 (FAD2) is responsible for the high content of linoleic acid by inserting a double bond at the delta-12 (omega-6) position of oleic acid. In this study, sixteen FAD2 and FAD2-2 protein sequences from oilseeds were analyzed by computational tools including two databases of the NCBI and EXPASY and data management tools such as SignalP, TMHMM, Psort, ProtParam, TargetP, PLACE and PlantCARE. These services were used to predict the protein properties such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. The polypeptide sequences were aligned and a neighbour-joining tree was constructed using MEGA5.1 to elucidate phylogenetic relationships among FAD2 genes. Based on the phylogenetic analysis species with high similarity in FAD2 sequence grouped together. FAD2 proteins include highly conserved histidine-rich motifs (HECGHH, HRRHH and HV[A/C/T]HH) that are located by three to five transmembrane anchors. For further investigations Sesamum indicum FAD2 was selected and analyzed by bioinformatics tools. Analysis showed no N-terminal signal peptide for probable localization of FAD2 protein in cytoplasmic organelles such as chloroplast, mitochondria and Golgi. Instead the C-terminal signaling motif YNNKL, Y(K/N)NKF or YRNKI allows FAD2 protein to selectively bind to and embed in the endoplasmic reticulum. FAD2 promoter contains different cis-regulatory elements involve in the biotic and abiotic stresses response or control of gene expression specifically in seeds. PMID:24816719

  16. Increase of eicosapentaenoic acid in thraustochytrids through thraustochytrid ubiquitin promoter-driven expression of a fatty acid {delta}5 desaturase gene.

    PubMed

    Kobayashi, Takumi; Sakaguchi, Keishi; Matsuda, Takanori; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2011-06-01

    Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C(16:0)), n - 6 docosapentaenoic acid (DPA) (C(22:5)(n) (- 6)), and docosahexaenoic acid (DHA) (C(22:6)(n) (- 3)), with eicosapentaenoic acid (EPA) (C(20:5)(n) (- 3)) and arachidonic acid (AA) (C(20:4)(n) (- 6)) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C(20:4)(n) (- 3)) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C(20:3)(n) (- 6)) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs. PMID:21478316

  17. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    PubMed Central

    2010-01-01

    Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should

  18. Sialic Acid-Mediated Gene Expression in Streptococcus pneumoniae and Role of NanR as a Transcriptional Activator of the nan Gene Cluster

    PubMed Central

    Afzal, Muhammad; Shafeeq, Sulman; Ahmed, Hifza

    2015-01-01

    In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to sialic acid (N-acetylneuraminic acid [Neu5Ac]). Transcriptome comparison of wild-type D39 grown in M17 medium with and without sialic acid revealed the elevated expression of various genes and operons, including the nan gene cluster (nan operon I and nanA gene). Our microarray analysis and promoter-lacZ fusion studies showed that the transcriptional regulator NanR acts as a transcriptional activator of nan operon I and the nanA gene in the presence of sialic acid. The putative regulatory site of NanR in the promoter region of nan operon I is predicted and confirmed by promoter truncation experiments. Furthermore, the role of CcpA in the regulation of the nan gene cluster is demonstrated through microarray analysis and promoter-lacZ fusion studies, suggesting that in the presence of sialic acid and glucose, CcpA represses the expression of nan operon I while the expression of the nanA gene is CcpA independent. PMID:25724955

  19. Stress-Survival Gene Identification From an Acid Mine Drainage Algal Mat Community

    NASA Astrophysics Data System (ADS)

    Urbina-Navarrete, J.; Fujishima, K.; Paulino-Lima, I. G.; Rothschild-Mancinelli, B.; Rothschild, L. J.

    2014-12-01

    Microbial communities from acid mine drainage environments are exposed to multiple stressors to include low pH, high dissolved metal loads, seasonal freezing, and desiccation. The microbial and algal communities that inhabit these niche environments have evolved strategies that allow for their ecological success. Metagenomic analyses are useful in identifying species diversity, however they do not elucidate the mechanisms that allow for the resilience of a community under these extreme conditions. Many known or predicted genes encode for protein products that are unknown, or similarly, many proteins cannot be traced to their gene of origin. This investigation seeks to identify genes that are active in an algal consortium during stress from living in an acid mine drainage environment. Our approach involves using the entire community transcriptome for a functional screen in an Escherichia coli host. This approach directly targets the genes involved in survival, without need for characterizing the members of the consortium.The consortium was harvested and stressed with conditions similar to the native environment it was collected from. Exposure to low pH (< 3.2), high metal load, desiccation, and deep freeze resulted in the expression of stress-induced genes that were transcribed into messenger RNA (mRNA). These mRNA transcripts were harvested to build complementary DNA (cDNA) libraries in E. coli. The transformed E. coli were exposed to the same stressors as the original algal consortium to select for surviving cells. Successful cells incorporated the transcripts that encode survival mechanisms, thus allowing for selection and identification of the gene(s) involved. Initial selection screens for freeze and desiccation tolerance have yielded E. coli that are 1 order of magnitude more resistant to freezing (0.01% survival of control with no transcript, 0.2% survival of E. coli with transcript) and 3 orders of magnitude more resistant to desiccation (0.005% survival of

  20. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

    PubMed Central

    Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

    2015-01-01

    ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many

  1. Study of Lateral Gene Transfer in an Acid Mine Drainage Community Enabled by Comparative Genomics

    NASA Astrophysics Data System (ADS)

    Hugenholtz, P.; Croft, L.; Tyson, G. W.; Baker, B. J.; Detter, C.; Richardson, P. M.; Banfield, J. F.

    2002-12-01

    Lateral gene transfer (LGT) is thought to play a crucial role in the ecology and evolution of prokaryotes. We are investigating the role of LGT in an acid mine drainage community hosted in a pyrite-dominated metal sulfide deposit at the Richmond mine at Iron Mountain, CA. Due to biologically-mediated pyrite dissolution, the prevailing conditions within the mine are extremely low pH (< 1.0), very high ionic concentrations (molar concentrations of iron sulfate and mM concentrations of arsenic, copper and zinc), and moderate to high temperatures (30 to >50 C). These conditions are thought to largely isolate the community from potential external gene donors since naked DNA, phage and prokaryotes native to neutral pH habitats do not persist at pH <1.0 precluding an external influx of genes by transformation, transduction and conjugation, respectively. Microbial communities exist in several distinct habitats within Richmond mine including biofilms (subaqueous slime streamers and subaerial slimes) and cells attached directly to pyrite granules. This, however, belies an unusual simplicity in community composition. All communities investigated to date comprise only a handful of phylogenetically distinct organisms, typically dominated by the iron-oxidizing genera Leptospirillum and Ferroplasma. We have undertaken a community genomics analysis of a subaerial biofilm dominated by a Leptospirillum population to facilitate the study of LGT in this type of environment. The genome of Ferroplasma acidarmanus fer1, a minor component of the target community (but a major component of other Richmond mine communities), has been sequenced. Comparative genome analyses indicate that F. acidarmanus and the ancestor of two acidophilic Thermoplasma species belonging to the Euryarchaeota have traded many genes with phylogenetically remote acidophilic Sulfolobus species (Crenarchaeota). The putatively transferred sets of Sulfolobus genes in Ferroplasma and the Thermoplasma ancestor are distinct

  2. Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3.

    PubMed

    Akbas, F; Aydin, Z

    2012-01-01

    Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels. PMID:22576912

  3. Expression of genes controlling unsaturated fatty acids biosynthesis and oil deposition in developing seeds of Sacha inchi (Plukenetia volubilis L.).

    PubMed

    Wang, Xiaojuan; Liu, Aizhong

    2014-10-01

    Sacha inchi (Plukenetia volubilis L., Euphorbiaceae) seed oil is rich in α-linolenic acid, a kind of n-3 fatty acids with many health benefits. To discover the mechanism underlying α-linolenic acid accumulation in sacha inchi seeds, preliminary research on sacha inchi seed development was carried out from one week after fertilization until maturity, focusing on phenology, oil content, and lipid profiles. The results suggested that the development of sacha inchi seeds from pollination to mature seed could be divided into three periods. In addition, investigations on the effect of temperature on sacha inchi seeds showed that total oil content decreased in the cool season, while unsaturated fatty acid and linolenic acid concentrations increased. In parallel, expression profiles of 17 unsaturated fatty acid related genes were characterized during seed development and the relationships between gene expression and lipid/unsaturated fatty acid accumulation were discussed. PMID:25119487

  4. Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa.

    PubMed Central

    Hungerer, C; Troup, B; Römling, U; Jahn, D

    1995-01-01

    The general tetrapyrrole precursor 5-aminolevulinic acid is formed in bacteria via two different biosynthetic pathways. Members of the alpha group of the proteobacteria use 5-aminolevulinic acid synthase for the condensation of succinyl-coenzyme A and glycine, while other bacteria utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-dependent pathway, involving the enzymes glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by hemL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vinelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginosa was cloned by complementation of an Escherichia coli hemA mutant. The hemA gene was mapped to the SpeI A fragment and the DpnIL fragment of the P. aeruginosa chromosome corresponding to min 24.1 to 26.8. The cloned hemA gene, coding for a protein of 423 amino acids with a calculated molecular mass of 46,234 Da, forms an operon with the gene for protein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre codons. Since the cloned hemA gene did not possess one of the appropriate stop codons, an autoregulatory mechanism such as that postulated for the enterobacterial system was ruled out. Three open reading frames of unknown function transcribed in the opposite direction to the hemA gene were found. hemM/orf1 and orf2 were found to be homologous to open reading frames located in the 5' region of enterobacterial hemA genes. Utilization of both transcription start sites was changed in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr analog), indicating the involvement of the transcription factor in hemA expression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined

  5. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids. PMID:26945769

  6. Effect of dietary fatty acids on inflammatory gene expression in healthy humans.

    PubMed

    Weaver, Kelly L; Ivester, Priscilla; Seeds, Michael; Case, L Douglas; Arm, Jonathan P; Chilton, Floyd H

    2009-06-01

    Over the past 100 years, changes in the food supply in Western nations have resulted in alterations in dietary fatty acid consumption, leading to a dramatic increase in the ratio of omega-6 (omega6) to omega3 polyunsaturated fatty acids (PUFA) in circulation and in tissues. Increased omega6/omega3 ratios are hypothesized to increase inflammatory mediator production, leading to higher incidence of inflammatory diseases, and may impact inflammatory gene expression. To determine the effect of reducing the omega6/omega3 ratio on expression of inflammatory pathway genes in mononuclear cells, healthy humans were placed on a controlled diet for 1 week, then given fish oil and borage oil for an additional 4 weeks. Serum and neutrophil fatty acid composition and ex vivo leukotriene B(4) production from stimulated neutrophils were measured at the start and end of the supplementation period and after a 2-week washout. RNA was isolated from mononuclear cells and expression of PI3K, Akt, NFkappaB, and inflammatory cytokines was measured by real-time PCR. A marked increase was seen in serum and neutrophil levels of long-chain omega3 PUFA concomitant with a reduction in the omega6/omega3 PUFA ratio (40%). The ex vivo capacity of stimulated neutrophils to produce leukotriene B(4) was decreased by 31%. Expression of PI3Kalpha and PI3Kgamma and the quantity of PI3Kalpha protein in mononuclear cells was reduced after supplementation, as was the expression of several proinflammatory cytokines. These data reveal that PUFA may exert their clinical effects via their capacity to regulate the expression of signal transduction genes and genes for proinflammatory cytokines. PMID:19359242

  7. Hepatocyte nuclear factor-4alpha and bile acids regulate human concentrative nucleoside transporter-1 gene expression.

    PubMed

    Klein, Kerstin; Kullak-Ublick, Gerd A; Wagner, Martin; Trauner, Michael; Eloranta, Jyrki J

    2009-04-01

    The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogs used in anticancer and antiviral therapy. Thus expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4alpha (HNF-4alpha) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4alpha in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4alpha specifically binds to two conserved direct repeat-1 motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha further increased, whereas the bile acid-inducible corepressor small heterodimer partner reduced, HNF-4alpha-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice. PMID:19228884

  8. Influence of phenolic acids on indole acetic acid production and on the type III secretion system gene transcription in food-associated Pseudomonas fluorescens KM05.

    PubMed

    Myszka, Kamila; Schmidt, Marcin T; Olejnik-Schmidt, Agnieszka K; Leja, Katarzyna; Czaczyk, Katarzyna

    2014-12-01

    The purpose of these investigations was to evaluate the reduction capability of phenolic acids (ferulic, chlorogenic, gallic, and p-coumaric acids) on indole acetic acid synthesis by food-associated Pseudomonas fluorescens KM05. Specific genetic primer for the type III secretion system (TTSS) in P. fluorescens KM05 was designed and the influence of phenolic acids on its expression was investigated. In the work the ferulic and chlorogenic acids at the concentration of 0.02 and 0.04 μg/ml affected on bacterial growth pattern and the signal molecules production. The phenolic acids, that were appreciable effective against P. fluorescens KM05 indole acetic acid production, significantly suppressed TTSS gene. PMID:24994472

  9. Intron-exon organization of the gene for the multifunctional animal fatty acid synthase.

    PubMed Central

    Amy, C M; Williams-Ahlf, B; Naggert, J; Smith, S

    1992-01-01

    The complete intron-exon organization of the gene encoding a multifunctional mammalian fatty acid synthase has been elucidated, and specific exons have been assigned to coding sequences for the component domains of the protein. The rat gene is interrupted by 42 introns and the sequences bordering the splice-site junctions universally follow the GT/AG rule. However, of the 41 introns that interrupt the coding region of the gene, 23 split the reading frame in phase I, 14 split the reading frame in phase 0, and only 4 split the reading frame in phase II. Remarkably, 46% of the introns interrupt codons for glycine. With only one exception, boundaries between the constituent enzymes of the multifunctional polypeptide coincide with the location of introns in the gene. The significance of the predominance of phase I introns, the almost uniformly short length of the 42 introns and the overall small size of the gene, is discussed in relation to the evolution of multifunctional proteins. Images PMID:1736293

  10. Expression of salicylic acid-related genes in Brassica oleracea var. capitata during Plasmodiophora brassicae infection.

    PubMed

    Manoharan, Ranjith Kumar; Shanmugam, Ashokraj; Hwang, Indeok; Park, Jong-In; Nou, Ill-Sup

    2016-06-01

    Brassica oleracea var. capitata (cabbage) is an important vegetable crop in Asian countries such as Korea, China, and Japan. Cabbage production is severely affected by clubroot disease caused by the soil-borne plant pathogen Plasmodiophora brassicae. During clubroot development, methyl salicylate (MeSA) is biosynthesized from salicylic acid (SA) by methyltransferase. In addition, methyl salicylate esterase (MES) plays a major role in the conversion of MeSA back into free SA. The interrelationship between MES and methytransferases during clubroot development has not been fully explored. To begin to examine these relationships, we investigated the expression of MES genes in disease-susceptible and disease-resistant plants during clubroot development. We identified three MES-encoding genes potentially involved in the defense against pathogen attack. We found that SS1 was upregulated in both the leaves and roots of B. oleracea during P. brassicae infection. These results support the conclusion that SA biosynthesis is suppressed during pathogen infection in resistant plants. We also characterized the expression of a B. oleracea BSMT gene, which appears to be involved in glycosylation rather than MeSA biosynthesis. Our results provide insight into the functions and interactions of genes for MES and methyltransferase during infection. Taken together, our findings indicate that MES genes are important candidates for use to control clubroot diseases. PMID:27171821

  11. Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene.

    PubMed Central

    Cabrera, H L; Barrio, R; Arribas, C

    1992-01-01

    Ubiquitin belongs to a multigene family. In Drosophila two members of this family have been previously described. We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library. This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein. There are no introns interrupting the coding sequence. Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis. The present results show that the 5' regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions. Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified. Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis. The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries. In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1381584

  12. The GLUT9 Gene Is Associated with Serum Uric Acid Levels in Sardinia and Chianti Cohorts

    PubMed Central

    Maschio, Andrea; Busonero, Fabio; Usala, Gianluca; Mulas, Antonella; Lai, Sandra; Dei, Mariano; Orrù, Marco; Albai, Giuseppe; Bandinelli, Stefania; Schlessinger, David; Lakatta, Edward; Scuteri, Angelo; Najjar, Samer S; Guralnik, Jack; Naitza, Silvia; Crisponi, Laura; Cao, Antonio; Abecasis, Gonçalo; Ferrucci, Luigi; Uda, Manuela; Chen, Wei-Min; Nagaraja, Ramaiah

    2007-01-01

    High serum uric acid levels elevate pro-inflammatory–state gout crystal arthropathy and place individuals at high risk for cardiovascular morbidity and mortality. Genome-wide scans in the genetically isolated Sardinian population identified variants associated with serum uric acid levels as a quantitative trait. They mapped within GLUT9, a Chromosome 4 glucose transporter gene predominantly expressed in liver and kidney. SNP rs6855911 showed the strongest association (p = 1.84 × 10−16), along with eight others (p = 7.75 × 10−16 to 6.05 × 10−11). Individuals homozygous for the rare allele of rs6855911 (minor allele frequency = 0.26) had 0.6 mg/dl less uric acid than those homozygous for the common allele; the results were replicated in an unrelated cohort from Tuscany. Our results suggest that polymorphisms in GLUT9 could affect glucose metabolism and uric acid synthesis and/or renal reabsorption, influencing serum uric acid levels over a wide range of values. PMID:17997608

  13. Aminoaciduria and altered renal expression of luminal amino acid transporters in mice lacking novel gene collectrin.

    PubMed

    Malakauskas, Sandra M; Quan, Hui; Fields, Timothy A; McCall, Shannon J; Yu, Ming-Jiun; Kourany, Wissam M; Frey, Campbell W; Le, Thu H

    2007-02-01

    Defects in renal proximal tubule transport manifest in a number of human diseases. Although variable in clinical presentation, disorders such as Hartnup disease, Dent's disease, and Fanconi syndrome are characterized by wasting of solutes commonly recovered by the proximal tubule. One common feature of these disorders is aminoaciduria. There are distinct classes of amino acid transporters located in the apical and basal membranes of the proximal tubules that reabsorb >95% of filtered amino acids, yet few details are known about their regulation. We present our physiological characterization of a mouse line with targeted deletion of the gene collectrin that is highly expressed in the kidney. Collectrin-deficient mice display a reduced urinary concentrating capacity due to enhanced solute clearance resulting from profound aminoaciduria. The aminoaciduria is generalized, characterized by loss of nearly every amino acid, and results in marked crystalluria. Furthermore, in the kidney, collectrin-deficient mice have decreased plasma membrane populations of amino acid transporter subtypes B(0)AT1, rBAT, and b(0,+)AT, as well as altered cellular distribution of EAAC1. Our data suggest that collectrin is a novel mediator of renal amino acid transport and may provide further insight into the pathogenesis of a number of human disease correlates. PMID:16985211

  14. Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria

    PubMed Central

    Bron, Peter A.; Benchimol, Marcos G.; Lambert, Jolanda; Palumbo, Emmanuelle; Deghorain, Marie; Delcour, Jean; de Vos, Willem M.; Kleerebezem, Michiel; Hols, Pascal

    2002-01-01

    Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of d-alanine and l-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Δalr) showed auxotrophy for d-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented d-alanine auxotrophy in the L. plantarum Δalr and L. lactis Δalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to d-cycloserine, a competitive inhibitor of Alr (600 and 200 μg/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that d-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Δalr. The resulting strain could grow in the absence of d-alanine only when expression of the alr gene was induced with nisin. PMID:12406763

  15. Development of marker genes for jasmonic acid signaling in shoots and roots of wheat.

    PubMed

    Liu, Hongwei; Carvalhais, Lilia Costa; Kazan, Kemal; Schenk, Peer M

    2016-05-01

    The jasmonic acid (JA) signaling pathway plays key roles in a diverse array of plant development, reproduction, and responses to biotic and abiotic stresses. Most of our understanding of the JA signaling pathway derives from the dicot model plant Arabidopsis thaliana, while corresponding knowledge in wheat is somewhat limited. In this study, the expression of 41 genes implicated in the JA signaling pathway has been assessed on 10 day-old bread wheat seedlings, 24 h, 48 h, and 72 h after methyl-jasmonate (MeJA) treatment using quantitative real-time PCR. The examined genes have been previously reported to be involved in JA biosynthesis and catabolism, JA perception and signaling, and pathogen defense in wheat shoots and roots. This study provides evidence to suggest that the effect of MeJA treatment is more prominent in shoots than roots of wheat seedlings, and substantial regulation of the JA pathway-dependent defense genes occurs at 72 h after MeJA treatment. Results show that the expression of 22 genes was significantly affected by MeJA treatment in wheat shoots. However, only PR1.1 and PR3 were significantly differentially expressed in wheat roots, both at 24 h post-MeJA treatment, with other genes showing large variation in their gene expression in roots. While providing marker genes on JA signaling in wheat, future work may focus on elucidating the regulatory function of JA-modulated transcription factors, some of which have well-studied potential orthologs in Arabidopsis. PMID:27115051

  16. The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.

    PubMed

    Shulami, S; Gat, O; Sonenshein, A L; Shoham, Y

    1999-06-01

    A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of alpha-D-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular alpha-glucuronidase (aguA) and a beta-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

  17. Molecular and biochemical characterization of the jasmonic acid methyltransferase gene from black cottonwood (Populus trichocarpa)

    SciTech Connect

    Zhao, Nan; Yao, Jianzhuang; Chaiprasongsuk, Minta; Li, Guanglin; Guan, Ju; Tschaplinski, Timothy J; Guo, Hong; Chen, Feng

    2013-01-01

    Methyl jasmonate is a metabolite known to be produced by many plants and has roles in diverse biological processes. It is biosynthesized by the action of S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH family of methyltransferases. Herein is reported the isolation and biochemical characterization of a JMT gene from black cottonwood (Populus trichocarpa). The genome of P. trichocarpa contains 28 SABATH genes (PtSABATH1 to PtSABATH28). Recombinant PtSABATH3 expressed in Escherichia coli showed the highest level of activity with jasmonic acid (JA) among carboxylic acids tested. It was therefore renamed PtJMT1. PtJMT1 also displayed activity with benzoic acid (BA), with which the activity was about 22% of that with JA. PtSABATH2 and PtSABATH4 were most similar to PtJMT1 among all PtSABATHs. However, neither of them had activity with JA. The apparent Km values of PtJMT1 using JA and BA as substrate were 175 lM and 341 lM, respectively. Mutation of Ser-153 and Asn-361, two residues in the active site of PtJMT1, to Tyr and Ser respectively, led to higher specific activity with BA than with JA. Homology-based structural modeling indicated that substrate alignment, in which Asn-361 is involved, plays a role in determining the substrate specificity of PtJMT1. In the leaves of young seedlings of black cottonwood, the expression of PtJMT1 was induced by plant defense signal molecules methyl jasmonate and salicylic acid and a fungal elicitor alamethicin, suggesting that PtJMT1 may have a role in plant defense against biotic stresses. Phylogenetic analysis suggests that PtJMT1 shares a common ancestor with the Arabidopsis JMT, and functional divergence of these two apparent JMT orthologs has occurred since the split of poplar and Arabidopsis lineages.

  18. Folic-Acid-Targeted Self-Assembling Supramolecular Carrier for Gene Delivery.

    PubMed

    Liao, Rongqiang; Yi, Shouhui; Liu, Manshuo; Jin, Wenling; Yang, Bo

    2015-07-27

    A targeting gene carrier for cancer-specific delivery was successfully developed through a "multilayer bricks-mortar" strategy. The gene carrier was composed of adamantane-functionalized folic acid (FA-AD), an adamantane-functionalized poly(ethylene glycol) derivative (PEG-AD), and β-cyclodextrin-grafted low-molecular-weight branched polyethylenimine (PEI-CD). Carriers produced by two different self-assembly schemes, involving either precomplexation of the PEI-CD with the FA-AD and PEG-AD before pDNA condensation (Method A) or pDNA condensation with the PEI-CD prior to addition of the FA-AD and PEG-AD to engage host-guest complexation (Method B) were investigated for their ability to compact pDNA into nanoparticles. Cell viability studies show that the material produced by the Method A assembly scheme has lower cytotoxicity than branched PEI 25 kDa (PEI-25KD) and that the transfection efficiency is maintained. These findings suggest that the gene carrier, based on multivalent host-guest interactions, could be an effective, targeted, and low-toxicity carrier for delivering nucleic acid to target cells. PMID:26032689

  19. Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation

    PubMed Central

    Gao, Wei-wei; Xiao, Rong-quan; Peng, Bing-ling; Xu, Huan-teng; Shen, Hai-feng; Huang, Ming-feng; Shi, Tao-tao; Yi, Jia; Zhang, Wen-juan; Wu, Xiao-nan; Gao, Xiang; Lin, Xiang-zhi; Dorrestein, Pieter C.; Rosenfeld, Michael G.; Liu, Wen

    2015-01-01

    Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control. PMID:26080448

  20. General roles of abscisic and jasmonic acids in gene activation as a result of mechanical wounding.

    PubMed Central

    Hildmann, T; Ebneth, M; Peña-Cortés, H; Sánchez-Serrano, J J; Willmitzer, L; Prat, S

    1992-01-01

    Exogenous application of abscisic acid (ABA) has been shown to induce a systemic pattern of proteinase inhibitor II (pin2) mRNA accumulation identical to that induced by mechanical wounding. Evidence is presented that the ABA-specific response is not restricted to pin2 genes but appears to be part of a general reaction to wound stress. Four other wound-induced, ABA-responsive genes that encode two additional proteinase inhibitors, the proteolytic enzyme leucine aminopeptidase, and the biosynthetic enzyme threonine deaminase were isolated from potato plants. Wounding or treatment with ABA resulted in a pattern of accumulation of these mRNAs very similar to that of pin2. ABA-deficient plants did not accumulate any of the mRNAs upon wounding, although they showed normal levels of expression upon ABA treatment. Also, application of methyl jasmonate (MeJA) induced a strong accumulation of these transcripts, both in wild-type and in ABA-deficient plants, thus supporting a role for jasmonic acid as an intermediate in the signaling pathway that leads from ABA accumulation in response to wounding to the transcriptional activation of the genes. PMID:1392612

  1. Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage.

    PubMed

    Mirete, Salvador; de Figueras, Carolina G; González-Pastor, Jose E

    2007-10-01

    Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment. PMID:17675438

  2. Bidirectional CLOCK/BMAL1-dependent circadian gene regulation by retinoic acid in vitro

    SciTech Connect

    Shirai, Hidenori; Oishi, Katsutaka; Ishida, Norio . E-mail: n.ishida@aist.go.jp

    2006-12-15

    A central circadian clock located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus entrains peripheral clocks through both neural and humoral factors. Although candidates for entrainment factors have been described, their details remain obscure. Here, we screened ligands for nuclear receptors that affect CLOCK/BMAL1-dependent transactivation of the mouse Period1 (mPer1) gene in NIH3T3 cells. We found that retinoic acids (RAs) significantly up-regulate mPer1 expression in an E-box-dependent manner. We also found that RAs up-regulate the expression of other E-box-dependent circadian genes such as mPer2, arginine vasopressin (mAVP), and peroxisome proliferator-activated receptor {alpha} (mPPAR{alpha}). Surprisingly, the effect of RAs on CLOCK/BMAL1 (E-box)-dependent mRNA expression was bidirectional and depended on the presence of exogenous retinoic acid receptor {alpha} (RAR{alpha}). These results suggest that RAs regulate the CLOCK/BMAL1-dependent transcription of circadian genes in a complex manner.

  3. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

    PubMed

    Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

    2016-01-01

    The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed. PMID:27187420

  4. Niche specific amino acid features within the core genes of the genus Shewanella.

    PubMed

    Banerjee, Rachana; Mukhopadhyay, Subhasis

    2012-01-01

    Shewanella species are found to dwell in various ecological niches. The widespread habitation where they live requires specific adaptations. Recent advances in genomic approaches, such as in sequencing technologies, generate huge amount of genomic data that lend support towards understanding the microbial evolution and diversity through comparative study. In this manuscript, we discuss a comparative analysis of core genes of phylogenetically related twelve members from the genus Shewanella. Phylogenetic analysis based on the core genes, differentiated two subgroups of the genus, one group comprises of species characterized as highpressure cold-adapted while the other group is characterized as mesophilic pressure-sensitive species. By analyzing the differences of amino acid composition of these two groups, we have identified the specific trend of amino acid usage that has been adopted by the psychro-peizo-tolerant Shewanella species. The functional categories have also been recognized which are responsible for rendering the particular amino acid compositional pattern in psychropeizophilic Shewanella species facilitating their niche adaptation. PMID:23144554

  5. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

    PubMed Central

    Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

    2016-01-01

    The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed. PMID:27187420

  6. Gene expression signature of DMBA-induced hamster buccal pouch carcinomas: modulation by chlorophyllin and ellagic acid.

    PubMed

    Vidya Priyadarsini, Ramamurthi; Kumar, Neeraj; Khan, Imran; Thiyagarajan, Paranthaman; Kondaiah, Paturu; Nagini, Siddavaram

    2012-01-01

    Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy. PMID:22485181

  7. Gene Expression Signature of DMBA-Induced Hamster Buccal Pouch Carcinomas: Modulation by Chlorophyllin and Ellagic Acid

    PubMed Central

    Vidya Priyadarsini, Ramamurthi; Kumar, Neeraj; Khan, Imran; Thiyagarajan, Paranthaman; Kondaiah, Paturu; Nagini, Siddavaram

    2012-01-01

    Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy. PMID:22485181

  8. Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores.

    PubMed Central

    Connors, M J; Mason, J M; Setlow, P

    1986-01-01

    Three Bacillus subtilis genes (termed sspA, sspB, and sspD) which code for small, acid-soluble spore proteins (SASPs) have been cloned, and their complete nucleotide sequence has been determined. The amino acid sequences of the SASPs coded for by these genes are similar to each other and to those of the SASP-1 of B. subtilis (coded for by the sspC gene) and the SASP-A/C family of B. megaterium. The sspA and sspB genes are expressed only in sporulation, in parallel with each other and with the sspC gene. Two regions upstream of the postulated transcription start sites for the sspA and B genes have significant homology with the analogous regions of the sspC gene and the SASP-A/C gene family. Purification of two of the three major B, subtilis SASPs (alpha and beta) and determination of their amino-terminal sequences indicated that the sspA gene codes for SASP-alpha and that the sspB gene codes for SASP-beta. This was confirmed by the introduction of deletion mutations into the cloned sspA and sspB genes and transfer of these deletions into the B. subtilis chromosome with concomitant loss of the wild-type gene. Images PMID:3009398

  9. Genomic Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185.

    PubMed

    Zhao, Xianming; Dauenpen, Meesapyodsuk; Qu, Cunmin; Qiu, Xiao

    2016-09-01

    Thraustochytrium sp. 26185 is a marine protist that can produce a large amount of docosahexaenoic acid (DHA, 22:6n-3), an ω3 very long chain polyunsaturated fatty acid (VLCPUFA) of nutritional importance. However, the mechanism of how this fatty acid is synthesized and assembled into the storage lipid triacylglycerol is unclear. Here we report sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Genome sequencing produced a total of 2,418,734,139 bp clean sequences with about 62 fold genome coverage. Annotation of the genome sequences revealed 10,797 coding genes. Among them, 10,216 genes could be assigned into 25 KOG classes where 451 genes were specifically assigned to the group of lipid transport and metabolism. Detailed analysis of these genes revealed co-existence of both aerobic pathway and anaerobic pathways for the biosynthesis of DHA in this species. However, in the aerobic pathway, a key gene encoding stearate Δ9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. Genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine:diacylglycerol cholinephosphotransferase, an important enzyme in bridging two types of glycerolipids, diacylglycerols (DAG) and phosphatidylcholines (PtdCho). These results shed new insight on the biosynthesis and assembly of VLCPUFA in the Thraustochytrium. PMID:27514858

  10. Enhanced Acid Tolerance in Bifidobacterium longum by Adaptive Evolution: Comparison of the Genes between the Acid-Resistant Variant and Wild-Type Strain.

    PubMed

    Jiang, Yunyun; Ren, Fazheng; Liu, Songling; Zhao, Liang; Guo, Huiyuan; Hou, Caiyun

    2016-03-28

    Acid stress can affect the viability of probiotics, especially Bifidobacterium. This study aimed to improve the acid tolerance of Bifidobacterium longum BBMN68 using adaptive evolution. The stress response, and genomic differences of the parental strain and the variant strain were compared by acid stress. The highest acid-resistant mutant strain (BBMN68m) was isolated from more than 100 asexual lines, which were adaptive to the acid stress for 10(th), 20(th), 30(th), 40(th), and 50(th) repeats, respectively. The variant strain showed a significant increase in acid tolerance under conditions of pH 2.5 for 2 h (from 7.92 to 4.44 log CFU/ml) compared with the wildtype strain (WT, from 7.87 to 0 log CFU/ml). The surface of the variant strain was also smoother. Comparative whole-genome analysis showed that the galactosyl transferase D gene (cpsD, bbmn68_1012), a key gene involved in exopolysaccharide (EPS) synthesis, was altered by two nucleotides in the mutant, causing alteration in amino acids, pI (from 8.94 to 9.19), and predicted protein structure. Meanwhile, cpsD expression and EPS production were also reduced in the variant strain (p < 0.05) compared with WT, and the exogenous WT-EPS in the variant strain reduced its acid-resistant ability. These results suggested EPS was related to acid responses of BBMN68. PMID:26608165

  11. Effect of maternal folic acid supplementation on hepatic one-carbon unit associated gene expressions in newborn piglets.

    PubMed

    Liu, Jing-Bo; Chen, Dai-Wen; Yu, Bing; Mao, Xiang-bing

    2011-08-01

    Intrauterine growth retardation (IUGR) induces alterations to hepatic gene expressions which might program poor postnatal growth and health status. Maternal folic acid supplementation was administered in gilt diets to test whether hepatic mRNA expressions of some important genes induced by IUGR could be rescued by folic acid supplementation. Thirty-two Yorkshire gilts were allotted to two treatment groups of control (C folic acid 1.3 mg/kg) or folic acid supplementation (FS folic acid 30 mg/kg) after mating, to study the effects of maternal folic acid supplementation on the mRNA expression of methionine adenosyltransferase (MAT), cystathionine-β-synthase (CBS), methylenetetrahydrofolate reductase (MTHFR), DNA methyltransferase1 (DNMT1), peroxisomal proliferator-activated receptor (PPARγ), glucocorticoid receptor (GR), obesity receptor (ob-R) and Acyl-CoA oxidase (AOX) in the liver of IUGR and NBW piglets. Blood and liver samples were collected for determinations of serum folic acid and gene expressions. The total number of born piglets, number of piglets born alive, average birth weight and 21 days average weight were not affected by dietary treatment (P>0.05), and serum folic acid concentration of piglets was greater in FS than C groups (P<0.05). Real-time PCR indicated that gene expression of MAT1A, MAT2A and DNMT1 were lower in IUGR piglets but could be elevated by maternal folic acid supplementation. Transcript expression levels of PPARγ, GR and AOX were higher in IUGR piglets, but were decreased to the level of normal piglets by maternal folic acid supplementation. Our results suggested that maternal folic acid supplementation be an effective way to rescue the gene expressions negatively induced by IUGR. PMID:21108044

  12. Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose1[W

    PubMed Central

    Le Roy, Katrien; Vergauwen, Rudy; Struyf, Tom; Yuan, Shuguang; Lammens, Willem; Mátrai, Janka; De Maeyer, Marc; Van den Ende, Wim

    2013-01-01

    Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs. PMID:23447526

  13. Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

    PubMed

    Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

    2015-09-30

    Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways. PMID:26343778

  14. Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes.

    PubMed

    Yuan, Yuan; Wang, Zhouyong; Jiang, Chao; Wang, Xumin; Huang, Luqi

    2014-01-25

    Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species. PMID:23085319

  15. Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency.

    PubMed

    Ichi, Ikuyo; Kono, Nozomu; Arita, Yuka; Haga, Shizuka; Arisawa, Kotoko; Yamano, Misato; Nagase, Mana; Fujiwara, Yoko; Arai, Hiroyuki

    2014-01-01

    In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9. PMID:24184513

  16. Phase Variation in the Helicobacter pylori Phospholipase A Gene and Its Role in Acid Adaptation

    PubMed Central

    Tannæs, Tone; Dekker, Niek; Bukholm, Geir; Bijlsma, Jetta J. E.; Appelmelk, Ben J.

    2001-01-01

    Previously, we have shown that Helicobacter pylori can spontaneously and reversibly change its membrane lipid composition, producing variants with low or high content of lysophospholipids. The “lyso” variant contains a high percentage of lysophospholipids, adheres better to epithelial cells, and releases more proteins such as urease and VacA, compared to the “normal” variant, which has a low content of lysophospholipids. Prolonged growth of the normal variant at pH 3.5, but not under neutral conditions, leads to enrichment of lyso variant colonies, suggesting that the colony switch is relevant to acid adaptation. In this study we show that the change in membrane lipid composition is due to phase variation in the pldA gene. A change in the (C) tract length of this gene results in reversible frameshifts, translation of a full-length or truncated pldA, and the production of active or inactive outer membrane phospholipase A (OMPLA). The role of OMPLA in determining the colony morphology was confirmed by the construction of an OMPLA-negative mutant. Furthermore, variants with an active OMPLA were able to survive acidic conditions better than variants with the inactive form. This explains why the lyso variant is selected at low pH. Our studies demonstrate that phase variation in the pldA gene, resulting in an active form of OMPLA, is important for survival under acidic conditions. We also demonstrated the active OMPLA genotype in fresh isolates of H. pylori from patients referred to gastroscopy for dyspepsia. PMID:11705905

  17. Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli.

    PubMed Central

    Gibello, A; Ferrer, E; Sanz, J; Martin, M

    1995-01-01

    The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin. PMID:8534083

  18. Cationic lioposomes with folic acid as targeting ligand for gene delivery.

    PubMed

    Cui, Shao-Hui; Zhi, De-Fu; Zhao, Yi-Nan; Chen, Hui-Ying; Meng, Yao; Zhang, Chuan-Min; Zhang, Shu-Biao

    2016-08-15

    In our previous Letter, we have carried out the synthesis of a novel DDCTMA cationic lipid which was formulated with DOPE for gene delivery. Herein, we used folic acid (FA) as targeting ligand and cholesterol (Chol) as helper lipid instead of DOPE for enhancing the stability of the liposomes. These liposomes were characterized by dynamic laser scattering (DLS), transmission electron microscopy (TEM) and agarose gel electrophoresis assays of pDNA binding affinity. The lipoplexes were prepared by using different weight ratios of DDCTMA/Chol (1:1, 2:1, 3:1, 4:1) liposomes and different concentrations of FA (50-200μg/mL) combining with pDNA. The transfection efficiencies of the lipoplexes were evaluated using pGFP-N2 and pGL3 plasmid DNA against NCI-H460 cells in vitro. Among them, the optimum gene transfection efficiency with DDCTMA/Chol (3:1)/FA (100μg/mL) was obtained. The results showed that FA could improve the gene transfection efficiencies of DDCTMA/Chol cationic liposome. Our results also convincingly demonstrated FA (100μg/mL)-coated DDCTMA/Chol (3:1) cationic liposome could serve as a promising candidate for the gene delivery. PMID:27426864

  19. Genome-Wide Identification, Classification, and Expression Analysis of Amino Acid Transporter Gene Family in Glycine Max

    PubMed Central

    Cheng, Lin; Yuan, Hong-Yu; Ren, Ren; Zhao, Shi-Qi; Han, Ya-Peng; Zhou, Qi-Ying; Ke, Dan-Xia; Wang, Ying-Xiang; Wang, Lei

    2016-01-01

    Amino acid transporters (AATs) play important roles in transporting amino acid across cellular membranes and are essential for plant growth and development. To date, the AAT gene family in soybean (Glycine max L.) has not been characterized. In this study, we identified 189 AAT genes from the entire soybean genomic sequence, and classified them into 12 distinct subfamilies based upon their sequence composition and phylogenetic positions. To further investigate the functions of these genes, we analyzed the chromosome distributions, gene structures, duplication patterns, phylogenetic tree, tissue expression patterns of the 189 AAT genes in soybean. We found that a large number of AAT genes in soybean were expanded via gene duplication, 46 and 36 GmAAT genes were WGD/segmental and tandemly duplicated, respectively. Further comprehensive analyses of the expression profiles of GmAAT genes in various stages of vegetative and reproductive development showed that soybean AAT genes exhibited preferential or distinct expression patterns among different tissues. Overall, our study provides a framework for further analysis of the biological functions of AAT genes in either soybean or other crops. PMID:27148336

  20. Genome-Wide Identification, Classification, and Expression Analysis of Amino Acid Transporter Gene Family in Glycine Max.

    PubMed

    Cheng, Lin; Yuan, Hong-Yu; Ren, Ren; Zhao, Shi-Qi; Han, Ya-Peng; Zhou, Qi-Ying; Ke, Dan-Xia; Wang, Ying-Xiang; Wang, Lei

    2016-01-01

    Amino acid transporters (AATs) play important roles in transporting amino acid across cellular membranes and are essential for plant growth and development. To date, the AAT gene family in soybean (Glycine max L.) has not been characterized. In this study, we identified 189 AAT genes from the entire soybean genomic sequence, and classified them into 12 distinct subfamilies based upon their sequence composition and phylogenetic positions. To further investigate the functions of these genes, we analyzed the chromosome distributions, gene structures, duplication patterns, phylogenetic tree, tissue expression patterns of the 189 AAT genes in soybean. We found that a large number of AAT genes in soybean were expanded via gene duplication, 46 and 36 GmAAT genes were WGD/segmental and tandemly duplicated, respectively. Further comprehensive analyses of the expression profiles of GmAAT genes in various stages of vegetative and reproductive development showed that soybean AAT genes exhibited preferential or distinct expression patterns among different tissues. Overall, our study provides a framework for further analysis of the biological functions of AAT genes in either soybean or other crops. PMID:27148336

  1. Polyethylenimine-polyacrylic acid nanocomposites: Type of bonding does influence the gene transfer efficacy and cytotoxicity.

    PubMed

    Tripathi, Sushil K; Ahmadi, Zeba; Gupta, Kailash C; Kumar, Pradeep

    2016-04-01

    The main aim of the current study is to compare the physicochemical properties, cytotoxicity and gene-transfer ability of electrostatically and covalently linked nanocomposites of polyethylenimine (PEI) and polyacrylic acid (PAA) on mammalian cells. Two series of nanocomposites, ionic PEI-PAA (iPP) and covalent PEI-PAA (cPP), were synthesized by varying the amounts of polyacrylic acid (PAA). Physicochemical characterization revealed that iPP nanopcomposites were of bigger sized than cPP nanocomposites with zeta potential almost comparable. Nucleic acid binding assay displayed that iPP and cPP nanocomposites, having sufficient cationic charge, efficiently interacted with plasmid DNA and completely retarded its electrophoretic mobility on agarose gel. In vitro MTT assay showed slightly higher cell viability of cPP/pDNA complexes over their ionic counterparts. Both the series of nanocomposite/pDNA complexes exhibited considerably higher transfection efficacy compared to pDNA complexes of native bPEI and the standard transfection reagent, Lipofectamine, with cPP/pDNA complexes performed much better than iPP/pDNA complexes. Flow cytometry further confirmed these findings where cPP-4/pDNA complex showed transfection in ∼85% HEK293 cells, while iPP-2/pDNA complex transfected ∼67% HEK293 cells. Lipofectamine/pDNA and bPEI/pDNA complexes could transfect just ∼35% and ∼26% HEK293 cells. All these results demonstrate the superiority of covalently linked nanocomposites (cPP) which could be used as efficient carriers for nucleic acids in future gene delivery applications. PMID:26745638

  2. Iron-biofortification in rice by the introduction of three barley genes participated in mugineic acid biosynthesis with soybean ferritin gene

    PubMed Central

    Masuda, Hiroshi; Kobayashi, Takanori; Ishimaru, Yasuhiro; Takahashi, Michiko; Aung, May S.; Nakanishi, Hiromi; Mori, Satoshi; Nishizawa, Naoko K.

    2013-01-01

    Iron deficiency is a serious problem around the world, especially in developing countries. The production of iron-biofortified rice will help ameliorate this problem. Previously, expression of the iron storage protein, ferritin, in rice using an endosperm-specific promoter resulted in a two-fold increase in iron concentration in the resultant transgenic seeds. However, further over expression of ferritin did not produce an additional increase in the seed iron concentration, and symptoms of iron deficiency were noted in the leaves of the transgenic plants. In the present study, we aimed to further increase the iron concentration in rice seeds without increasing the sensitivity to iron deficiency by enhancing the uptake and transport of iron via a ferric iron chelator, mugineic acid. To this end, we introduced the soybean ferritin gene (SoyferH2) driven by two endosperm-specific promoters, along with the barley nicotianamine synthase gene (HvNAS1), two nicotianamine aminotransferase genes (HvNAAT-A and -B), and a mugineic acid synthase gene (IDS3) to enhance mugineic acid production in rice plants. A marker-free vector was utilized as a means of increasing public acceptance. Representative lines were selected from 102 transformants based on the iron concentration in polished seeds and ferritin accumulation in the seeds. These lines were grown in both commercially supplied soil (iron-sufficient conditions) and calcareous soil (iron-deficient conditions). Lines expressing both ferritin and mugineic acid biosynthetic genes showed signs of iron-deficiency tolerance in calcareous soil. The iron concentration in polished T3 seeds was increased by 4 and 2.5 times, as compared to that in non-transgenic lines grown in normal and calcareous soil, respectively. These results indicate that the concomitant introduction of the ferritin gene and mugineic acid biosynthetic genes effectively increased the seed iron level without causing iron sensitivity under iron-limited conditions

  3. Differential effects of omega-3 and omega-6 Fatty acids on gene expression in breast cancer cells.

    PubMed

    Hammamieh, Rasha; Chakraborty, Nabarun; Miller, Stacy-Ann; Waddy, Edward; Barmada, Mohsen; Das, Rina; Peel, Sheila A; Day, Agnes A; Jett, Marti

    2007-01-01

    Essential fatty acids have long been identified as possible oncogenic factors. Existing reports suggest omega-6 (omega-6) essential fatty acids (EFA) as pro-oncogenic and omega-3 (omega-3) EFA as anti-oncogenic factors. The omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), inhibit the growth of human breast cancer cells while the omega-6 fatty acids induces growth of these cells in animal models and cell lines. In order to explore likely mechanisms for the modulation of breast cancer cell growth by omega-3 and omega-6 fatty acids, we examined the effects of arachidonic acid (AA), linoleic acid (LA), EPA and DHA on human breast cancer cell lines using cDNA microarrays and quantitative polymerase chain reaction. MDA-MB-231, MDA-MB-435s, MCF-7 and HCC2218 cell lines were treated with the selected fatty acids for 6 and 24 h. Microarray analysis of gene expression profiles in the breast cancer cells treated with both classes of fatty acids discerned essential differences among the two classes at the earlier time point. The differential effects of omega-3 and omega-6 fatty acids on the breast cancer cells were lessened at the late time point. Data mining and statistical analyses identified genes that were differentially expressed between breast cancer cells treated with omega-3 and omega-6 fatty acids. Ontological investigations have associated those genes to a broad spectrum of biological functions, including cellular nutrition, cell division, cell proliferation, metastasis and transcription factors etc., and thus presented an important pool of biomarkers for the differential effect of omega-3 and omega-6EFAs. PMID:16823509

  4. Genetic alterations in fatty acid transport and metabolism genes are associated with metastatic progression and poor prognosis of human cancers.

    PubMed

    Nath, Aritro; Chan, Christina

    2016-01-01

    Reprogramming of cellular metabolism is a hallmark feature of cancer cells. While a distinct set of processes drive metastasis when compared to tumorigenesis, it is yet unclear if genetic alterations in metabolic pathways are associated with metastatic progression of human cancers. Here, we analyzed the mutation, copy number variation and gene expression patterns of a literature-derived model of metabolic genes associated with glycolysis (Warburg effect), fatty acid metabolism (lipogenesis, oxidation, lipolysis, esterification) and fatty acid uptake in >9000 primary or metastatic tumor samples from the multi-cancer TCGA datasets. Our association analysis revealed a uniform pattern of Warburg effect mutations influencing prognosis across all tumor types, while copy number alterations in the electron transport chain gene SCO2, fatty acid uptake (CAV1, CD36) and lipogenesis (PPARA, PPARD, MLXIPL) genes were enriched in metastatic tumors. Using gene expression profiles, we established a gene-signature (CAV1, CD36, MLXIPL, CPT1C, CYP2E1) that strongly associated with epithelial-mesenchymal program across multiple cancers. Moreover, stratification of samples based on the copy number or expression profiles of the genes identified in our analysis revealed a significant effect on patient survival rates, thus confirming prominent roles of fatty acid uptake and metabolism in metastatic progression and poor prognosis of human cancers. PMID:26725848

  5. Genetic alterations in fatty acid transport and metabolism genes are associated with metastatic progression and poor prognosis of human cancers

    PubMed Central

    Nath, Aritro; Chan, Christina

    2016-01-01

    Reprogramming of cellular metabolism is a hallmark feature of cancer cells. While a distinct set of processes drive metastasis when compared to tumorigenesis, it is yet unclear if genetic alterations in metabolic pathways are associated with metastatic progression of human cancers. Here, we analyzed the mutation, copy number variation and gene expression patterns of a literature-derived model of metabolic genes associated with glycolysis (Warburg effect), fatty acid metabolism (lipogenesis, oxidation, lipolysis, esterification) and fatty acid uptake in >9000 primary or metastatic tumor samples from the multi-cancer TCGA datasets. Our association analysis revealed a uniform pattern of Warburg effect mutations influencing prognosis across all tumor types, while copy number alterations in the electron transport chain gene SCO2, fatty acid uptake (CAV1, CD36) and lipogenesis (PPARA, PPARD, MLXIPL) genes were enriched in metastatic tumors. Using gene expression profiles, we established a gene-signature (CAV1, CD36, MLXIPL, CPT1C, CYP2E1) that strongly associated with epithelial-mesenchymal program across multiple cancers. Moreover, stratification of samples based on the copy number or expression profiles of the genes identified in our analysis revealed a significant effect on patient survival rates, thus confirming prominent roles of fatty acid uptake and metabolism in metastatic progression and poor prognosis of human cancers. PMID:26725848

  6. Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.

    PubMed Central

    Abe, H; Yamaguchi-Shinozaki, K; Urao, T; Iwasaki, T; Hosokawa, D; Shinozaki, K

    1997-01-01

    In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) and requires protein biosynthesis for ABA-dependent gene expression. Previous experiments established that a 67-bp DNA fragment of the rd22 promoter is sufficient for dehydration- and ABA-induced gene expression and that this DNA fragment contains two closely located putative recognition sites for the basic helix-loop-helix protein MYC and one putative recognition site for MYB. We have carefully analyzed the 67-bp region of the rd22 promoter in transgenic tobacco plants and found that both the first MYC site and the MYB recognition site function as cis-acting elements in the dehydration-induced expression of the rd22 gene. A cDNA encoding a MYC-related DNA binding protein was isolated by DNA-ligand binding screening, using the 67-bp region as a probe, and designated rd22BP1. The rd22BP1 cDNA encodes a 68-kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors. The rd22BP1 protein binds specifically to the first MYC recognition site in the 67-bp fragment. RNA gel blot analysis revealed that transcription of the rd22BP1 gene is induced by dehydration stress and ABA treatment, and its induction precedes that of rd22. We have reported a drought- and ABA-inducible gene that encodes the MYB-related protein ATMYB2. In a transient transactivation experiment using Arabidopsis leaf protoplasts, we demonstrated that both the rd22BP1 and ATMYB2 proteins activate transcription of the rd22 promoter fused to the beta-glucuronidase reporter gene. These results indicate that both the rd22BP1 (MYC) and ATMYB2 (MYB) proteins function as transcriptional activators in the dehydration- and ABA-inducible expression of the rd22 gene. PMID:9368419

  7. Intracellular calcium-release and protein kinase C-activation stimulate sonic hedgehog gene expression during gastric acid secretion

    PubMed Central

    El-Zaatari, Mohamad; Zavros, Yana; Tessier, Art; Waghray, Meghna; Lentz, Steve; Gumucio, Deborah; Todisco, Andrea; Merchant, Juanita L.

    2010-01-01

    Introduction Hypochlorhydria during Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2+i-dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)-activation. Method We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1 (sHip-1), a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, EGTA+BAPTA, PKC-overexpressing adenoviruses, and PKC-inhibitors were used to modulate Ca2+i-release, PKC-activity and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H+/K+-β-cholera-toxin overexpressing mice (Ctox). Results Mice that expressed sHip-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression was also repressed in the hyperchlorhydric Ctox model with elevated cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca2+i-release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin- and carbachol-mediated release of Ca2+i induced Shh expression. Ca2+-chelation with BAPTA+EGTA reduced Shh expression. Overexpression of PKC-α, -β and -δ (but not PKC-ε) induced Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Conclusion Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca2+i-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin. PMID:20816837

  8. Gene expression profiles in zebrafish (Danio rerio) liver after acute exposure to okadaic acid.

    PubMed

    Zhang, Nai-sheng; Li, Hong-ye; Liu, Jie-sheng; Yang, Wei-dong

    2014-03-01

    Okadaic acid (OA), a main component of diarrheic shellfish poisoning (DSP) toxins, is a strong and specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. However, not all of the OA-induced effects can be explained by this phosphatase inhibition, and controversial results on OA are increasing. To provide clues on potential mechanisms of OA other than phosphatase inhibition, here, acute toxicity of OA was evaluated in zebrafish, and changes in gene expression in zebrafish liver tissues upon exposure to OA were observed by microarray. The i.p. ED50 (6 h) of OA on zebrafish was 1.54 μg OA/g body weight (bw). Among the genes analyzed on the zebrafish array, 55 genes were significantly up-regulated and 36 down-regulated in the fish liver tissue upon exposure to 0.176 μg OA/g bw (low-dose group, LD) compared with the low ethanol control (LE). However, there were no obvious functional clusters for them. On the contrary, fish exposure to 1.760 μg OA/g bw (high-dose group, HD) yielded a great number of differential expressed genes (700 up and 285 down) compared with high ethanol control (HE), which clustered in several functional terms such as p53 signaling pathway, Wnt signaling pathway, glutathione metabolism and protein processing in endoplasmic reticulum, etc. These genes were involved in protein phosphatase activity, translation factor activity, heat shock protein binding, as well as transmembrane transporter activity. Our findings may give some useful information on the pathways of OA-induced injury in fish. PMID:24637248

  9. Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp. ICE-L under salt stress.

    PubMed

    An, Meiling; Mou, Shanli; Zhang, Xiaowen; Zheng, Zhou; Ye, Naihao; Wang, Dongsheng; Zhang, Wei; Miao, Jinlai

    2013-12-01

    The Antarctic ice microalgae Chlamydomonas sp. ICE-L which is highly resistant to salt stress holds promise in providing an alternative species for the production of microalgal oil. We studied the effects of the alga in confrontation with NaCl stress on the growth, oil yield and expression of fatty acid desaturase genes. The growth rate of Chlamydomonas sp. ICE-L decreased with the gradual increase in NaCl concentration. Interestingly, we found that the highest lipid content was achieved at 16‰ NaCl, reaching 23% (w/w). Meanwhile, the expression of Δ9ACPCiFAD increased rapidly while Δ12CiFAD, ω3CiFAD2 and Δ6CiFAD showed a delayed elevation in response to altered salt stress. C18:3 was the dominant PUFA, which account for about 75% TFA in Chlamydomonas sp. ICE-L. Under 96‰ and 128‰ NaCl stress, the content of C20:5 almost approached that of C18:3. In contrast, low salinity enhanced the dominance of C18:3 at the expense of C20:3 and C20:5. PMID:24084208

  10. Interactions Between Fatty Acid Transport Proteins, Genes That Encode for Them, and Exercise: A Systematic Review.

    PubMed

    Jayewardene, Avindra F; Mavros, Yorgi; Reeves, Anneliese; Hancock, Dale P; Gwinn, Tom; Rooney, Kieron B

    2016-08-01

    Long-chain fatty acid (LCFA) movement into skeletal muscle involves a highly mediated process in which lipid rafts are utilized in the cellular membrane, involving numerous putative plasma membrane-associated LCFA transport proteins. The process of LCFA uptake and oxidation is of particular metabolic significance both at rest and during light to moderate exercise. A comprehensive systematic search of electronic databases was conducted to investigate whether exercise alters protein and/or gene expression of putative LCFA transport proteins. There were 31 studies meeting all eligibility criteria, of these 13 utilized an acute exercise protocol and 18 examined chronic exercise adaptations. Seventeen involved a study design incorporating an exercise stimulus, while the remaining 14 incorporated a combined exercise and diet stimulus. Divergent data relating to acute exercise, as well as prolonged exercise training (≥3 weeks), on protein content (PC) response was identified for proteins CD36, FABPpm and CAV1. Messenger ribonucleic acid (mRNA) data did not always correspond to functional PC, supporting previous suggestions of a disconnect due to potentially limiting factors post gene expression. The large array of study designs, cohorts, and primary dependent variables within the studies included in the present review elucidate the complexity of the interaction between exercise and LCFA transport proteins. Summary of the results in the present review validate the need for further targeted investigation within this topic, and provide an important information base for such research. J. Cell. Physiol. 231: 1671-1687, 2016. © 2015 Wiley Periodicals, Inc. PMID:26638980

  11. Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy

    PubMed Central

    Firozi, P; Zhang, W; Chen, L; Quiocho, FA; Worley, KC; Templeton, NS

    2012-01-01

    Polysaccharide contaminants in plasmid DNA, including current good manufacturing practices (cGMP) clinical preparations, must be removed to provide the greatest safety and efficacy for use in gene therapy and other clinical applications. We developed assays and methods for the detection and removal of these polysaccharides, our Super Clean DNA (SC-DNA) process, and have shown that these contaminants in plasmid DNA preparations are responsible for toxicity observed post-injection in animals. Furthermore, these contaminants limit the efficacy of low and high doses of plasmid DNA administered by numerous delivery routes. In particular, colanic acid (CA) that is mainly long-chained, branched and has high molecular weight (MW) is most refractory when complexed to cationic delivery vehicles and injected intravenously (IV). Because CA is often extremely large and tightly intertwined with DNA, it must be degraded, in order, to be effectively removed. We have produced a recombinant, truncated colanic acid degrading enzyme (CAE) that successfully accomplishes this task. Initially, we isolated a newly identified CAE from a bacteriophage that required truncation for proper folding while retaining its full enzymatic activity during production. Any plasmid DNA preparation can be digested with CAE and further purified, providing a critical advance to non-viral gene therapy. PMID:20664542

  12. Amino acid-based cationic lipids with α-tocopherol hydrophobic tail for efficient gene delivery.

    PubMed

    Yi, Wen-Jing; Zheng, Li-Ting; Su, Rong-Chuan; Liu, Qiang; Zhao, Zhi-Gang

    2015-11-01

    In this work, three amino acid-based cationic lipids L1-L3 bearing the same α-tocopherol moiety and biodegradable ester bond linkage, but differing in the polar head-group, were prepared and applied as non-viral gene delivery vectors. The physicochemical properties such as size, zeta-potential, stability, and cellular uptake of the lipoplexes formed from lipids L1-L3 as well as the transfection efficacy (TE) were investigated. The results showed that the chemical composition of the cationic head-group clearly affects the physicochemical parameters of the amino acid-based lipids, especially the TE. Besides their low cytotoxicity, these lipoplexes also showed comparable TE to commercially available lipofectamine 2000. In particular, dipeptide lipid L3 gave excellent TE, which was 1.8 times higher than bPEI 25k in the presence of 10% serum in Hela cells. These results demonstrate the promising use of novel dipeptide lipids for safe and efficient gene delivery. PMID:25973654

  13. Mycobacterium tuberculosis gene Rv2136c is dispensable for acid resistance and virulence in mice

    PubMed Central

    Darby, Crystal M.; Venugopal, Aditya; Ehrt, Sabine; Nathan, Carl F.

    2011-01-01

    Summary The gene Rv2136c is annotated to encode the Mycobacterium tuberculosis (Mtb) homologue of Escherichia coli’s undecaprenyl pyrophosphate phosphatase. In previous work, a genetic screen of 10,100 Mtb transposon mutants identified Rv2136c as being involved in acid resistance in Mtb. The Rv2136c:Tn strain was also sensitive to sodium dodecyl sulfate, lipophilic antibiotics, elevated temperature and reactive oxygen and nitrogen intermediates and was attenuated for growth and persistence in mice. However, none of these phenotypes could be genetically complemented, leading us to generate an Rv2136c knockout strain to test its role in Mtb pathogenicity. Genetic deletion revealed that Rv2136c is not responsible for any of the phenotypes observed in the transposon mutant strain. An independent genomic mutation is likely to have accounted for the extreme attenuation of this strain. Identification of the mutated gene will further our understanding of acid resistance mechanisms in Mtb and may offer a target for anti-tuberculosis chemotherapy. PMID:21778115

  14. Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity

    PubMed Central

    McMahon, Derek; Dinh, Anna; Kurz, Daniel; Shah, Dharika; Han, Gil-Soo; Carman, George M.; Brasaemle, Dawn L.

    2014-01-01

    Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/β hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli. Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity. PMID:24879803

  15. Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts.

    PubMed

    Seiliez, Iban; Gabillard, Jean-Charles; Riflade, Marine; Sadoul, Bastien; Dias, Karine; Avérous, Julien; Tesseraud, Sophie; Skiba, Sandrine; Panserat, Stéphane

    2012-03-01

    Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing. PMID:22252009

  16. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis.

    PubMed

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  17. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    PubMed Central

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  18. Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy.

    PubMed

    Martens, Thomas F; Remaut, Katrien; Deschout, Hendrik; Engbersen, Johan F J; Hennink, Wim E; van Steenbergen, Mies J; Demeester, Jo; De Smedt, Stefaan C; Braeckmans, Kevin

    2015-03-28

    Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular

  19. Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

  20. Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes.

    PubMed

    Huang, Jianzhong; Aki, Tsunehiro; Yokochi, Toshihiro; Nakahara, Toro; Honda, Daiske; Kawamoto, Seiji; Shigeta, Seiko; Ono, Kazuhisa; Suzuki, Osamu

    2003-01-01

    Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids. PMID:14730428

  1. Fatty Acid Desaturase 1 (FADS1) Gene Polymorphisms Control Human Hepatic Lipid Composition

    PubMed Central

    Wang, Libo; Athinarayanan, Shaminie; Jiang, Guanglong; Chalasani, Naga; Zhang, Min; Liu, Wanqing

    2014-01-01

    Fatty Acid Desaturase (FADS) genes and their variants have been associated with multiple metabolic phenotypes including liver enzymes and hepatic fat accumulation but the detailed mechanism remains unclear. We aimed to delineate the role of FADSs in modulating lipid composition in human liver. We performed a targeted lipidomic analysis of a variety of phospholipids, sphingolipids and ceramides among 154 human liver tissue samples. The associations between previously Genome-wide Association Studies (GWAS)-identified six FADS single nucleotide polymorphisms (SNPs) and these lipid levels as well as total hepatic fat content (HFC) were tested. The potential function of these SNPs in regulating transcription of 3 FADS genes (FADS1, FADS2 and FADS3) in the locus was also investigated. We found that while these SNPs were in high linkage disequilibrium (r2 >0.8), the rare alleles of these SNPs were consistently and significantly associated with the accumulation of multiple very-long-chain fatty acids (VLCFAs), with C47H85O13P (C36:4), a phosphatidylinositol (PI) and C43H80O8PN (C38:3), a phosphatidylethanolamine (PE) reached the Bonferroni corrected significance (p<3×10−4). Meanwhile, these SNPs were significantly associated with increased ratios between the more saturated and relatively less saturated forms of VLCFAs, especially between PEs, PIs and phosphatidylcholines (PCs) (p≤3.5×10−6). These alleles were also associated with increased total HFC (p<0.05). Further analyses revealed that these alleles were associated with decreased hepatic expression of FADS1 (p=0.0018 for rs174556), but not FADS2 or FADS3 (p>0.05). Conclusion Our findings revealed critical insight into the mechanism underlying FADS1 and its polymorphisms in modulating hepatic lipid deposition by altering gene transcription and controlling lipid composition in human livers. PMID:25123259

  2. The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

    PubMed Central

    Pédrono, Frédérique; Blanchard, Hélène; Kloareg, Maela; D'andréa, Sabine; Daval, Stéphanie; Rioux, Vincent; Legrand, Philippe

    2010-01-01

    In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176–183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Δ5- and Δ6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3. PMID:19752397

  3. Characterization of the promoter region of the gene for the rat neutral and basic amino acid transporter and chromosomal localization of the human gene.

    PubMed Central

    Yan, N; Mosckovitz, R; Gerber, L D; Mathew, S; Murty, V V; Tate, S S; Udenfriend, S

    1994-01-01

    The promoter region of the rat kidney neutral and basic amino acid transporter (NBAT) gene has been isolated and sequenced. The major transcription initiation site was mapped by primer extension. The entire promoter region and a set of 5' deletions within it were expressed at a high level in LLC-PK1 cells using the luciferase indicator gene. Positive and negative regulatory elements in the promoter region were observed. A human genomic clone of the transporter was also obtained and was used to localize the NBAT gene at the p21 region of chromosome 2. Images PMID:8052618

  4. X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster.

    PubMed

    Elkins, Jonathan M; Kershaw, Nadia J; Schofield, Christopher J

    2005-01-15

    The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates. PMID:15352873

  5. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

    PubMed

    Li, Huan-Jun; He, Yi-Long; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Li, Na; Xu, Jun-Wei

    2016-06-10

    The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs. PMID:27080449

  6. Effect of anthranilic acid on the catabolite repression of a Drosophila amylase gene in E. coli

    SciTech Connect

    Stevens, S.M.; Moehring, J.M.; Chernin, M.I.

    1987-05-01

    A Drosophila pseudoobscura amylase pseudogene cloned in Escherichia coli is expressed at high levels. The expression of this pseudogene is repressed when glucose (0.5% final conc) is added to a starch minimal medium culture of E. coli cells that contain the amylase plasmid pAMY17F. Addition of anthranilic acid (7 mM final conc.) to catabolite repressed cells acts like adenosine 3',5' cyclic monophosphate (cAMP) by derepressing the amylase pseudogene at the promoter. This is consistent with the Metabolite Gene Regulation (MGR) model proposed by Kline et al. which suggests that small molecules can circumvent the necessity for cAMP. Catabolite repression of the amylase structural gene of D. pseudoobscura has been previously shown. This would suggest that the amylase pseudogene expression in E. coli is either from a Drosophila structural gene promoter co-cloned with the pseudogene or a catabolite repressible E. coli promoter placed in the proper orientation and reading frame during the rearrangement of pAMY17F.

  7. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid.

    PubMed

    Kashyap, Vasundhra; Gudas, Lorraine J; Brenet, Fabienne; Funk, Patricia; Viale, Agnes; Scandura, Joseph M

    2011-02-01

    Retinoic acid (RA) regulates clustered Hox gene expression during embryogenesis and is required to establish the anterior-posterior body plan. Using mutant embryonic stem cell lines deficient in the RA receptor γ (RARγ) or Hoxa1 3'-RA-responsive element, we studied the kinetics of transcriptional and epigenomic patterning responses to RA. RARγ is essential for RA-induced Hox transcriptional activation, and deletion of its binding site in the Hoxa1 enhancer attenuates transcriptional and epigenomic activation of both Hoxa and Hoxb gene clusters. The kinetics of epigenomic reorganization demonstrate that complete erasure of the polycomb repressive mark H3K27me3 is not necessary to initiate Hox transcription. RARγ is not required to establish the bivalent character of Hox clusters, but RA/RARγ signaling is necessary to erase H3K27me3 from activated Hox genes during embryonic stem cell differentiation. Highly coordinated, long range epigenetic Hox cluster reorganization is closely linked to transcriptional activation and is triggered by RARγ located at the Hoxa1 3'-RA-responsive element. PMID:21087926

  8. Fatty Acid Esters of Phloridzin Induce Apoptosis of Human Liver Cancer Cells through Altered Gene Expression

    PubMed Central

    Nair, Sandhya V. G.; Ziaullah; Rupasinghe, H. P. Vasantha

    2014-01-01

    Phloridzin (phlorizin or phloretin 2′-O-glucoside) is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin) using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA) ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2), growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR) and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK), cell cycle machinery (CDKs, TERT, TOP2A, TOP2B) as well as epigenetics regulators (HDACs). These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects mediated

  9. Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapa.

    PubMed

    Duan, Weike; Song, Xiaoming; Liu, Tongkun; Huang, Zhinan; Ren, Jun; Hou, Xilin; Du, Jianchang; Li, Ying

    2015-01-01

    Ascorbic acid (AsA) is an important antioxidant in plants and an essential vitamin for humans. Extending the study of AsA-related genes from Arabidopsis thaliana to Brassica rapa could shed light on the evolution of AsA in plants and inform crop breeding. In this study, we conducted whole-genome annotation, molecular-evolution and gene-expression analyses of all known AsA-related genes in B. rapa. The nucleobase-ascorbate transporter (NAT) gene family and AsA l-galactose pathway genes were also compared among plant species. Four important insights gained are that: 1) 102 AsA-related gene were identified in B. rapa and they mainly diverged 12-18 Ma accompanied by the Brassica-specific genome triplication event; 2) during their evolution, these AsA-related genes were preferentially retained, consistent with the gene dosage hypothesis; 3) the putative proteins were highly conserved, but their expression patterns varied; and 4) although the number of AsA-related genes is higher in B. rapa than in A. thaliana, the AsA contents and the numbers of expressed genes in leaves of both species are similar, the genes that are not generally expressed may serve as substitutes during emergencies. In summary, this study provides genome-wide insights into evolutionary history and mechanisms of AsA-related genes following whole-genome triplication in B. rapa. PMID:25552535

  10. Genetic Variants in the FADS Gene: Implications for Dietary Recommendations for Fatty Acid Intake.

    PubMed

    Mathias, Rasika A; Pani, Vrindarani; Chilton, Floyd H

    2014-06-01

    Unequivocally, genetic variants within the fatty acid desaturase (FADS) cluster are determinants of long chain polyunsaturated fatty acid (LC-PUFA) levels in circulation, cells and tissues. A recent series of papers have addressed these associations in the context of ancestry; evidence clearly supports that the associations are robust to ethnicity. However ∼80% of African Americans carry two copies of the alleles associated with increased levels of arachidonic acid, compared to only ∼45% of European Americans raising important questions of whether gene-PUFA interactions induced by a modern western diet are differentially driving the risk of diseases of inflammation in diverse populations, and are these interactions leading to health disparities. We highlight an important aspect thus far missing in the debate regarding dietary recommendations; we content that current evidence from genetics strongly suggest that an individual's, or at the very least the population from which an individual is sampled, genetic architecture must be factored into dietary recommendations currently in place. PMID:24977108

  11. Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.

    PubMed

    Yelin, Ronit; Schyr, Racheli Ben-Haroush; Kot, Hadas; Zins, Sharon; Frumkin, Ayala; Pillemer, Graciela; Fainsod, Abraham

    2005-03-01

    Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer. PMID:15708568

  12. Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: The aim of this research was to examine relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA, in O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes after adaptation to pineapple juice (PJ) and subsequently to determine survival with e...

  13. Suppressors of ssy1 and ptr3 null mutations define novel amino acid sensor-independent genes in Saccharomyces cerevisiae.

    PubMed Central

    Forsberg, H; Hammar, M; Andréasson, C; Molinér, A; Ljungdahl, P O

    2001-01-01

    Ssy1p and Ptr3p are components of the yeast plasma membrane SPS amino acid sensor. In response to extracellular amino acids this sensor initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and amino acid permeases (AAPs). As a result of diminished leucine uptake capabilities, ssy1Delta leu2 and ptr3Delta leu2 mutant strains are unable to grow on synthetic complete medium (SC). Genes affecting the functional expression of AAPs were identified by selecting spontaneous suppressing mutations in amino acid sensor-independent (ASI) genes that restore growth on SC. The suppressors define 11 recessive (asi) complementation groups and 5 dominant (ASI) linkage groups. Strains with mutations in genes assigned to these 16 groups fall into two phenotypic classes. Mutations in the class I genes (ASI1, ASI2, ASI3, TUP1, SSN6, ASI13) derepress the transcription of AAP genes. ASI1, ASI2, and ASI3 encode novel membrane proteins, and Asi1p and Asi3p are homologous proteins that have conserved ubiquitin ligase-like RING domains at their extreme C termini. Several of the class II genes (DOA4, UBA1, BRO1, BUL1, RSP5, VPS20, VPS36) encode proteins implicated in controlling aspects of post-Golgi endosomal-vacuolar protein sorting. The results from genetic and phenotypic analysis indicate that SPS sensor-initiated signals function positively to facilitate amino acid uptake and that two independent ubiquitin-mediated processes negatively modulate amino acid uptake. PMID:11454748

  14. Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

    PubMed

    Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

    2015-03-01

    Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae. PMID:25698512

  15. A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

    PubMed Central

    Subbarao, E K; London, W; Murphy, B R

    1993-01-01

    The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses. PMID:8445709

  16. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter

    PubMed Central

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  17. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter.

    PubMed

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  18. Clusters of genes encoding fructan biosynthesizing enzymes in wheat and barley.

    PubMed

    Huynh, Bao-Lam; Mather, Diane E; Schreiber, Andreas W; Toubia, John; Baumann, Ute; Shoaei, Zahra; Stein, Nils; Ariyadasa, Ruvini; Stangoulis, James C R; Edwards, James; Shirley, Neil; Langridge, Peter; Fleury, Delphine

    2012-10-01

    Fructans are soluble carbohydrates with health benefits and possible roles in plant adaptation. Fructan biosynthetic genes were isolated using comparative genomics and physical mapping followed by BAC sequencing in barley. Genes encoding sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT) and sucrose:fructan 6-fructosyltransferase (6-SFT) were clustered together with multiple copies of vacuolar invertase genes and a transposable element on two barley BAC. Intron-exon structures of the genes were similar. Phylogenetic analysis of the fructosyltransferases and invertases in the Poaceae showed that the fructan biosynthetic genes may have evolved from vacuolar invertases. Quantitative real-time PCR was performed using leaf RNA extracted from three wheat cultivars grown under different conditions. The 1-SST, 1-FFT and 6-SFT genes had correlated expression patterns in our wheat experiment and in existing barley transcriptome database. Single nucleotide polymorphism (SNP) markers were developed and successfully mapped to a major QTL region affecting wheat grain fructan accumulation in two independent wheat populations. The alleles controlling high- and low- fructan in parental lines were also found to be associated in fructan production in a diverse set of 128 wheat lines. To the authors' knowledge, this is the first report on the mapping and sequencing of a fructan biosynthetic gene cluster and in particular, the isolation of a novel 1-FFT gene from barley. PMID:22864927

  19. Acid Ceramidase (ASAH1) Is a Global Regulator of Steroidogenic Capacity and Adrenocortical Gene Expression

    PubMed Central

    Lucki, Natasha C.; Bandyopadhyay, Sibali; Wang, Elaine; Merrill, Alfred H.

    2012-01-01

    In H295R human adrenocortical cells, ACTH rapidly activates ceramide (Cer) and sphingosine (SPH) turnover with a concomitant increase in SPH-1-phosphate secretion. These bioactive lipids modulate adrenocortical steroidogenesis, primarily by acting as second messengers in the protein kinase A/cAMP-dependent pathway. Acid ceramidase (ASAH1) directly regulates the intracellular balance of Cer, SPH, and SPH-1-phosphate by catalyzing the hydrolysis of Cer into SPH. ACTH/cAMP signaling stimulates ASAH1 transcription and activity, supporting a role for this enzyme in glucocorticoid production. Here, the role of ASAH1 in regulating steroidogenic capacity was examined using a tetracycline-inducible ASAH1 short hairpin RNA H295R human adrenocortical stable cell line. We show that ASAH1 suppression increases the transcription of multiple steroidogenic genes, including Cytochrome P450 monooxygenase (CYP)17A1, CYP11B1/2, CYP21A2, steroidogenic acute regulatory protein, hormone-sensitive lipase, 18-kDa translocator protein, and the melanocortin-2 receptor. Induced gene expression positively correlated with enhanced histone H3 acetylation at target promoters. Repression of ASAH1 expression also induced the expression of members of the nuclear receptor nuclear receptor subfamily 4 (NR4A) family while concomitantly suppressing the expression of dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1. ASAH1 knockdown altered the expression of genes involved in sphingolipid metabolism and changed the cellular amounts of distinct sphingolipid species. Finally, ASAH1 silencing increased basal and cAMP-dependent cortisol and dehydroepiandrosterone secretion, establishing ASAH1 as a pivotal regulator of steroidogenic capacity in the human adrenal cortex. PMID:22261821

  20. Genome-Wide Methylation and Gene Expression Changes in Newborn Rats following Maternal Protein Restriction and Reversal by Folic Acid

    PubMed Central

    Stupka, Elia; Clark, Adrian J. L.; Langley-Evans, Simon

    2013-01-01

    A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures. PMID:24391732

  1. Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

    PubMed Central

    Martín, M. Cruz; Alonso, Juan C.; Suárez, Juan E.; Alvarez, Miguel A.

    2000-01-01

    The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation. PMID:10831443

  2. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  3. Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

    PubMed

    Shin, John J; Aftab, Qurratulain; Austin, Pamela; McQueen, Jennifer A; Poon, Tak; Li, Shu Chen; Young, Barry P; Roskelley, Calvin D; Loewen, Christopher J R

    2016-09-01

    A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C-COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain

  4. Identification and functional characterization of genes encoding omega-3 polyunsaturated fatty acid biosynthetic activities from unicellular microalgae.

    PubMed

    Vaezi, Royah; Napier, Johnathan A; Sayanova, Olga

    2013-12-01

    In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative "front-end" desaturases (Δ6 and Δ4) from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs) in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15). These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in marine microalgae. PMID:24351909

  5. Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

    PubMed Central

    Vaezi, Royah; Napier, Johnathan A.; Sayanova, Olga

    2013-01-01

    In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4) from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs) in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15). These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in marine microalgae. PMID:24351909

  6. Polymorphism in the ELOVL6 Gene Is Associated with a Major QTL Effect on Fatty Acid Composition in Pigs

    PubMed Central

    Corominas, Jordi; Ramayo-Caldas, Yuliaxis; Puig-Oliveras, Anna; Pérez-Montarelo, Dafne; Noguera, Jose L.; Folch, Josep M.; Ballester, Maria

    2013-01-01

    Background The ELOVL fatty acid elongase 6 (ELOVL6), the only elongase related to de novo lipogenesis, catalyzes the rate-limiting step in the elongation cycle by controlling the fatty acid balance in mammals. It is located on pig chromosome 8 (SSC8) in a region where a QTL affecting palmitic, and palmitoleic acid composition was previously detected, using an Iberian x Landrace intercross. The main goal of this work was to fine-map the QTL and to evaluate the ELOVL6 gene as a positional candidate gene affecting the percentages of palmitic and palmitoleic fatty acids in pigs. Methodology and Principal Findings The combination of a haplotype-based approach and single-marker analysis allowed us to identify the main, associated interval for the QTL, in which the ELOVL6 gene was identified and selected as a positional candidate gene. A polymorphism in the promoter region of ELOVL6, ELOVL6:c.-533C>T, was highly associated with the percentage of palmitic and palmitoleic acids in muscle and backfat. Significant differences in ELOVL6 gene expression were observed in backfat when animals were classified by the ELOVL6:c.-533C>T genotype. Accordingly, animals carrying the allele associated with a decrease in ELOVL6 gene expression presented an increase in C16:0 and C16:1(n-7) fatty acid content and a decrease of elongation activity ratios in muscle and backfat. Furthermore, a SNP genome-wide association study with ELOVL6 relative expression levels in backfat showed the strongest effect on the SSC8 region in which the ELOVL6 gene is located. Finally, different potential genomic regions associated with ELOVL6 gene expression were also identified by GWAS in liver and muscle, suggesting a differential tissue regulation of the ELOVL6 gene. Conclusions and Significance Our results suggest ELOVL6 as a potential causal gene for the QTL analyzed and, subsequently, for controlling the overall balance of fatty acid composition in pigs. PMID:23341976

  7. Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration.

    PubMed Central

    Rothan, C.; Duret, S.; Chevalier, C.; Raymond, P.

    1997-01-01

    High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2. PMID:12223703

  8. Indole-3-acetic acid (IAA) induced changes in oil content, fatty acid profiles and expression of four fatty acid biosynthetic genes in Chlorella vulgaris at early stationary growth phase.

    PubMed

    Jusoh, Malinna; Loh, Saw Hong; Chuah, Tse Seng; Aziz, Ahmad; Cha, Thye San

    2015-03-01

    Microalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the β-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3. PMID:25583439

  9. Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

    PubMed Central

    2013-01-01

    Background Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important

  10. Identification of the Bile Acid Transporter Slco1a6 as a Candidate Gene That Broadly Affects Gene Expression in Mouse Pancreatic Islets.

    PubMed

    Tian, Jianan; Keller, Mark P; Oler, Angie T; Rabaglia, Mary E; Schueler, Kathryn L; Stapleton, Donald S; Broman, Aimee Teo; Zhao, Wen; Kendziorski, Christina; Yandell, Brian S; Hagenbuch, Bruno; Broman, Karl W; Attie, Alan D

    2015-11-01

    We surveyed gene expression in six tissues in an F2 intercross between mouse strains C57BL/6J (abbreviated B6) and BTBR T(+) tf/J (abbreviated BTBR) made genetically obese with the Leptin(ob) mutation. We identified a number of expression quantitative trait loci (eQTL) affecting the expression of numerous genes distal to the locus, called trans-eQTL hotspots. Some of these trans-eQTL hotspots showed effects in multiple tissues, whereas some were specific to a single tissue. An unusually large number of transcripts (∼8% of genes) mapped in trans to a hotspot on chromosome 6, specifically in pancreatic islets. By considering the first two principal components of the expression of genes mapping to this region, we were able to convert the multivariate phenotype into a simple Mendelian trait. Fine mapping the locus by traditional methods reduced the QTL interval to a 298-kb region containing only three genes, including Slco1a6, one member of a large family of organic anion transporters. Direct genomic sequencing of all Slco1a6 exons identified a nonsynonymous coding SNP that converts a highly conserved proline residue at amino acid position 564 to serine. Molecular modeling suggests that Pro564 faces an aqueous pore within this 12-transmembrane domain-spanning protein. When transiently overexpressed in HEK293 cells, BTBR organic anion transporting polypeptide (OATP)1A6-mediated cellular uptake of the bile acid taurocholic acid (TCA) was enhanced compared to B6 OATP1A6. Our results suggest that genetic variation in Slco1a6 leads to altered transport of TCA (and potentially other bile acids) by pancreatic islets, resulting in broad gene regulation. PMID:26385979

  11. Cloning and identification of the human LPAAT-zeta gene, a novel member of the lysophosphatidic acid acyltransferase family.

    PubMed

    Li, Dan; Yu, Long; Wu, Hai; Shan, Yuxi; Guo, Jinhu; Dang, Yongjun; Wei, Youheng; Zhao, Shouyuan

    2003-01-01

    Lysophosphatidic acid (LPA) is a naturally occurring component of phospholipid and plays a critical role in the regulation of many physiological and pathophysiological processes including cell growth, survival, and pro-angiogenesis. LPA is converted to phosphatidic acid by the action of lysophosphatidic acid acyltransferase (LPAAT). Five members of the LPAAT gene family have been detected in humans to date. Here, we report the identification of a novel LPAAT member, which is designated as LPAAT-zeta. LPAAT-zeta was predicted to encode a protein consisting of 456 amino acid residues with a signal peptide sequence and the acyltransferase domain. Northern blot analysis showed that LPAAT-zeta was ubiquitously expressed in all 16 human tissues examined, with levels in the skeletal muscle, heart, and testis being relatively high and in the lung being relatively low. The human LPAAT-zeta gene consisted of 13 exons and is positioned at chromosome 8p11.21. PMID:12938015

  12. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati.

    PubMed

    Yaacob, Norhayati; Mohamad Ali, Mohd Shukuri; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  13. Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

    PubMed

    Ito, Tomoko; Iida-Tanaka, Naoko; Koyama, Yoshiyuki

    2008-05-01

    Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells. PMID:18446606

  14. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati

    PubMed Central

    Yaacob, Norhayati; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  15. Does Short-Term Dietary Omega-3 Fatty Acid Supplementation Influence Brain Hippocampus Gene Expression of Zinc Transporter-3?

    PubMed

    Sopian, Nur Farhana Ahmad; Ajat, Mokrish; Shafie, Nurul' Izzati; Noor, Mohd Hezmee Mohd; Ebrahimi, Mehdi; Rajion, Mohamed Ali; Meng, Goh Yong; Ahmad, Hafandi

    2015-01-01

    Dietary omega-3 fatty acids have been recognized to improve brain cognitive function. Deficiency leads to dysfunctional zinc metabolism associated with learning and memory impairment. The objective of this study is to explore the effect of short-term dietary omega-3 fatty acids on hippocampus gene expression at the molecular level in relation to spatial recognition memory in mice. A total of 24 male BALB/c mice were randomly divided into four groups and fed a standard pellet as a control group (CTL, n = 6), standard pellet added with 10% (w/w) fish oil (FO, n = 6), 10% (w/w) soybean oil (SO, n = 6) and 10% (w/w) butter (BT, n = 6). After 3 weeks on the treatment diets, spatial-recognition memory was tested on a Y-maze. The hippocampus gene expression was determined using a real-time PCR. The results showed that 3 weeks of dietary omega-3 fatty acid supplementation improved cognitive performance along with the up-regulation of α-synuclein, calmodulin and transthyretin genes expression. In addition, dietary omega-3 fatty acid deficiency increased the level of ZnT3 gene and subsequently reduced cognitive performance in mice. These results indicate that the increased the ZnT3 levels caused by the deficiency of omega-3 fatty acids produced an abnormal zinc metabolism that in turn impaired the brain cognitive performance in mice. PMID:26184176

  16. Role of Acinus in Regulating Retinoic Acid-Responsive Gene Pre-mRNA Splicing

    PubMed Central

    Wang, Fang; Soprano, Kenneth J.; Soprano, Dianne Robert

    2014-01-01

    Acinus-S’ is a co-repressor for retinoic acid receptor (RAR)-dependent gene transcription and has been suggested to be involved in RNA processing. In this study the role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S’, with the activity of Acinus-L higher than that of Acinus-S’, increase the splicing of a retinoic acid (RA)-responsive minigene containing a weak 5′ splice site but not a RA-responsive minigene containing a strong 5′ splice site. RA treatment further enhances the splicing of the weak 5′ splice site by Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The RRM domain is necessary for the RA-dependent splicing activity of Acinus and the RA-independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5′ splice site of these two genes in a RA-dependent manner for RARβ and a RA-independent manner for Bcl-x. Taken together, these studies demonstrate that Acinus functions in both RAR-dependent splicing and RAR-dependent transcription. PMID:25205379

  17. Conversion of 5-formyltetrahydrofolic acid to 5-methyltetrahydrofolic acid is unimpaired in folate-adequate persons homozygous for the C677T mutation in the methylenetetrahydrofolate reductase gene.

    PubMed

    Stern, L L; Bagley, P J; Rosenberg, I H; Selhub, J

    2000-09-01

    Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolic acid (5-CH(3)-H(4) folic acid), the methyl donor for the formation of methionine from homocysteine. A common C677T transition in the MTHFR gene results in a variant with a lower specific activity and a greater sensitivity to heat than the normal enzyme, as measured in vitro. This study was undertaken to determine the capacity of homozygotes for the MTHFR C677T transition to convert 5-formyltetrahydrofolic acid (5-HCO-H(4) folic acid) to 5-CH(3)-H(4) folic acid, a process that requires the action of MTHFR. Six subjects homozygous for the C677T transition (T/T) and 6 subjects with wild-type MTHFR (C/C) were given a 5-mg oral dose of (6R:,S:)-5-HCO-H(4) folic acid. Plasma and urine were analyzed for 5-CH(3)-H(4) folic acid concentrations using affinity/HPLC coupled with fluorescence or UV detection. The mean areas under the curves created by the rise and fall of plasma 5-CH(3)-H(4) folic acid after the oral dose did not differ between the two genotypes, 424.5 +/- 140.3 (T/T) vs. 424.1+/- 202.4 h.nmol/L (C/C). There also was no significant difference in the mean cumulative 7-h urinary excretion of 5-CH(3)-H(4) folic acid between the T/T (2.5 +/- 1.4 micromol) and C/C (1.9 +/- 1.0 micromol) genotypes. Under the conditions employed, the conversion of oral 5-HCO-H(4) folic acid to 5-CH(3)-H(4) folic acid is not impaired in persons with the T/T MTHFR genotype. Possible reasons for these findings are discussed. PMID:10958818

  18. Identification of nuclear genes affecting 2-Deoxyglucose resistance in Schizosaccharomyces pombe.

    PubMed

    Vishwanatha, Akshay; Rallis, Charalampos; Bevkal Subramanyaswamy, Shubha; D'Souza, Cletus Joseph Michael; Bähler, Jürg; Schweingruber, Martin Ernst

    2016-09-01

    2-Deoxyglucose (2-DG) is a toxic glucose analog. To identify genes involved in 2-DG toxicity in Schizosaccharomyces pombe, we screened a wild-type overexpression library for genes which render cells 2-DG resistant. A gene we termed odr1, encoding an uncharacterized hydrolase, led to strong resistance and altered invertase expression when overexpressed. We speculate that Odr1 neutralizes the toxic form of 2-DG, similar to the Saccharomyces cerevisiae Dog1 and Dog2 phosphatases which dephosphorylate 2-DG-6-phosphate synthesized by hexokinase. In a complementary approach, we screened a haploid deletion library to identify 2-DG-resistant mutants. This screen identified the genes snf5, ypa1, pas1 and pho7 In liquid medium, deletions of these genes conferred 2-DG resistance preferentially under glucose-repressed conditions. The deletion mutants expressed invertase activity more constitutively than the control strain, indicating defects in the control of glucose repression. No S. cerevisiae orthologs of the pho7 gene is known, and no 2-DG resistance has been reported for any of the deletion mutants of the other genes identified here. Moreover, 2-DG leads to derepressed invertase activity in S. pombe, while in S. cerevisiae it becomes repressed. Taken together, these findings suggest that mechanisms involved in 2-DG resistance differ between budding and fission yeasts. PMID:27481777

  19. Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

    PubMed Central

    Kitagawa, Wataru; Takami, Sachiko; Miyauchi, Keisuke; Masai, Eiji; Kamagata, Yoichi; Tiedje, James M.; Fukuda, Masao

    2002-01-01

    The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC. PMID:11751829

  20. Cloning and characterization of a benzoic acid/salicylic acid carboxyl methyltransferase gene involved in floral scent production from lily (Lilium 'Yelloween').

    PubMed

    Wang, H; Sun, M; Li, L L; Xie, X H; Zhang, Q X

    2015-01-01

    In lily flowers, the volatile ester methyl benzoate is one of the major and abundant floral scent compounds; however, knowledge regarding the biosynthesis of methyl benzoate remains unknown for Lilium. In this study, we isolated a benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) gene, LiBSMT, from petals of Lilium 'Yelloween'. The gene has an open reading frame of 1083 base pairs (bp) and encodes a protein of 41.05 kDa. Sequence alignment and phylogenetic analyses of LiBSMT revealed 40-50% similarity with other known benzenoid carboxyl methyltransferases in other plant species, and revealed homology to BSMT of Oryza sativa. Heterologous expression of this gene in Escherichia coli yielded an enzyme responsible for catalyzing benzoic acid and salicylic acid to methyl benzoate and methyl salicylate, respectively. Quantitative real-time polymerase chain reaction analysis showed that LiBSMT was preferentially expressed in petals. Moreover, the expression of LiBSMT in petals was developmentally regulated. These expression patterns correlate well with the emission of methyl benzoate. Our results indicate that LiBSMT plays an important role in floral scent methyl benzoate production and emission in lily flowers. PMID:26600510

  1. Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

    PubMed Central

    Boini, Krishna M.; Xia, Min; Abais, Justine M.; Xu, Ming; Li, Cai-xia; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs+/− and Asm+/− mice. Given that the homozygotes of Cbs−/−/Asm−/− mice could not survive for 3 weeks. Cbs+/−/Asm+/+, Cbs+/−/Asm+/− and Cbs+/−/Asm−/− as well as their Cbs wild type littermates were used to study the role of Asm−/− under a background of Cbs+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs+/−) mice with different copies of Asm gene compared to Cbs+/+ mice with different Asm gene copies. Cbs+/−/Asm+/+ mice had significantly increased renal Asm activity, ceramide production and O2.− level compared to Cbs+/+/Asm+/+, while Cbs+/−/Asm−/− mice showed significantly reduced renal Asm activity, ceramide production and O2.− level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs+/−/Asm−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O2.− production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or

  2. [Cephalosporin-Acid Synthetase of Escherichia coli Strain VKPM B-10182: Genomic Context, Gene Identification, Producer Strain Production].

    PubMed

    Eldarov M, A; Sklyarenko, A V; Mardanov, A V; Beletsky, A V; Zhgun, A A; Dumina, M V; Medvedeva, N V; Satarova, D E; Ravin, N V; Yarockii, S V

    2015-01-01

    An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of β-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and β-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA. PMID:26596082

  3. Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol.

    PubMed

    Mienda, Bashir Sajo; Shamsir, Mohd Shahir; Md Illias, Rosli

    2016-08-01

    Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli. PMID:26513379

  4. Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

    PubMed Central

    Penninckx, I A; Eggermont, K; Terras, F R; Thomma, B P; De Samblanx, G W; Buchala, A; Métraux, J P; Manners, J M; Broekaert, W F

    1996-01-01

    A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response. PMID:8989885

  5. A screen for genes that function in abscisic acid signaling in Arabidopsis thaliana.

    PubMed Central

    Nambara, Eiji; Suzuki, Masaharu; Abrams, Suzanne; McCarty, Donald R; Kamiya, Yuji; McCourt, Peter

    2002-01-01

    The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development under a diverse range of environmental conditions. To identify genes functioning in ABA signaling, we have carried out a screen for mutants that takes advantage of the ability of wild-type Arabidopsis seeds to respond to (-)-(R)-ABA, an enantiomer of the natural (+)-(S)-ABA. The premise of the screen was to identify mutations that preferentially alter their germination response in the presence of one stereoisomer vs. the other. Twenty-six mutants were identified and genetic analysis on 23 lines defines two new loci, designated CHOTTO1 and CHOTTO2, and a collection of new mutant alleles of the ABA-insensitive genes, ABI3, ABI4, and ABI5. The abi5 alleles are less sensitive to (+)-ABA than to (-)-ABA. In contrast, the abi3 alleles exhibit a variety of differences in response to the ABA isomers. Genetic and molecular analysis of these alleles suggests that the ABI3 transcription factor may perceive multiple ABA signals. PMID:12136027

  6. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery.

    PubMed

    Liu, Ya; Kong, Ming; Cheng, Xiao Jie; Wang, Qian Qian; Jiang, Li Ming; Chen, Xi Guang

    2013-04-15

    The present work described nanoparticles (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) using coacervation process as novel potential carriers for gene delivery. An N/P ratio of 5 and OCMCS/HA weight ratio of 4 were the optimal conditions leading to the smallest (164.94 nm), positive charged (+14.2 mV) and monodispersed NPs. OCMCS-HA/DNA (OHD) NPs showed higher in vitro DNA release rates and increased cellular uptake by Caco-2 cells due to the HA involved in NPs. The MTT survival assay indicated no significant cytotoxicity. The transfection efficiency of OHD NPs was 5-fold higher than OCMCS/DNA (OD) NPs; however, it decreased significantly in the presence of excess free HA. The results indicated that OHD NPs internalized in Caco-2 cells were mediated by the hyaluronan receptor CD44. The data obtained in the present research gave evidence of the potential of OHD NPs for the targeting and further transfer of genes to the epithelial cells. PMID:23544543

  7. Fibroblasts behavior after N-acetylcysteine and amino acids exposure: extracellular matrix gene expression.

    PubMed

    Avantaggiato, Anna; Palmieri, Annalisa; Bertuzzi, Gianluigi; Carinci, Francesco

    2014-06-01

    Reactive oxygen species (ROS) are chemically reactive molecules with impaired electrons that make them unstable and able to react easily with a great variety of molecules. The main targets of ROS are DNA, proteins, and membrane phospholipids. In the skin, ROS are able to affect the production of collagen and elastin, the main components of the extracellular matrix (ECM). This action contributes to the skin's aging. N-Acetylcysteine (NAC) is an acetylated cysteine residue with excellent anti-oxidant activity that boosts glutathione (GSH) levels. This study evaluates the effect of a solution of NAC and amino acids, which is used in aesthetic medicine as an intra-dermal injective treatment, on fibroblast behavior. To this aim, the expression levels of some ECM-related genes (HAS1, HYAL1 ELN, ELANE, MMP2, MMP3, MMP13, COL1A1, COL3A1) were analyzed on cultured dermal fibroblasts using real-time reverse transcription polymerase chain reaction (RT-PCR). All but two collagen genes were up-regulated after 24 hr of treatment. PMID:24438160

  8. Abscisic acid regulation of DC8, a carrot embryonic gene. [Daucus carota

    SciTech Connect

    Hatzopoulos, P.; Fong, F.; Sung, Z.R. Texas A M Univ., College Station )

    1990-10-01

    DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage embryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 {times} 10{sup {minus}7} molar ABA while 10{sup {minus}6} molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.

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

    PubMed

    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. PMID:25381910

  10. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  11. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  12. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences. PMID:18397498

  13. Differential expression of two 1-aminocyclopropane-1-carboxylic acid oxidase genes in broccoli after harvest.

    PubMed Central

    Pogson, B J; Downs, C G; Davies, K M

    1995-01-01

    Broccoli (Brassica oleracea L.) floral tissues rapidly differentiate and grow before harvest and then senesce rapidly after harvest. Associated with this postharvest deterioration is an increase in ethylene production by florets. Two cDNA clones having high nucleotide identity to sequences encoding 1-amino-cyclopropane-1-carboxylic acid (ACC) oxidase were isolated from senescing florets. The cDNAs, ACC Ox1 and ACC Ox2, apparently encode mRNAs from different genes. ACC Ox1 transcripts were found at low levels in whole florets at the time of harvest and increased markedly in abundance after harvest. ACC Ox1 transcript abundance also increased in sepals after harvest and in excised yellowing leaves. Transcripts corresponding to ACC Ox2 were found exclusively within the reproductive structures. These ACC Ox2 transcripts were absent at harvest but started to increase in abundance within 2 h of harvest and then accumulated to high levels. Hormone treatment did not alter the abundance of ACC Ox1 transcripts, whereas ACC Ox2 transcripts increased in abundance after treatment with abscisic acid and propylene. Wounding did not affect the levels of ACC Ox1 or Ox2 transcripts after harvest. At harvest, individual broccoli florets were closed and remained unpollinated. We propose a model whereby the rapid increase in ACC Ox1 and Ox2 transcript abundance after harvest contributes to increased ethylene production by florets. This ethylene may regulate aspects of postharvest senescence, in particular chlorophyll loss. PMID:7610162

  14. Acid-Labile Poly(glycidyl methacrylate)-Based Star Gene Vectors.

    PubMed

    Yang, Yan-Yu; Hu, Hao; Wang, Xing; Yang, Fei; Shen, Hong; Xu, Fu-Jian; Wu, De-Cheng

    2015-06-10

    It was recently reported that ethanolamine-functionalized poly(glycidyl methacrylate) (PGEA) possesses great potential applications in gene therapy due to its good biocompatibility and high transfection efficiency. Importing responsivity into PGEA vectors would further improve their performances. Herein, a series of responsive star-shaped vectors, acetaled β-cyclodextrin-PGEAs (A-CD-PGEAs) consisting of a β-CD core and five PGEA arms linked by acid-labile acetal groups, were proposed and characterized as therapeutic pDNA vectors. The A-CD-PGEAs owned abundant hydroxyl groups to shield extra positive charges of A-CD-PGEAs/pDNA complexes, and the star structure could decrease charge density. The incorporation of acetal linkers endowed A-CD-PGEAs with pH responsivity and degradation. In weakly acidic endosome, the broken acetal linkers resulted in decomposition of A-CD-PGEAs and morphological transformation of A-CD-PGEAs/pDNA complexes, lowering cytotoxicity and accelerating release of pDNA. In comparison with control CD-PGEAs without acetal linkers, A-CD-PGEAs exhibited significantly better transfection performances. PMID:25993557

  15. cGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone.

    PubMed Central

    Penson, S. P.; Schuurink, R. C.; Fath, A.; Gubler, F.; Jacobsen, J. V.; Jones, R. L.

    1996-01-01

    The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell. PMID:12239379

  16. Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.

    PubMed Central

    Widom, R L; Rhee, M; Karathanasis, S K

    1992-01-01

    The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha

  17. Validation of reference genes for quantitative real-time PCR in valproic acid rat models of autism.

    PubMed

    Zhou, Jinlong; Zhang, Xiaozheng; Ren, Junrong; Wang, Ping; Zhang, Junfeng; Wei, Zhaoming; Tian, Yingfang

    2016-08-01

    Autism is a neurodevelopmental disorder, and embryonic exposure to valproic acid (VPA) in rodents is the most frequently studied environmentally triggered autism models. Valproic acid can affect gene transcription as a histone deacetylase inhibitor, and thus may alter the expression of the most genes including reference genes. The aim of the current study is to validate suitable reference genes for quantitative real-time PCR (qPCR) quantification in prefrontal cortex and hippocampus of VPA rat models of autism. Female rats received a single intraperitoneal injection of 400 mg/kg sodium VPA at day 12.5 post-conception and controls were injected with saline. Male offspring were used to observe the expression of nine commonly used reference genes by qPCR, and the data were analyzed by four commonly used reference selection program including geNorm, BestKeeper, NormFinder and RefFinder. The results showed that VPA affected the expression of these commonly used reference genes in prefrontal cortex and hippocampus on postnatal 3, 5 weeks and 80 days, Gapdh and Actin, two very frequently used reference genes, were identified as the least stable genes in VPA group. Hprt1 was selected as the most stable gene, and Hmbs and Tbp were the optimum gene pair in prefrontal cortex and hippocampus across all VPA and controls. Problematically, the use of unstable reference genes results in calculation of different PGRN mRNA expression levels. The results suggest that selection of suitable references is critical for accurate mRNA quantification, and specifically in VPA induced rat models of autism. PMID:27287459

  18. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to ...

  19. Induction of liver alpha-1 acid glycoprotein gene expression involves both positive and negative transcription factors.

    PubMed Central

    Lee, Y M; Tsai, W H; Lai, M Y; Chen, D S; Lee, S C

    1993-01-01

    Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutagenesis coupled with transfection analysis indicated that AGP/EBP binding to all of these sites resulted in positive regulation of the promoter. Dose-response data suggest that AGP/EBP binding to these sites results in the cooperative activation of the promoter. In contrast, functional assays showed that element B is a negative regulatory element; this element is recognized by heat-stable DNA-binding factors which are found in many cells and tissues. The regulation of these binding proteins was studied in rat liver treated with lipopolysaccharide (LPS), which induced an acute-phase reaction. We found that LPS treatment resulted in a two- to threefold increase in AGP/EBP activity and a severalfold decrease in the activity of factors that bind to element B in the liver. These results indicate that expression of the AGP gene can be regulated by both positive and negative factors and that the modulation of these factors can account for the LPS induction of the AGP gene. Images PMID:8417341

  20. Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

    PubMed

    Chen, Ming; Liu, Minghua; Yu, Lili; Cai, Guoru; Chen, Qinghai; Wu, Rong; Wang, Feng; Zhang, Bo; Jiang, Tianlun; Fu, Welling

    2005-08-01

    A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples. PMID:16193990

  1. Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato.

    PubMed

    Yu, Chao; Wang, Hua-Sen; Yang, Sha; Tang, Xian-Feng; Duan, Ming; Meng, Qing-Wei

    2009-01-01

    An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 degrees C), but inhibited by high temperature (40 degrees C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F(v)/F(m)) and the O(2) evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress. PMID:19648018

  2. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    SciTech Connect

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  3. Association of novel SNPs in the candidate genes affecting caprine milk fatty acids related to human health

    PubMed Central

    Dixit, S.P.; Sivalingam, Jayakumar; Tyagi, A.K.; Saroha, V.; Sharma, A.; Nagda, R.K.

    2015-01-01

    In the present investigation, 618 milk samples of Sirohi breed of goat were collected, and analyzed for conjugated linoleic acid (CLA, C18:2) and other fatty acids. The CLA in studied goat milk samples was 4.87 mg/g of milk fat and C18:2 cis-9, trans-11 contributes 2.9 mg/g of milk fat and trans10 cis12 contributes 0.82 mg/g of milk fat. The saturated fatty acids in the milk accounted for 69.55% and unsaturated fatty acid accounted for 28.50%. The unsaturated fatty acid was constituted by monounsaturated fatty acid (24.57%) and polyunsaturated fatty acids (3.96%.). The major contribution (45.56%) in total fatty acid was of C12:0, C14:0 and C16:0. C18:0 and short chain ones (C4:0, C6:0, C8:0, and C10:0) have a neutral or cholesterol-decreasing effect. The DNA sequence analysis of the genes (DGAT1, SCAP, PPARG, OLR, FABP3 and PRL) in a random panel of 8 Sirohi goats revealed 38 SNPs across the targeted regions. Out of the studied SNPs (38) across these genes, 22 SNPs had significant effect on one or a group of fatty acids including CLA. The genotypes at these loci showed significant differences in the least square means of a particular fatty acid or a group of fatty acids including CLA and its isomers. PMID:25853060

  4. Association of novel SNPs in the candidate genes affecting caprine milk fatty acids related to human health.

    PubMed

    Dixit, S P; Sivalingam, Jayakumar; Tyagi, A K; Saroha, V; Sharma, A; Nagda, R K

    2015-06-01

    In the present investigation, 618 milk samples of Sirohi breed of goat were collected, and analyzed for conjugated linoleic acid (CLA, C18:2) and other fatty acids. The CLA in studied goat milk samples was 4.87 mg/g of milk fat and C18:2 cis-9, trans-11 contributes 2.9 mg/g of milk fat and trans10 cis12 contributes 0.82 mg/g of milk fat. The saturated fatty acids in the milk accounted for 69.55% and unsaturated fatty acid accounted for 28.50%. The unsaturated fatty acid was constituted by monounsaturated fatty acid (24.57%) and polyunsaturated fatty acids (3.96%.). The major contribution (45.56%) in total fatty acid was of C12:0, C14:0 and C16:0. C18:0 and short chain ones (C4:0, C6:0, C8:0, and C10:0) have a neutral or cholesterol-decreasing effect. The DNA sequence analysis of the genes (DGAT1, SCAP, PPARG, OLR, FABP3 and PRL) in a random panel of 8 Sirohi goats revealed 38 SNPs across the targeted regions. Out of the studied SNPs (38) across these genes, 22 SNPs had significant effect on one or a group of fatty acids including CLA. The genotypes at these loci showed significant differences in the least square means of a particular fatty acid or a group of fatty acids including CLA and its isomers. PMID:25853060

  5. Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

    PubMed

    Xie, Yun; Zhao, Jun-Long; Wang, Chuan-Wei; Yu, Ai-Xin; Liu, Niu; Chen, Li; Lin, Fei; Xu, Han-Hong

    2016-05-18

    Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values <27) were analyzed at 1, 3, and 6 h after GlyF treatment by RT-qPCR. The results demonstrated that expression levels of four AAT genes, RcLHT6, RcANT15, RcProT2, and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional

  6. Type III Secretion System Genes of Dickeya dadantii 3937 Are Induced by Plant Phenolic Acids

    PubMed Central

    Yang, Shihui; Peng, Quan; San Francisco, Michael; Wang, Yongjun; Zeng, Quan; Yang, Ching-Hong

    2008-01-01

    Background Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many Gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified. Methodology/Principal Findings In this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a σ54-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA. Conclusion/Significance The induction of T3SS expression by OCA and TCA is moderated through the rsmB-RsmA pathway. This is the first report of plant phenolic compounds that induce the expression T3SS genes of plant pathogenic bacteria. PMID:18698421

  7. Gene cloning and functional analysis of a second delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus.

    PubMed

    Sakuradani, Eiji; Shimizu, Sakayu

    2003-04-01

    We demonstrated that Mortierella alpina 1S-4 has two delta 6-desaturases, which are involved in the desaturation of linoleic acid to gamma-linolenic acid. For one of the two delta 6-desaturases, designated as delta 6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two delta 6-desaturases, designated as delta 6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella delta 6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast delta 9-desaturase, which has the corresponding domain at the C-terminus. The full-length delta 6II cDNA was expressed in A. oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quantity of delta 6I RNA was 2.4-, 9-, and 17-fold higher than that of delta 6II RNA on 2, 3, and 4 days in M. alpina 1S-4, respectively. M. alpina 1S-4 is the first fungus to be confirmed to have two functional delta 6-desaturase genes. PMID:12784608

  8. Differential regulation of ParaHox genes by retinoic acid in the invertebrate chordate amphioxus (Branchiostoma floridae).

    PubMed

    Osborne, Peter W; Benoit, Gérard; Laudet, Vincent; Schubert, Michael; Ferrier, David E K

    2009-03-01

    The ParaHox cluster is the evolutionary sister to the Hox cluster. Like the Hox cluster, the ParaHox cluster displays spatial and temporal regulation of the component genes along the anterior/posterior axis in a manner that correlates with the gene positions within the cluster (a feature called collinearity). The ParaHox cluster is however a simpler system to study because it is composed of only three genes. We provide a detailed analysis of the amphioxus ParaHox cluster and, for the first time in a single species, examine the regulation of the cluster in response to a single developmental signalling molecule, retinoic acid (RA). Embryos treated with either RA or RA antagonist display altered ParaHox gene expression: AmphiGsx expression shifts in the neural tube, and the endodermal boundary between AmphiXlox and AmphiCdx shifts its anterior/posterior position. We identified several putative retinoic acid response elements and in vitro assays suggest some may participate in RA regulation of the ParaHox genes. By comparison to vertebrate ParaHox gene regulation we explore the evolutionary implications. This work highlights how insights into the regulation and evolution of more complex vertebrate arrangements can be obtained through studies of a simpler, unduplicated amphioxus gene cluster. PMID:19103191

  9. Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

    PubMed Central

    Wang, Ju; Lin, Jianxun; Zhao, Minyan

    2015-01-01

    Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO. PMID:25815733

  10. Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

    SciTech Connect

    Zhang, P.; Shanklin, J.; Burton, J. W.; Upchurch, R. G.; Whittle, E.; Dewey, R. E.

    2008-11-01

    Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoform of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.

  11. The role of invertases in plant compensatory responses to simulated herbivory

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of a plant to recover from mammalian herbivory by exhibiting enhanced growth and reproduction compared to unharmed plants, is called compensation. Although it is clear that genetic variation for compensation exists, little is known about the specific genes underpinnings leading this fitn...

  12. Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza

    PubMed Central

    Xu, Zhichao; Luo, Hongmei; Ji, Aijia; Zhang, Xin; Song, Jingyuan; Chen, Shilin

    2016-01-01

    Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. PMID:26904067

  13. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

    PubMed

    Sirikantaramas, Supaart; Morimoto, Satoshi; Shoyama, Yoshinari; Ishikawa, Yu; Wada, Yoshiko; Shoyama, Yukihiro; Taura, Futoshi

    2004-09-17

    Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis. PMID:15190053

  14. Oleic acid induces specific alterations in the morphology, gene expression and steroid hormone production of cultured bovine granulosa cells.

    PubMed

    Yenuganti, Vengala Rao; Viergutz, Torsten; Vanselow, Jens

    2016-06-01

    After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation. PMID:27118706

  15. Integrated Systems Biology Analysis of Transcriptomes Reveals Candidate Genes for Acidity Control in Developing Fruits of Sweet Orange (Citrus sinensis L. Osbeck)

    PubMed Central

    Huang, Dingquan; Zhao, Yihong; Cao, Minghao; Qiao, Liang; Zheng, Zhi-Liang

    2016-01-01

    Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). Fruits of these varieties at 45 days post anthesis (DPA), which corresponds to Stage I (cell division), had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion). Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis). Our network analysis shows that the acid-correlated genes belong to three distinct network modules. Several of these candidate fruit acidity genes encode regulatory proteins involved in transport (such as AHA10), degradation (such as APD2) and transcription (such as AIL6) and act as hubs in the citrate accumulation gene networks. Taken together, our integrated systems biology analysis has provided new insights into the fruit citrate accumulation gene network and led to the identification of candidate genes likely associated with the fruit acidity control. PMID:27092171

  16. Long-range regulation by shared retinoic acid response elements modulates dynamic expression of posterior Hoxb genes in CNS development.

    PubMed

    Ahn, Youngwook; Mullan, Hillary E; Krumlauf, Robb

    2014-04-01

    Retinoic acid (RA) signaling plays an important role in determining the anterior boundary of Hox gene expression in the neural tube during embryogenesis. In particular, RA signaling is implicated in a rostral expansion of the neural expression domain of 5׳ Hoxb genes (Hoxb9-Hoxb5) in mice. However, underlying mechanisms for this gene regulation have remained elusive due to the lack of RA responsive element (RARE) in the 5׳ half of the HoxB cluster. To identify cis-regulatory elements required for the rostral expansion, we developed a recombineering technology to serially label multiple genes with different reporters in a single bacterial artificial chromosome (BAC) vector containing the mouse HoxB cluster. This allowed us to simultaneously monitor the expression of multiple genes. In contrast to plasmid-based reporters, transgenic BAC reporters faithfully recapitulated endogenous gene expression patterns of the Hoxb genes including the rostral expansion. Combined inactivation of two RAREs, DE-RARE and ENE-RARE, in the BAC completely abolished the rostral expansion of the 5׳ Hoxb genes. Knock-out of endogenous DE-RARE lead to significantly reduced expression of multiple Hoxb genes and attenuated Hox gene response to exogenous RA treatment in utero. Regulatory potential of DE-RARE was further demonstrated by its ability to anteriorize 5׳ Hoxa gene expression in the neural tube when inserted into a HoxA BAC reporter. Our data demonstrate that multiple RAREs cooperate to remotely regulate 5׳ Hoxb genes during CNS development, providing a new insight into the mechanisms for gene regulation within the Hox clusters. PMID:24525295

  17. Long-term storage of low-invertase Katahdin at different temperature set points

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Successful storage of processing potatoes balances the risk of crop loss caused by pathogen-dependent spoilage with the need to maintain the low tuber reducing sugar contents that are required to produce light-colored fried products. Potato varieties with very low expression of the vacuolar acid inv...

  18. Fatty acid-binding protein (fabp) genes of spotted green pufferfish (Tetraodon nigroviridis): comparative genomics and spatial transcriptional regulation.

    PubMed

    Thirumaran, Aruloli; Wright, Jonathan M

    2014-05-01

    The fatty acid-binding protein (fabp) genes belong to the multigene family of intracellular lipid-binding proteins. To date, 12 different FABPs have been identified in vertebrate genomes. Owing to the teleost-specific genome duplication event, many fishes have duplicated copies of the fabp genes. Here, we identified and characterized the fabp genes of spotted green pufferfish (Tetraodon nigroviridis). Seven fabp genes were identified, out of which, two were retained in the pufferfish genome as duplicated copies. Each putative pufferfish Fabp protein shares greatest sequence identity and similarity with their teleost and tetrapod orthologs, and clustered together as a distinct clade in phylogenetic analysis. Conserved gene synteny was evident between the pufferfish fabp genes and the orthologs of human, zebrafish, three-spined stickleback, and medaka FABP/fabp genes, providing evidence that the duplicated copies of pufferfish fabp genes most likely arose as a result of the teleost-specific genome duplication event. The differential tissue-specific distribution of pufferfish fabp transcripts suggests divergent spatial regulation of duplicated pairs of fabp genes. PMID:25153522

  19. Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid.

    PubMed

    Stevenson, G; Andrianopoulos, K; Hobbs, M; Reeves, P R

    1996-08-01

    Colanic acid (CA) is an extracellular polysaccharide produced by most Escherichia coli strains as well as by other species of the family Enterobacteriaceae. We have determined the sequence of a 23-kb segment of the E. coli K-12 chromosome which includes the cluster of genes necessary for production of CA. The CA cluster comprises 19 genes. Two other sequenced genes (orf1.3 and galF), which are situated between the CA cluster and the O-antigen cluster, were shown to be unnecessary for CA production. The CA cluster includes genes for synthesis of GDP-L-fucose, one of the precursors of CA, and the gene for one of the enzymes in this pathway (GDP-D-mannose 4,6-dehydratase) was identified by biochemical assay. Six of the inferred proteins show sequence similarity to glycosyl transferases, and two others have sequence similarity to acetyl transferases. Another gene (wzx) is predicted to encode a protein with multiple transmembrane segments and may function in export of the CA repeat unit from the cytoplasm into the periplasm in a process analogous to O-unit export. The first three genes of the cluster are predicted to encode an outer membrane lipoprotein, a phosphatase, and an inner membrane protein with an ATP-binding domain. Since homologs of these genes are found in other extracellular polysaccharide gene clusters, they may have a common function, such as export of polysaccharide from the cell. PMID:8759852

  20. Identification of differences in human and great ape phytanic acid metabolism that could influence gene expression profiles and physiological functions

    PubMed Central

    2010-01-01

    Background It has been proposed that anatomical differences in human and great ape guts arose in response to species-specific diets and energy demands. To investigate functional genomic consequences of these differences, we compared their physiological levels of phytanic acid, a branched chain fatty acid that can be derived from the microbial degradation of chlorophyll in ruminant guts. Humans who accumulate large stores of phytanic acid commonly develop cerebellar ataxia, peripheral polyneuropathy, and retinitis pigmentosa in addition to other medical conditions. Furthermore, phytanic acid is an activator of the PPAR-alpha transcription factor that influences the expression of genes relevant to lipid metabolism. Results Despite their trace dietary phytanic acid intake, all great ape species had elevated red blood cell (RBC) phytanic acid levels relative to humans on diverse diets. Unlike humans, chimpanzees showed sexual dimorphism in RBC phytanic acid levels, which were higher in males relative to females. Cultured skin fibroblasts from all species had a robust capacity to degrade phytanic acid. We provide indirect evidence that great apes, in contrast to humans, derive significant amounts of phytanic acid from the hindgut fermentation of plant materials. This would represent a novel reduction of metabolic activity in humans relative to the great apes. Conclusion We identified differences in the physiological levels of phytanic acid in humans and great apes and propose this is causally related to their gut anatomies and microbiomes. Phytanic acid levels could contribute to cross-species and sex-specific differences in human and great ape transcriptomes, especially those related to lipid metabolism. Based on the medical conditions caused by phytanic acid accumulation, we suggest that differences in phytanic acid metabolism could influence the functions of human and great ape nervous, cardiovascular, and skeletal systems. PMID:20932325

  1. Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat

    PubMed Central

    Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation. PMID:27148345

  2. Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat.

    PubMed

    Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation. PMID:27148345

  3. Evolutionary Distance of Amino Acid Sequence Orthologs across Macaque Subspecies: Identifying Candidate Genes for SIV Resistance in Chinese Rhesus Macaques

    PubMed Central

    Ross, Cody T.; Roodgar, Morteza; Smith, David Glenn

    2015-01-01

    We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIVmac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIVmac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIVmac in Chinese rhesus macaques. PMID:25884674

  4. Evolutionary distance of amino acid sequence orthologs across macaque subspecies: identifying candidate genes for SIV resistance in Chinese rhesus macaques.

    PubMed

    Ross, Cody T; Roodgar, Morteza; Smith, David Glenn

    2015-01-01

    We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIVmac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIVmac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIVmac in Chinese rhesus macaques. PMID:25884674

  5. EFFECTS OF HEAT AND BROMOCHLOROACETIC ACID ON MALE REPRODUCTION IN HEAT SHOCK FACTOR-1 GENE KNOCKOUT MICE

    EPA Science Inventory

    Effects of heat and bromochloroacetic acid on male reproduction in heat shock factor-1 gene knockout mice.
    Luft JC1, IJ Benjamin2, JB Garges1 and DJ Dix1. 1Reproductive Toxicology Division, USEPA, RTP, NC, 27711 and 2Dept of Internal Medicine, Univ.of Texas Southwestern Med C...

  6. Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.

    PubMed

    Ehara, Tatsuya; Kamei, Yasutomi; Yuan, Xunmei; Takahashi, Mayumi; Kanai, Sayaka; Tamura, Erina; Tsujimoto, Kazutaka; Tamiya, Takashi; Nakagawa, Yoshimi; Shimano, Hitoshi; Takai-Igarashi, Takako; Hatada, Izuho; Suganami, Takayoshi; Hashimoto, Koshi; Ogawa, Yoshihiro

    2015-03-01

    The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage-specific DNA demethylation of a particular metabolic pathway. PMID:25311726

  7. Adaptation to an automated platform of algorithmic combinations of advantageous mutations in genes generated using amino acid scanning mutational strategy.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent mutational strategies for generating and screening of genes for optimized traits, including directed evolution, domain shuffling, random mutagenesis, and site-directed mutagenesis, have been adapted for automated platforms. Here we discuss the amino acid scanning mutational strategy and its ...

  8. [Cloning and expression of a delta6-fatty acid desaturase gene from Rhizopus stolonifer in Saccharomyces cervisiae].

    PubMed

    Lu, He; Chai, You-rong; Zhang, Xue-kun; Lei, Tian-gang; Li, Jia-na

    2007-02-01

    Fatty acid composition of fungi is analysed through the gas chromatography( GC) technique. With specific activity a novel enzyme delta6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R. stolonifer was identified as a fungal species that produced plentiful gamma-linolenic acid. A 1475bp full-length cDNA, designated as RnD6D here, with high homology to fungal delta6-fatty acid desaturase genes was isolated from R. stolonifer using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1380bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. To elucidate the function of this novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to gamma-linolenic acid(GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced delta6-fatty acid desaturase activity of RnD6D which led to the accumulation of gamma-linolenic acid. PMID:17436625

  9. Supplementation of essential fatty acids to Holstein calves during late uterine life and first month of life alters hepatic fatty acid profile and gene expression.

    PubMed

    Garcia, M; Greco, L F; Lock, A L; Block, E; Santos, J E P; Thatcher, W W; Staples, C R

    2016-09-01

    Linoleic acid is an essential dietary fatty acid (FA). However, how the supplementation of linoleic acid during uterine and early life may modify the FA profile and transcriptome regulation of the liver, and performance of preweaned dairy calves is unknown. Our objective was to evaluate the effect of supplementation of essential FA to Holstein calves during late uterine and early life on their hepatic FA profile and global gene expression at 30 d of age. During the last 8 wk of pregnancy, Holstein cattle (n=96) were fed either no fat supplement (control), a saturated FA supplement enriched with C18:0, or an unsaturated FA supplement enriched with linoleic acid. Male calves (n=40) born from these dams were fed a milk replacer (MR) with either low (LLA) or high linoleic acid (HLA) concentration as the sole feedstuff during the first 30 d. Liver biopsy was performed at 30 d of age, and microarray analysis was performed on 18 liver samples. Total concentration of FA in liver were greater in calves fed LLA compared with those fed HLA MR (8.2 vs. 7.1%), but plasma concentrations of total FA did not differ due to MR diets. The FA profiles of plasma and liver of calves were affected differently by the prepartum diets. Specifically, the FA profile in liver was affected moderately by the feeding of fat prepartum, but the profiles did not differ due to the type of FA fed prepartum. The type of MR fed during the first 30 d of life had major effects on both plasma and liver FA profiles, resembling the type of fat fed. Plasma and liver of calves fed LLA MR had greater percentage of medium-chain FA (C12:0 and C14:0), whereas plasma and liver from calves fed HLA MR had greater percentages of linoleic and α-linolenic acids. Dams fed fat or a specific type of FA modified the expression of some genes in liver of calves, particularly those genes involved in biological functions and pathways related to upregulation of lipid metabolism and downregulation of inflammatory responses

  10. Uncovering co-expression gene network regulating fruit acidity in diverse apples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that encodes an Aluminum-activated Malate Transporter1 (...

  11. Evaluation of FABP2 as candidate gene for a fatty acid composition QTL in porcine chromosome 8.

    PubMed

    Estellé, J; Mercadé, A; Pérez-Enciso, M; Pena, R N; Silió, L; Sánchez, A; Folch, J M

    2009-02-01

    The objective of this work was to analyse the porcine Fatty acid binding protein 2, intestinal (FABP2) gene as a candidate gene for a fatty acid composition quantitative trait loci (QTL) previously described on porcine chromosome 8 in an Iberian by Landrace F(2) cross (IBMAP). Re-sequencing of the porcine FABP2 gene in three Iberian and eight Landrace parental animals resulted in the identification of three single-nucleotide polymorphisms, all of them localized in intron 1. The polymorphism FABP2:g.412T>C, localized in intron 1, and two additional microsatellites were genotyped in the IBMAP population in order to perform an association test of the FABP2 gene and to better define the QTL position previously described. Association analyses of the FABP2:g.412T>C with the fatty acid composition traits were not significant in simple association and marker-assisted association tests, suggesting that the FABP2 region sequenced is not responsible for the QTL. However, the addition of three new markers to the pedigree allowed us to define the S0144-SW61 marker interval as the most likely QTL position, facilitating the future study of other candidate genes for this QTL. PMID:19207930

  12. Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer.

    PubMed

    Walters, R W; Yi, S M; Keshavjee, S; Brown, K E; Welsh, M J; Chiorini, J A; Zabner, J

    2001-06-01

    Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors. PMID:11262413

  13. Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

    PubMed Central

    James, D W; Lim, E; Keller, J; Plooy, I; Ralston, E; Dooner, H K

    1995-01-01

    The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues. PMID:7734965

  14. Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply

    PubMed Central

    Zeng, Mu-Heng; Liu, Sheng-Hong; Yang, Miao-Xian; Zhang, Ya-Jun; Liang, Jia-Yong; Wan, Xiao-Rong; Liang, Hong

    2013-01-01

    Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures. PMID:23880865

  15. Influence of ω-3 fatty acid eicosapentaenoic acid on IGF-1 and COX-2 gene expression in granulosa cells of PCOS women

    PubMed Central

    Shahnazi, Vahideh; Zaree, Mina; Nouri, Mohammad; Mehrzad-Sadaghiani, Mahzad; Fayezi, Shabnam; Darabi, Maryam; Khani, Sajjad; Darabi, Masoud

    2015-01-01

    Background: The omega-3 (ω-3) fatty acid eicosapentaenoic acid (EPA) is currently used in the clinic as a nutritional supplement to improve infertility, particularly in women with polycystic ovarian syndrome (PCOS). Objective: The present study was designed to investigate the effect of EPA on insulin-like growth factor 1 (IGF-1) and cyclooxygenase 2 (COX-2) gene expression in primary cultured granulosa cells from patients undergoing in vitro fertilization (IVF), and also to compare this effect with those in granulosa cells of PCOS patients. Materials and Methods: In this experimental study, human granulosa cells were isolated from follicular fluid of normal and PCOS women undergoing IVF by hyaluronidase digestions, followed by Percoll gradient centrifugation. Cells were cultured in vitro, exposed to a range of concentrations of the EPA (25-100 µM) for 24 hr, and investigated with respect to COX-2 and IGF-1 gene expression by real time-PCR. Results: In both groups, all doses of the EPA significantly induced IGF-1 mRNA gene expression compared to the untreated control. High doses of EPA in the presence of recombinant (r) FSH produced a stimulatory effect on IGF-1 and a suppressive effect (p=0.01) on the COX-2 gene expression, which were more pronounced in granulosa cells from PCOS patients. Conclusion: EPA affect diversely the gene expression of IGF-1 and COX-2 in granulosa cells, which were more pronounced in PCOS compared to control. These findings represent the possible underlying molecular mechanisms for the positive impact of the ω-3 fatty acids on reproduction, especially in patients with PCOS. PMID:25999995

  16. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae.

    PubMed

    Didion, T; Regenberg, B; Jørgensen, M U; Kielland-Brandt, M C; Andersen, H A

    1998-02-01

    Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D-leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium. PMID:9489675

  17. Acid detergent lignin, lodging resistance index, and expression of the caffeic acid O-methyltransferase gene in brown midrib-12 sudangrass

    PubMed Central

    Li, Yuan; Liu, Guibo; Li, Jun; You, Yongliang; Zhao, Haiming; Liang, Huan; Mao, Peisheng

    2015-01-01

    Understanding the relationship between acid detergent lignin (ADL) and lodging resistance index (LRI) is essential for breeding new varieties of brown midrib (bmr) sudangrass (Sorghum sudanense (Piper) Stapf.). In this study, bmr-12 near isogenic lines and their wild-types obtained by back cross breeding were used to compare relevant forage yield and quality traits, and to analyze expression of the caffeic acid O-methyltransferase (COMT) gene using quantitative real time-PCR. The research showed that the mean ADL content of bmr-12 mutants (20.94 g kg−1) was significantly (P < 0.05) lower than measured in N-12 lines (43.45 g kg−1), whereas the LRI of bmr-12 mutants (0.29) was significantly (P < 0.05) higher than in N-12 lines (0.22). There was no significant correlation between the two indexes in bmr-12 materials (r = −0.44, P > 0.05). Sequence comparison of the COMT gene revealed two point mutations present in bmr-12 but not in the wild-type, the second mutation changed amino acid 129 of the protein from Gln (CAG) to a stop codon (UAG). The relative expression level of COMT gene was significantly reduced, which likely led to the decreased ADL content observed in the bmr-12 mutant. PMID:26366111

  18. Changes in PTGS1 and ALOX12 Gene Expression in Peripheral Blood Mononuclear Cells Are Associated with Changes in Arachidonic Acid, Oxylipins, and Oxylipin/Fatty Acid Ratios in Response to Omega-3 Fatty Acid Supplementation

    PubMed Central

    Berthelot, Claire C.; Kamita, Shizuo George; Sacchi, Romina; Yang, Jun; Nording, Malin L.; Georgi, Katrin; Hegedus Karbowski, Christine; German, J. Bruce; Weiss, Robert H.; Hogg, Ronald J.; Hammock, Bruce D.; Zivkovic, Angela M.

    2015-01-01

    Introduction There is a high degree of inter-individual variability among people in response to intervention with omega-3 fatty acids (FA), which may partly explain conflicting results on the effectiveness of omega-3 FA for the treatment and prevention of chronic inflammatory diseases. In this study we sought to evaluate whether part of this inter-individual variability in response is related to the regulation of key oxylipin metabolic genes in circulating peripheral blood mononuclear cells (PBMCs). Methods Plasma FA and oxylipin profiles from 12 healthy individuals were compared to PBMC gene expression profiles following six weeks of supplementation with fish oil, which delivered 1.9 g/d eicosapentaenoic acid (EPA) and 1.5 g/d docosahexaenoic acid (DHA). Fold changes in gene expression were measured by a quantitative polymerase chain reaction (qPCR). Results Healthy individuals supplemented with omega-3 FA had differential responses in prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), and interleukin 8 (IL-8) gene expression in isolated PBMCs. In those individuals for whom plasma arachidonic acid (ARA) in the phosphatidylethanolamine (PE) lipid class decreased in response to omega-3 intervention, there was a corresponding decrease in gene expression for PTGS1 and ALOX12. Several oxylipin product/FA precursor ratios (e.g. prostaglandin E2 (PGE2)/ARA for PTGS1 and 12-hydroxyeicosatetraenoic acid (12-HETE)/ARA for ALOX12) were also associated with fold change in gene expression, suggesting an association between enzyme activity and gene expression. The fold-change in PTGS1 gene expression was highly positively correlated with ALOX12 gene expression but not with PTGS2, whereas IL-8 and PTGS2 were positively correlated. Conclusions The regulation of important oxylipin metabolic genes in PBMCs varied with the extent of change in ARA concentrations in the case of PTGS1 and ALOX12

  19. Exploiting natural variation of secondary metabolism identifies a gene controlling the glycosylation diversity of dihydroxybenzoic acids in Arabidopsis thaliana.

    PubMed

    Li, Xu; Svedin, Elisabeth; Mo, Huaping; Atwell, Susanna; Dilkes, Brian P; Chapple, Clint

    2014-11-01

    Plant secondary metabolism is an active research area because of the unique and important roles the specialized metabolites have in the interaction of plants with their biotic and abiotic environment, the diversity and complexity of the compounds and their importance to human medicine. Thousands of natural accessions of Arabidopsis thaliana characterized with increasing genomic precision are available, providing new opportunities to explore the biochemical and genetic mechanisms affecting variation in secondary metabolism within this model species. In this study, we focused on four aromatic metabolites that were differentially accumulated among 96 Arabidopsis natural accessions as revealed by leaf metabolic profiling. Using UV, mass spectrometry, and NMR data, we identified these four compounds as different dihydroxybenzoic acid (DHBA) glycosides, namely 2,5-dihydroxybenzoic acid (gentisic acid) 5-O-β-D-glucoside, 2,3-dihydroxybenzoic acid 3-O-β-D-glucoside, 2,5-dihydroxybenzoic acid 5-O-β-D-xyloside, and 2,3-dihydroxybenzoic acid 3-O-β-D-xyloside. Quantitative trait locus (QTL) mapping using recombinant inbred lines generated from C24 and Col-0 revealed a major-effect QTL controlling the relative proportion of xylosides vs. glucosides. Association mapping identified markers linked to a gene encoding a UDP glycosyltransferase gene. Analysis of Transfer DNA (T-DNA) knockout lines verified that this gene is required for DHBA xylosylation in planta and recombinant protein was able to xylosylate DHBA in vitro. This study demonstrates that exploiting natural variation of secondary metabolism is a powerful approach for gene function discovery. PMID:25173843

  20. Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation

    PubMed Central

    Fu, Kai-Yun; Guo, Wen-Chao; Ahmat, Tursun; Li, Guo-Qing

    2015-01-01

    A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis. PMID:26657797

  1. Contrasting microbial functional genes in two distinct saline-alkali and slightly acidic oil-contaminated sites.

    PubMed

    Liang, Yuting; Zhao, Huihui; Zhang, Xu; Zhou, Jizhong; Li, Guanghe

    2014-07-15

    To compare the functional gene structure and diversity of microbial communities in saline-alkali and slightly acidic oil-contaminated sites, 40 soil samples were collected from two typical oil exploration sites in North and South China and analyzed with a comprehensive functional gene array (GeoChip 3.0). The overall microbial pattern was significantly different between the two sites, and a more divergent pattern was observed in slightly acidic soils. Response ratio was calculated to compare the microbial functional genes involved in organic contaminant degradation and carbon, nitrogen, phosphorus, and sulfur cycling. The results indicated a significantly low abundance of most genes involved in organic contaminant degradation and in the cycling of nitrogen and phosphorus in saline-alkali soils. By contrast, most carbon degradation genes and all carbon fixation genes had similar abundance at both sites. Based on the relationship between the environmental variables and microbial functional structure, pH was the major factor influencing the microbial distribution pattern in the two sites. This study demonstrated that microbial functional diversity and heterogeneity in oil-contaminated environments can vary significantly in relation to local environmental conditions. The limitation of nitrogen and phosphorus and the low degradation capacity of organic contaminant should be carefully considered, particularly in most oil-exploration sites with saline-alkali soils. PMID:24784752

  2. Retinoid acid-related orphan receptor γ, RORγ, participates in diurnal transcriptional regulation of lipid metabolic genes

    PubMed Central

    Takeda, Yukimasa; Kang, Hong Soon; Lih, Fred B.; Jiang, Hongfeng; Blaner, William S.; Jetten, Anton M.

    2014-01-01

    The hepatic circadian clock plays a pivotal role in regulating major aspects of energy homeostasis and lipid metabolism. In this study, we show that RORγ robustly regulates the rhythmic expression of several lipid metabolic genes, including the insulin-induced gene 2a, Insig2a, elongation of very long chain fatty acids-like 3, Elovl3 and sterol 12α-hydroxylase, Cyp8b1, by enhancing their expression at ZT20-4. The time-dependent increase in their expression correlates with the rhythmic expression pattern of RORγ. The enhanced recruitment of RORγ to ROREs in their promoter region, increased histone acetylation, and reporter and mutation analysis support the concept that RORγ regulates the transcription of several lipid metabolic genes directly by binding ROREs in their promoter regulatory region. Consistent with the disrupted expression of a number of lipid metabolic genes, loss of RORγ reduced the level of several lipids in liver and blood in a ZT-preferred manner. Particularly the whole-body bile acid pool size was considerably reduced in RORγ−/− mice in part through its regulation of several Cyp genes. Similar observations were made in liver-specific RORγ-deficient mice. Altogether, our study indicates that RORγ functions as an important link between the circadian clock and the transcriptional regulation of several metabolic genes. PMID:25143535

  3. MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

    PubMed

    Garzon, R; Pichiorri, F; Palumbo, T; Visentini, M; Aqeilan, R; Cimmino, A; Wang, H; Sun, H; Volinia, S; Alder, H; Calin, G A; Liu, C-G; Andreeff, M; Croce, C M

    2007-06-14

    MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively. PMID:17260024

  4. CsSAD: a fatty acid desaturase gene involved in abiotic resistance in Camellia sinensis (L.).

    PubMed

    Ding, Z T; Shen, J Z; Pan, L L; Wang, Y U; Li, Y S; Wang, Y; Sun, H W

    2016-01-01

    Tea (Camellia sinensis L.) is a thermophilic evergreen woody plant that has poor cold tolerance. The SAD gene plays a key role in regulating fatty acid synthesis and membrane lipid fluidity in response to temperature change. In this study, full-length SAD cDNA was cloned from tea leaves using rapid amplification of cDNA ends and polymerase chain reaction (PCR)-based methods. Sequence analysis demonstrated that CsSAD had a high similarity to other corresponding cDNAs. At 25°C, the CsSAD transcriptional level was highest in the leaf and lowest in the stem, but there was no obvious difference between the root and stem organs. CsSAD expression was investigated by reverse transcription-PCR, which showed that CsSAD was upregulated at 4° and -5°C. At 25°C, CsSAD was induced by polyethylene glycol, abscisic acid, and wounding, and a similar trend was observed at 4°C, but the mean expression level at 4°C was lower than that at 25°C. Under natural cold acclimation, the 'CsCr05' variety's CsSAD expression level increased before decreasing. The CsSAD expression level in variety 'CsCr06' showed no obvious change at first, but rapidly increased to a maximum when the temperature was very low. Our study demonstrates that CsSAD is upregulated in response to different abiotic conditions, and that it is important to study the stress resistance of the tea plant, particularly in response to low temperature, drought, and wounding. PMID:26985937

  5. Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I.

    PubMed

    Yan, Qi; Liu, Qin; Li, Meng-Meng; Li, Fang-Hui; Zhu, Bin; Wang, Jun-Zhong; Lu, Yin-Ping; Liu, Jia; Wu, Jun; Zheng, Xin; Lu, Meng-Ji; Wang, Bao-Ju; Yang, Dong-Liang

    2016-06-01

    Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection. PMID:27376800

  6. Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria.

    PubMed Central

    Platteeuw, C; Simons, G; de Vos, W M

    1994-01-01

    A transcriptional fusion vector, designated pNZ272, based on the promoterless beta-glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed beta-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific beta-glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied. Images PMID:8135517

  7. Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid1[W

    PubMed Central

    Sonnante, Gabriella; D'Amore, Rosalinda; Blanco, Emanuela; Pierri, Ciro L.; De Palma, Monica; Luo, Jie; Tucci, Marina; Martin, Cathie

    2010-01-01

    Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes. PMID:20431089

  8. Omega-3 Fatty Acid Enriched Chevon (Goat Meat) Lowers Plasma Cholesterol Levels and Alters Gene Expressions in Rats

    PubMed Central

    Rajion, Mohamed Ali; Meng, Goh Yong; Soleimani Farjam, Abdoreza

    2014-01-01

    In this study, control chevon (goat meat) and omega-3 fatty acid enriched chevon were obtained from goats fed a 50% oil palm frond diet and commercial goat concentrate for 100 days, respectively. Goats fed the 50% oil palm frond diet contained high amounts of α-linolenic acid (ALA) in their meat compared to goats fed the control diet. The chevon was then used to prepare two types of pellets (control or enriched chevon) that were then fed to twenty-male-four-month-old Sprague-Dawley rats (n = 10 in each group) for 12 weeks to evaluate their effects on plasma cholesterol levels, tissue fatty acids, and gene expression. There was a significant increase in ALA and docosahexaenoic acid (DHA) in the muscle tissues and liver of the rats fed the enriched chevon compared with the control group. Plasma cholesterol also decreased (P < 0.05) in rats fed the enriched chevon compared to the control group. The rat pellets containing enriched chevon significantly upregulated the key transcription factor PPAR-γ and downregulated SREBP-1c expression relative to the control group. The results showed that the omega-3 fatty acid enriched chevon increased the omega-3 fatty acids in the rat tissues and altered PPAR-γ and SREBP-1c genes expression. PMID:24719886

  9. Okadaic acid mimics multiple changes in early protein phosphorylation and gene expression induced by tumor necrosis factor or interleukin-1.

    PubMed

    Guy, G R; Cao, X; Chua, S P; Tan, Y H

    1992-01-25

    Okadaic acid, a phosphatase inhibitor from a marine organism, mimics tumor necrosis factor/interleukin-1 (TNF/IL-1) in inducing changes in early cellular protein phosphorylation. A total of approximately 116 proteins exhibit significant and concordant changes in phosphorylation or dephosphorylation within 15 min in human fibroblasts activated by either okadaic acid, TNF, or IL-1. The fidelity of this mimicry by okadaic acid extends to the phosphorylation of the 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, epidermal growth factor receptor, and other cdc2-kinase substrates (c-abl, RB, and p53). The okadaic acid-induced pattern of protein phosphorylation is distinct from that observed in cells treated with phorbol 12-myristate 13-acetate or with ligands like epidermal growth factor, cyclic AMP agonists, bradykinin, or interferons. Like TNF, okadaic acid also induces the transcription of immediate early response genes like c-jun and Egr-1 as well as the interleukin-6 genes. The overall early effects of okadaic acid uniquely parallel those of TNF/IL-1 and not those of other cytokines or ligands. Regulation of protein phosphatase inhibition is discussed as a mechanism for TNF/IL-1 signal transduction. PMID:1370482

  10. Omega-3 fatty acid enriched chevon (goat meat) lowers plasma cholesterol levels and alters gene expressions in rats.

    PubMed

    Ebrahimi, Mahdi; Rajion, Mohamed Ali; Meng, Goh Yong; Soleimani Farjam, Abdoreza

    2014-01-01

    In this study, control chevon (goat meat) and omega-3 fatty acid enriched chevon were obtained from goats fed a 50% oil palm frond diet and commercial goat concentrate for 100 days, respectively. Goats fed the 50% oil palm frond diet contained high amounts of α-linolenic acid (ALA) in their meat compared to goats fed the control diet. The chevon was then used to prepare two types of pellets (control or enriched chevon) that were then fed to twenty-male-four-month-old Sprague-Dawley rats (n = 10 in each group) for 12 weeks to evaluate their effects on plasma cholesterol levels, tissue fatty acids, and gene expression. There was a significant increase in ALA and docosahexaenoic acid (DHA) in the muscle tissues and liver of the rats fed the enriched chevon compared with the control group. Plasma cholesterol also decreased (P < 0.05) in rats fed the enriched chevon compared to the control group. The rat pellets containing enriched chevon significantly upregulated the key transcription factor PPAR-γ and downregulated SREBP-1c expression relative to the control group. The results showed that the omega-3 fatty acid enriched chevon increased the omega-3 fatty acids in the rat tissues and altered PPAR-γ and SREBP-1c genes expression. PMID:24719886

  11. rre37 Overexpression Alters Gene Expression Related to the Tricarboxylic Acid Cycle and Pyruvate Metabolism in Synechocystis sp. PCC 6803

    PubMed Central

    Iijima, Hiroko; Watanabe, Atsuko; Takanobu, Junko; Hirai, Masami Yokota; Osanai, Takashi

    2014-01-01

    The tricarboxylic acid (TCA) cycle and pyruvate metabolism of cyanobacteria are unique and important from the perspectives of biology and biotechnology research. Rre37, a response regulator induced by nitrogen depletion, activates gene expression related to sugar catabolism. Our previous microarray analysis has suggested that Rre37 controls the transcription of genes involved in sugar catabolism, pyruvate metabolism, and the TCA cycle. In this study, quantitative real-time PCR was used to measure the transcript levels of 12 TCA cycle genes and 13 pyruvate metabolism genes. The transcripts of 6 genes (acnB, icd, ppc, pyk1, me, and pta) increased after 4 h of nitrogen depletion in the wild-type GT strain but the induction was abolished by rre37 overexpression. The repression of gene expression of fumC, ddh, and ackA caused by nitrogen depletion was abolished by rre37 overexpression. The expression of me was differently affected by rre37 overexpression, compared to the other 24 genes. These results indicate that Rre37 differently controls the genes of the TCA cycle and pyruvate metabolism, implying the key reaction of the primary in this unicellular cyanobacterium. PMID:25614900

  12. A novel method based on physicochemical properties of amino acids and one class classification algorithm for disease gene identification.

    PubMed

    Yousef, Abdulaziz; Charkari, Nasrollah Moghadam

    2015-08-01

    Identifying the genes that cause disease is one of the most challenging issues to establish the diagnosis and treatment quickly. Several interesting methods have been introduced for disease gene identification for a decade. In general, the main differences between these methods are the type of data used as a prior-knowledge, as well as machine learning (ML) methods used for identification. The disease gene identification task has been commonly viewed by ML methods as a binary classification problem (whether any gene is disease or not). However, the nature of the data (since there is no negative data available for training or leaners) creates a major problem which affect the results. In this paper, sequence-based, one class classification method is introduced to assign genes to disease class (yes, no). First, to generate feature vector, the sequences of proteins (genes) are initially transformed to numerical vector using physicochemical properties of amino acid. Second, as there is no definite approach to define non-disease genes (negative data); we have attempted to model solely disease genes (positive data) to make a prediction by employing Support Vector Data Description algorithm. The experimental results confirm the efficiency of the method with precision, recall and F-measure of 79.3%, 82.6% and 80.9%, respectively. PMID:26146156

  13. Transcriptomic profiling of linolenic acid-responsive genes in ROS signaling from RNA-seq data in Arabidopsis

    PubMed Central

    Mata-Pérez, Capilla; Sánchez-Calvo, Beatriz; Begara-Morales, Juan C.; Luque, Francisco; Jiménez-Ruiz, Jaime; Padilla, María N.; Fierro-Risco, Jesús; Valderrama, Raquel; Fernández-Ocaña, Ana; Corpas, Francisco J.; Barroso, Juan B.

    2015-01-01

    Linolenic acid (Ln) released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA). The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signaling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC) with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2-fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs) and Allene oxide cyclases (AOCs). In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding), such as WRKY, JAZ, MYC, and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signaling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1), methionine sulfoxide reductase (MSR) and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signaling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity) and especially in stresses mediated by ROS. PMID

  14. Differences in growth, fillet quality, and fatty acid metabolism-related gene expression between juvenile male and female rainbow trout.

    PubMed

    Manor, Meghan L; Cleveland, Beth M; Kenney, P Brett; Yao, Jianbo; Leeds, Tim

    2015-04-01

    Sexual maturation occurs at the expense of stored energy and nutrients, including lipids; however, little is known regarding sex effects on nutrient regulatory mechanisms in rainbow trout prior to maturity. Thirty-two, 14-month-old, male and female rainbow trout were sampled for growth, carcass yield, fillet composition, and gene expression of liver, white muscle, and visceral adipose tissue. Growth parameters, including gonadosomatic index, were not affected by sex. Females had higher percent separable muscle yield, but there were no sex effects on fillet proximate composition. Fillet shear force indicated females produce firmer fillets than males. Male livers had greater expression of three cofactors within the mTOR signaling pathway that act to inhibit TORC1 assembly; mo25, rictor, and pras40. Male liver also exhibited increased expression of β-oxidation genes cpt1b and ehhadh. These findings are indicative of increased mitochondrial β-oxidation in male liver. Females exhibited increased expression of the mTOR cofactor raptor in white muscle and had higher expression levels of several genes within the fatty acid synthesis pathway, including gpat, srebp1, scd1, and cd36. Female muscle also had increased expression of β-oxidation genes cpt1d and cpt2. Increased expression of both fatty acid synthesis and β-oxidation genes suggests female muscle may have greater fatty acid turnover. Differences between sexes were primarily associated with variation of gene expression within the mTOR signaling pathway. Overall, data suggest there is differential regulation of gene expression in male and female rainbow trout tissues prior to the onset of sexual maturity that may lead to nutrient repartitioning during maturation. PMID:25673423

  15. Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

    PubMed

    Wang, Renxue; Liu, Lin; Sheps, Jonathan A; Forrest, Dana; Hofmann, Alan F; Hagey, Lee R; Ling, Victor

    2013-08-15

    The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice. PMID:23764895

  16. Deriving Gene Networks from SNP Associated with Triacylglycerol and Phospholipid Fatty Acid Fractions from Ribeyes of Angus Cattle

    PubMed Central

    Buchanan, Justin W.; Reecy, James M.; Garrick, Dorian J.; Duan, Qing; Beitz, Don C.; Koltes, James E.; Saatchi, Mahdi; Koesterke, Lars; Mateescu, Raluca G.

    2016-01-01

    The fatty acid profile of beef is a complex trait that can benefit from gene-interaction network analysis to understand relationships among loci that contribute to phenotypic variation. Phenotypic measures of fatty acid profile from triacylglycerol and phospholipid fractions of longissimus muscle, pedigree information, and Illumina 54 k bovine SNP genotypes were utilized to derive an annotated gene network associated with fatty acid composition in 1,833 Angus beef cattle. The Bayes-B statistical model was utilized to perform a genome wide association study to estimate associations between 54 k SNP genotypes and 39 individual fatty acid phenotypes within each fraction. Posterior means of the effects were estimated for each of the 54 k SNP and for the collective effects of all the SNP in every 1-Mb genomic window in terms of the proportion of genetic variance explained by the window. Windows that explained the largest proportions of genetic variance for individual lipids were found in the triacylglycerol fraction. There was almost no overlap in the genomic regions explaining variance between the triacylglycerol and phospholipid fractions. Partial correlations were used to identify correlated regions of the genome for the set of largest 1 Mb windows that explained up to 35% genetic variation in either fatty acid fraction. SNP were allocated to windows based on the bovine UMD3.1 assembly. Gene network clusters were generated utilizing a partial correlation and information theory algorithm. Results were used in conjunction with network scoring and visualization software to analyze correlated SNP across 39 fatty acid phenotypes to identify SNP of significance. Significant pathways implicated in fatty acid metabolism through GO term enrichment analysis included homeostasis of number of cells, homeostatic process, coenzyme/cofactor activity, and immunoglobulin. These results suggest different metabolic pathways regulate the development of different types of lipids found in

  17. Associations between variants of FADS genes and omega-3 and omega-6 milk fatty acids of Canadian Holstein cows

    PubMed Central

    2014-01-01

    Background Fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes code respectively for the enzymes delta-5 and delta-6 desaturases which are rate limiting enzymes in the synthesis of polyunsaturated omega-3 and omega-6 fatty acids (FAs). Omega-3 and-6 FAs as well as conjugated linoleic acid (CLA) are present in bovine milk and have demonstrated positive health effects in humans. Studies in humans have shown significant relationships between genetic variants in FADS1 and 2 genes with plasma and tissue concentrations of omega-3 and-6 FAs. The aim of this study was to evaluate the extent of sequence variations within these two genes in Canadian Holstein cows as well as the association between sequence variants and health promoting FAs in milk. Results Thirty three SNPs were detected within the studied regions of genes including a synonymous mutation (FADS1-07, rs42187261, 306Tyr > Tyr) in exon 8 of FADS1, a non-synonymous mutation (FADS2-14, rs211580559, 294Ala > Val) within FADS2 exon 7, a splice site SNP (FADS2-05, rs211263660), a 3′UTR SNP (FADS2-23, rs109772589), and another 3′UTR SNP with an effect on a microRNA binding site within FADS2 gene (FADS2-19, rs210169303). Association analyses showed significant relations between three out of seven tested SNPs and several FAs. Significant associations (FDR P < 0.05) were recorded between FADS2-23 (rs109772589) and two omega-6 FAs (dihomogamma linolenic acid [C20:3n6] and arachidonic acid [C20:4n6]), FADS1-07 (rs42187261) and one omega-3 FA (eicosapentaenoic acid, C20:5n3) and tricosanoic acid (C23:0), and one intronic SNP, FADS1-01 (rs136261927) and C20:3n6. Conclusion Our study has demonstrated positive associations between three SNPs within FADS1 and FADS2 genes (a SNP within the 3’UTR, a synonymous SNP and an intronic SNP), with three milk PUFAs of Canadian Holstein cows thus suggesting possible involvement of synonymous and non-coding region variants in FA synthesis. These SNPs may serve as

  18. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  19. Diet and gene interactions influence the skeletal response to polyunsaturated fatty acids.

    PubMed

    Bonnet, Nicolas; Somm, Emmanuel; Rosen, Clifford J

    2014-11-01

    Diets rich in omega-3s have been thought to prevent both obesity and osteoporosis. However, conflicting findings are reported, probably as a result of gene by nutritional interactions. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear receptor that improves insulin sensitivity but causes weight gain and bone loss. Fish oil is a natural agonist for PPARγ and thus may exert its actions through the PPARγ pathway. We examined the role of PPARγ in body composition changes induced by a fish or safflower oil diet using two strains of C57BL/6J (B6); i.e. B6.C3H-6T (6T) congenic mice created by backcrossing a small locus on Chr 6 from C3H carrying 'gain of function' polymorphisms in the Pparγ gene onto a B6 background, and C57BL/6J mice. After 9months of feeding both diets to female mice, body weight, percent fat and leptin levels were less in mice fed the fish oil vs those fed safflower oil, independent of genotype. At the skeletal level, fish oil preserved vertebral bone mineral density (BMD) and microstructure in B6 but not in 6T mice. Moreover, fish oil consumption was associated with an increase in bone marrow adiposity and a decrease in BMD, cortical thickness, ultimate force and plastic energy in femur of the 6T but not the B6 mice. These effects paralleled an increase in adipogenic inflammatory and resorption markers in 6T but not B6. Thus, compared to safflower oil, fish oil (high ratio omega-3/-6) prevents weight gain, bone loss, and changes in trabecular microarchitecture in the spine with age. These beneficial effects are absent in mice with polymorphisms in the Pparγ gene (6T), supporting the tenet that the actions of n-3 fatty acids on bone microstructure are likely to be genotype dependent. Thus caution must be used in interpreting dietary intervention trials with skeletal endpoints in mice and in humans. PMID:25088402

  20. New Insights into Roles of Cell Wall Invertase in Early Seed Development Revealed by Comprehensive Spatial and Temporal Expression Patterns of GhCWIN1 in Cotton1[W][OA

    PubMed Central

    Wang, Lu; Ruan, Yong-Ling

    2012-01-01

    Despite substantial evidence on the essential roles of cell wall invertase (CWIN) in seed filling, it remains largely unknown how CWIN exerts its regulation early in seed development, a critical stage that sets yield potential. To fill this knowledge gap, we systematically examined the spatial and temporal expression patterns of a major CWIN gene, GhCWIN1, in cotton (Gossypium hirsutum) seeds from prefertilization to prestorage phase. GhCWIN1 messenger RNA was abundant at the innermost seed coat cell layer at 5 d after anthesis but became undetectable at 10 d after anthesis, at the onset of its differentiation into transfer cells characterized by wall ingrowths, suggesting that CWIN may negatively regulate transfer cell differentiation. Within the filial tissues, GhCWIN1 transcript was detected in endosperm cells undergoing nuclear division but not in those cells at the cellularization stage, with similar results observed in Arabidopsis (Arabidopsis thaliana) endosperm for CWIN, AtCWIN4. These findings indicate a function of CWIN in nuclear division but not cell wall biosynthesis in endosperm, contrasting to the role proposed for sucrose synthase (Sus). Further analyses revealed a preferential expression pattern of GhCWIN1 and AtCWIN4 in the provascular region of the torpedo embryos in cotton and Arabidopsis seed, respectively, indicating a role of CWIN in vascular initiation. Together, these novel findings provide insights into the roles of CWIN in regulating early seed development spatially and temporally. By comparing with previous studies on Sus expression and in conjunction with the expression of other related genes, we propose models of CWIN- and Sus-mediated regulation of early seed development. PMID:22864582

  1. Improving cold storage and processing traits in potato through targeted gene knockout.

    PubMed

    Clasen, Benjamin M; Stoddard, Thomas J; Luo, Song; Demorest, Zachary L; Li, Jin; Cedrone, Frederic; Tibebu, Redeat; Davison, Shawn; Ray, Erin E; Daulhac, Aurelie; Coffman, Andrew; Yabandith, Ann; Retterath, Adam; Haun, William; Baltes, Nicholas J; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

    2016-01-01

    Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest shelf life. However, cold temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide--a potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference (RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. Because RNAi often results in incomplete gene silencing and requires the plant to be transgenic, here we used transcription activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele, and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome. These results provide a framework for using TALENs to quickly improve traits in commercially relevant autotetraploid potato lines. PMID:25846201

  2. Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: Application of ultra-microelectrode to enhance sucrose biosensor's sensitivity.

    PubMed

    Bagal-Kestwal, Dipali; Karve, Meena S; Kakade, Bhalchandra; Pillai, Vijayamohanan K

    2008-12-01

    We are reporting fabrication and characterization of electrochemical sucrose biosensor using ultra-microelectrode (UME) for the detection of heavy metal ions (Hg(II), Ag(I), Pb(II) and Cd(II)). The working UME, with 25 microm diameter, was modified with invertase (INV, EC: 3.2.1.26) and glucose oxidase (GOD, EC: 1.1.3.4) entrapped in agarose-guar gum. The hydrophilic character of the agarose-guar gum composite matrix was checked by water contact angle measurement. The atomic force microscopy (AFM) images of the membranes showed proper confinement of both the enzymes during co-immobilization. The dynamic range for sucrose biosensor was achieved in the range of 1 x 10(-10) to 1 x 10(-7)M with lower detection limit 1 x 10(-10)M at pH 5.5 with 9 cycles of reuse. The spectrophotometric and electrochemical studies showed linear relationship between concentration of heavy metal ions and degree of inhibition of invertase. The toxicity sequence for invertase using both methods was observed as Hg(2+)>Pb(2+)>Ag(+)>Cd(2+). The dynamic linear range for mercury using electrochemical biosensor was o