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Sample records for acid invertase genes

  1. Differential expression of acid invertase genes in roots of metallicolous and non-metallicolous populations of Rumex japonicus under copper stress.

    PubMed

    Huang, Wu-Xing; Cao, Yi; Huang, Li-Juan; Ren, Cong; Xiong, Zhi-Ting

    2011-09-01

    Recent evidence indicates that during copper (Cu) stress, the roots of metallicolous plants manifest a higher activity of acid invertase enzymes, which are rate-limiting in sucrose catabolism, than non-metallicolous plants. To test whether the higher activity of acid invertases is the result of higher expression of acid invertase genes, we isolated partial cDNAs for acid invertases from two populations of Rumex japonicus (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, and designed primers to measure changes in transcript levels during Cu stress. We also determined the growth of the plants' roots, Cu accumulation, and acid invertase activities. The seedlings of R. japonicus were exposed to control or 20 μM Cu(2+) for 6d under hydroponic conditions. The transcript level and enzyme activity of acid invertases in metallicolous plants were both significantly higher than those in non-metallicolous plants when treated with 20 μM. Under Cu stress, the root length and root biomass of metallicolous plants were also significantly higher than those of non-metallicolous plants. The results suggested that under Cu stress, the expression of acid invertase genes in metallicolous plants of R. japonicus differed from those in non-metallicolous plants. Furthermore, the higher acid invertase activities of metallicolous plants under Cu stress could be due in part to elevated expression of acid invertase genes.

  2. Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress.

    PubMed

    Zhang, Luan; Xiong, Zhi-ting; Xu, Zhong-rui; Liu, Chen; Cai, Shen-wen

    2014-06-01

    The roots of metallophytes serve as the key interface between plants and heavy metal-contaminated underground environments. It is known that the roots of metallicolous plants show a higher activity of acid invertase enzymes than those of non-metallicolous plants when under copper stress. To test whether the higher activity of acid invertases is the result of increased expression of acid invertase genes or variations in the amino acid sequences between the two population types, we isolated full cDNAs for acid invertases from two populations of Kummerowia stipulacea (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, expressed them in Pichia pastoris, and conducted real-time PCR to determine differences in transcript levels during Cu stress. Heterologous expression of acid invertase cDNAs in P. pastoris indicated that variations in the amino acid sequences of acid invertases between the two populations played no significant role in determining enzyme characteristics. Seedlings of K. stipulacea were exposed to 0.3µM Cu(2+) (control) and 10µM Cu(2+) for 7 days under hydroponics׳ conditions. The transcript levels of acid invertases in metallicolous plants were significantly higher than in non-metallicolous plants when under copper stress. The results suggest that the expression of acid invertase genes in metallicolous plants of K. stipulacea differed from those in non-metallicolous plants under such conditions. In addition, the sugars may play an important role in regulating the transcript level of acid invertase genes and acid invertase genes may also be involved in root/shoot biomass allocation.

  3. 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

  4. Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development.

    PubMed

    Yu, Xiyan; Wang, Xiufeng; Zhang, Wenqian; Qian, Tingting; Tang, Guimin; Guo, Yankui; Zheng, Chengchao

    2008-01-01

    To unravel the roles of soluble acid invertase in muskmelon (Cucumis melo L.), its activity in transgenic muskmelon plants was reduced by an antisense approach. For this purpose, a 1038 bp cDNA fragment of muskmelon soluble acid invertase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the stems were obviously thinner. Transmission electron microscopy revealed that degradation of the chloroplast membrane occurred in transgenic leaves and the number of grana in the chloroplast was significantly reduced, suggesting that the slow growth and weaker phenotype of the transgenic plants may be due to damage to the chloroplast ultrastructure, which in turn resulted in a decrease in net photosynthetic rate. The sucrose concentration increased and levels of acid invertase decreased in transgenic fruit, and the fruit size was 60% smaller than that of the control. In addition, transgenic fruit reached full-slip at 25 d after pollination (DAP), approximately 5 d before the control fruit (full-slip at 30 DAP), and this accelerated maturity correlated with a dramatic elevation of ethylene production at the later stages of fruit development. Together, these results suggest that soluble acid invertase not only plays an important role during muskmelon plant and fruit development but also controls the sucrose content in muskmelon fruit.

  5. 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

  6. 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

  7. 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.

  8. 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 Central

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

    2015-01-01

    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

  9. 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

  10. Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression.

    PubMed

    Wang, Li-Ting; Wang, Ai-Yu; Hsieh, Chang-Wen; Chen, Chih-Yu; Sung, Hsien-Yi

    2005-05-01

    Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.

  11. Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1

    PubMed Central

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

    2015-01-01

    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

  12. 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.

  13. 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

  14. Acid and Alkaline Invertases in Suspension Cultures of Sugar Beet Cells

    PubMed Central

    Masuda, Hiroshi; Takahashi, Toshimasa; Sugawara, Shiro

    1988-01-01

    Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar Km values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and Km values for sucrose. PMID

  15. A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

    PubMed

    Rae, Anne L; Casu, Rosanne E; Perroux, Jai M; Jackson, Mark A; Grof, Christopher P L

    2011-06-15

    Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.

  16. Invertases involved in the development of the parasitic plant Phelipanche ramosa: characterization of the dominant soluble acid isoform, PrSAI1.

    PubMed

    Draie, Rida; Péron, Thomas; Pouvreau, Jean-Bernard; Véronési, Christophe; Jégou, Sandrine; Delavault, Philippe; Thoiron, Séverine; Simier, Philippe

    2011-09-01

    Phelipanche ramosa L. parasitizes major crops, acting as a competitive sink for host photoassimilates, especially sucrose. An understanding of the mechanisms of sucrose utilization in parasites is an important step in the development of new control methods. Therefore, in this study, we characterized the invertase gene family in P. ramosa and analysed its involvement in plant development. Invertase-encoded cDNAs were isolated using degenerate primers corresponding to highly conserved regions of invertases. In addition to enzyme assays, gene expression was analysed using real-time quantitative reverse transcriptase-polymerase chain reaction during overall plant development. The dominant isoform was purified and sequenced using electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS). Five invertase-encoded cDNAs were thus characterized, including PrSai1 which encodes a soluble acid invertase (SAI). Of the five invertases, PrSai1 transcripts and SAI activity were dominant in growing organs. The most active invertase corresponded to the PrSai1 gene product. The purified PrSAI1 displayed low pI and optimal pH values, specificity for β-fructofuranosides and inhibition by metallic ions and competitive inhibition by fructose. PrSAI1 is a typical vacuolar SAI that is actively involved in growth following both germination and attachment to host roots. In addition, germinated seeds displayed enhanced cell wall invertase activity (PrCWI) in comparison with preconditioned seeds, suggesting the contribution of this activity in the sink strength of infected roots during the subsequent step of root penetration. Our results show that PrSAI1 and, possibly, PrCWI constitute good targets for the development of new transgenic resistance in host plants using proteinaceous inhibitors or silencing strategies. PMID:21726369

  17. 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

    2014-05-15

    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.

  18. 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

  19. Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase.

    PubMed

    Le Roy, Katrien; Lammens, Willem; Verhaest, Maureen; De Coninck, Barbara; Rabijns, Anja; Van Laere, André; Van den Ende, Wim

    2007-11-01

    Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.

  20. Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase1[C

    PubMed Central

    Le Roy, Katrien; Lammens, Willem; Verhaest, Maureen; De Coninck, Barbara; Rabijns, Anja; Van Laere, André; Van den Ende, Wim

    2007-01-01

    Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs. PMID:17873089

  1. 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

  2. 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

  3. 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

  4. Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison.

    PubMed

    Fahrendorf, T; Beck, E

    1990-01-01

    Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The Km values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity ('multiple forms'). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively. PMID:24201951

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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...

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

    PubMed

    Bhaskar, Pudota B; Wu, Lei; Busse, James S; Whitty, Brett R; Hamernik, Andy J; Jansky, Shelley H; Buell, C Robin; Bethke, Paul C; Jiang, Jiming

    2010-10-01

    Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

  13. Genes for alkaline/neutral invertase in rice: alkaline/neutral invertases are located in plant mitochondria and also in plastids.

    PubMed

    Murayama, Seiji; Handa, Hirokazu

    2007-04-01

    Two cDNA clones (OsNIN1 and OsNIN3) encoding an alkaline/neutral invertase localized in organelles were identified from rice. The deduced amino acid sequences of these cDNA clones showed high homology to other plant alkaline/neutral invertases. Semi-quantitative reverse transcription polymerase chain reaction revealed that the expression of OsNIN1 was constitutive and independent of organ difference, although its expression level was low. Analyses using five types of web software for the prediction of protein localization in the cell, Predotar, PSORT, Mitoprot, TargetP, and ChloroP, strongly supported the possibility that OsNIN1 is transported into the mitochondria and that OsNIN3 is transported into plastids. Transient expression of fusion proteins combining the amino terminal region of these two proteins with sGFP demonstrated that N-OsNIN1::GFP and N-OsNIN3::GFP fusion proteins were transported into the mitochondria and plastids, respectively. We expressed the OsNIN1 protein in vitro and revealed that the translated protein had an invertase activity. These results clearly indicate that some of alkaline/neutral invertases are located in plant organelles, mitochondria and plastids, and that they might have a novel physiological function in plant organelles.

  14. 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

  15. Invertases in Oat Seedlings

    PubMed Central

    Pressey, Russell; Avants, Jimmy K.

    1980-01-01

    The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters Km and Vmax of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg2+, with complete inhibition by 2 micromolar HgCl2. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl2 and 200 millimolar mannitol) also blocked the auxin effect on invertase loss. PMID:16661129

  16. Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits 1

    PubMed Central

    Hubbard, Natalie L.; Huber, Steven C.; Pharr, D. Mason

    1989-01-01

    Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 ± 0.3 micromole per hour per gram fresh weight and 2.2 ± 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality. PMID:16667212

  17. Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli.

    PubMed

    Pek, Han Bin; Klement, Maximilian; Ang, Kok Siong; Chung, Bevan Kai-Sheng; Ow, Dave Siak-Wei; Lee, Dong-Yup

    2015-01-01

    Various isoforms of invertases from prokaryotes, fungi, and higher plants has been expressed in Escherichia coli, and codon optimisation is a widely-adopted strategy for improvement of heterologous enzyme expression. Successful synthetic gene design for recombinant protein expression can be done by matching its translational elongation rate against heterologous host organisms via codon optimization. Amongst the various design parameters considered for the gene synthesis, codon context bias has been relatively overlooked compared to individual codon usage which is commonly adopted in most of codon optimization tools. In addition, matching the rates of transcription and translation based on secondary structure may lead to enhanced protein folding. In this study, we evaluated codon context fitness as design criterion for improving the expression of thermostable invertase from Thermotoga maritima in Escherichia coli and explored the relevance of secondary structure regions for folding and expression. We designed three coding sequences by using (1) a commercial vendor optimized gene algorithm, (2) codon context for the whole gene, and (3) codon context based on the secondary structure regions. Then, the codon optimized sequences were transformed and expressed in E. coli. From the resultant enzyme activities and protein yield data, codon context fitness proved to have the highest activity as compared to the wild-type control and other criteria while secondary structure-based strategy is comparable to the control. Codon context bias was shown to be a relevant parameter for enhancing enzyme production in Escherichia coli by codon optimization. Thus, we can effectively design synthetic genes within heterologous host organisms using this criterion. PMID:26047917

  18. 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...

  19. 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

  20. Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana.

    PubMed

    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

  1. Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana.

    PubMed

    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.

  2. 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.

  3. Regulation of arbuscular mycorrhization by apoplastic invertases: enhanced invertase activity in the leaf apoplast affects the symbiotic interaction.

    PubMed

    Schaarschmidt, Sara; Kopka, Joachim; Ludwig-Müller, Jutta; Hause, Bettina

    2007-08-01

    The effect of constitutive invertase overexpression on the arbuscular mycorrhiza (AM) is shown. The analysis of the enhanced potential for sucrose cleavage was performed with a heterozygous line of Nicotiana tabacum 35S::cwINV expressing a chimeric gene encoding apoplast-located yeast-derived invertase with the CaMV35S promoter. Despite the 35S promoter, roots of the transgenic plants showed no or only minor effects on invertase activity whereas the activity in leaves was increased at different levels. Plants with strongly elevated leaf invertase activity, which exhibited a strong accumulation of hexoses in source leaves, showed pronounced phenotypical effects like stunted growth and chlorosis, and an undersupply of the root with carbon. Moreover, transcripts of PR (pathogenesis related) genes accumulated in the leaves. In these plants, mycorrhization was reduced. Surprisingly, plants with slightly increased leaf invertase activity showed a stimulation of mycorrhization, particularly 3 weeks after inoculation. Compared with wild-type, a higher degree of mycorrhization accompanied by a higher density of all fungal structures and a higher level of Glomus intraradices-specific rRNA was detected. Those transgenic plants showed no accumulation of hexoses in the source leaves, minor phenotypical effects and no increased PR gene transcript accumulation. The roots had even lower levels of phenolic compounds (chlorogenic acid and scopolin), amines (such as tyramine, dopamine, octopamine and nicotine) and some amino acids (including 5-amino-valeric acid and 4-amino-butyric acid), as well as an increased abscisic acid content compared with wild-type. Minor metabolic changes were found in the leaves of these plants. The changes in metabolism and defense status of the plant and their putative role in the formation of an AM symbiosis are discussed. PMID:17521407

  4. 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

  5. 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

  6. 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.

  7. Phloem sugar flux and jasmonic acid-responsive cell wall invertase control extrafloral nectar secretion in Ricinus communis.

    PubMed

    Millán-Cañongo, Cynthia; Orona-Tamayo, Domancar; Heil, Martin

    2014-07-01

    Plants secrete extrafloral nectar (EFN) that attracts predators. The efficiency of the resulting anti-herbivore defense depends on the quantity and spatial distribution of EFN. Thus, according to the optimal defense hypothesis (ODH), plants should secrete EFN on the most valuable organs and when herbivore pressure is high. Ricinus communis plants secreted most EFN on the youngest (i.e., most valuable) leaves and after the simulation of herbivory via the application of jasmonic acid (JA). Here, we investigated the physiological mechanisms that might produce these seemingly adaptive spatiotemporal patterns. Cell wall invertase (CWIN; EC 3.2.1.26) was most active in the hours before peak EFN secretion, its decrease preceded the decrease in EFN secretion, and CWIN activity was inducible by JA. Thus, CWIN appears to be a central player in EFN secretion: its activation by JA is likely to cause the induction of EFN secretion after herbivory. Shading individual leaves decreased EFN secretion locally on these leaves with no effect on CWIN activity in the nectaries, which is likely to be because it decreased the content of sucrose, the substrate of CWIN, in the phloem. Our results demonstrate how the interplay of two physiological processes can cause ecologically relevant spatiotemporal patterns in a plant defense trait.

  8. Cell Wall Invertase in Tobacco Crown Gall Cells 1

    PubMed Central

    Weil, Marion; Rausch, Thomas

    1990-01-01

    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: Mr 63,000, pH optimum at 4.7, Km sucrose 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl2 but is insensitive to H2O2, 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. Images Figure 1 PMID:16667892

  9. 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.

  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. 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

  12. A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

    PubMed

    Maruta, Takanori; Miyazaki, Nozomi; Nosaka, Ryota; Tanaka, Hiroyuki; Padilla-Chacon, Daniel; Otori, Kumi; Kimura, Ayako; Tanabe, Noriaki; Yoshimura, Kazuya; Tamoi, Masahiro; Shigeoka, Shigeru

    2015-05-01

    Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression.

  13. 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

    2014-04-28

    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.

  14. 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

  15. 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.

  16. Aqueous two-phase (PEG4000/Na2SO4) extraction and characterization of an acid invertase from potato tuber (Solanum tuberosum).

    PubMed

    Yuzugullu, Yonca; Duman, Yonca Avcı

    2015-01-01

    Invertases are key metabolic enzymes that catalyze irreversible hydrolysis of sucrose into fructose and glucose. Plant invertases have essential roles in carbohydrate metabolism, plant development, and stress responses. To study their isolation and purification from potato, an attractive system useful for the separation of biological molecules, an aqueous two-phase system, was used. The influence of various system parameters such as type of phase-forming salts, polyethylene glycol (PEG) molecular mass, salt, and polymer concentration was investigated to obtain the highest recovery of enzyme. The PEG4000 (12.5%, w/w)/Na2SO4(15%, w/w) system was found to be ideal for partitioning invertase into the bottom salt-rich phase. The addition of 3% MnSO4 (w/w) at pH 5.0 increased the purity by 5.11-fold with the recovered activity of 197%. The Km and Vmax on sucrose were 3.95 mM and 0.143 U mL(-1) min(-1), respectively. Our data confirmed that the PEG4000/Na2SO4 aqueous two-phase system combined with the presence of MnSO4 offers a low-cost purification of invertase from readily available potato tuber in a single step. The biochemical characteristics of temperature and pH stability for potato invertase prepared from an ATPS make the enzyme a good candidate for its potential use in many research and industrial applications.

  17. 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

  18. 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...

  19. 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

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

    PubMed

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

    2015-06-01

    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

  1. Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

    PubMed Central

    Obenland, D M; Simmen, U; Boller, T; Wiemken, A

    1993-01-01

    Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal. PMID:8310063

  2. 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

  3. 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)

  4. Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolism.

    PubMed

    Kusch, Ute; Harms, Karsten; Rausch, Thomas; Greiner, Steffen

    2009-01-01

    Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action.

  5. Development of tuberous roots and sugar accumulation as related to invertase activity and mineral nutrition.

    PubMed

    Ricardo, C P; Sovia, D

    1974-03-01

    Sucrose storage in tuberous roots was not observed when the tissues had very high activities of acid invertase. High activities of the enzyme were always present in the roots at early stages of their development. In species where the activity of the enzyme decreased during root development, sucrose was stored. Thus, acid invertase was undetectable in mature roots of carrots (Daucus carota L.) where sucrose formed almost 80% of the dry matter. Conversely, radish (Raphanus sativus L.) and turnip (Brassica rapa L.) roots, in which the activity of the enzyme remained high until maturity, did not store appreciable amounts of sucrose (2% and 9%, respectively, of the dry matter in the mature roots), reducing sugars being the main reserve (more than 80% of the dry matter in mature turnips). The correlation between sucrose content and acid invertase activity was furthermore evident in both sucrose- and hexose-storing roots when the activity of this enzyme was affected by changes in the mineral nutrition. Deficiencies of nitrogen and sulphur reduced the activity of acid and alkaline invertases and led to increase in sucrose content and decrease in reducing sugars. However, the decline of alkaline invertase activity in tissues low in acid invertase had no clear effect on sugar content. Sodium chloride (10(-1)M) affected acid invertase and sugars in a manner similar to that of the two deficiencies, but had practically no effect on alkaline invertase. The changes in sugar content produced by the variations in mineral nutrition were small in hexose-storing roots in relation to those of sucrose-storing roots. It is possible that this result is related to the different levels of acid invertase in the two types of roots.

  6. Three-dimensional Structure of Saccharomyces Invertase

    PubMed Central

    Sainz-Polo, M. Angela; Ramírez-Escudero, Mercedes; Lafraya, Alvaro; González, Beatriz; Marín-Navarro, Julia; Polaina, Julio; Sanz-Aparicio, Julia

    2013-01-01

    Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition. PMID:23430743

  7. Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein gammaTIP.

    PubMed

    Balk, P A; de Boer, A D

    1999-09-01

    Many bulbous plants need a low-temperature treatment for flowering. Cold, for example, affects the elongation of the stalk, thereby influencing the quality of the cut flower. How the elongation of the stalk is promoted by cold and which physiological and biochemical mechanisms are involved have remained obscure. As invertase has been shown to be involved in the cold-induced elongation of the flower stalks of tulips (Lambrechts et al., 1994, Plant Physiol 104: 515-520), we further characterized this enzyme by cloning the cDNA and analysing its expression in various tissues of the tulip (Tulipa gesneriana L. cv. Apeldoorn) stalk. In addition, the role of sucrose synthase was investigated. Since turgor pressure is an important force driving cell elongation, the role of a water-channel protein (gammaTIP) was studied in relation to these two enzymes. The mRNA level of the invertase found was substantially up-regulated as a result of cold treatment. Analysis of the amino acid sequence of this invertase revealed the presence of a vacuolar targeting signal. Two different forms of sucrose synthase were found, the expression of one of them appeared to be restricted to the vascular tissue while the other form was present in the surrounding tissue. Both sucrose synthases were present in the stalk during the entire period of bulb storage and after planting, but their activities declined during stalk elongation. The expression of the gammaTIP gene was restricted mainly to the vascular tissue and its expression profile was identical to that of invertase. Simultaneous expression of invertase and gammaTIP possibly leads to an increase in osmotic potential and vacuolar water uptake, thus providing a driving force for stretching the stalk cells.

  8. The role of hexokinases from grape berries (Vitis vinifera L.) in regulating the expression of cell wall invertase and sucrose synthase genes.

    PubMed

    Wang, X Q; Li, L M; Yang, P P; Gong, C L

    2014-02-01

    In plants, hexokinase (HXK, EC 2.7.1.1) involved in hexose phosphorylation, plays an important role in sugar sensing and signaling. In this study, we found that at Phase I of grape berry development, lower hexose (glucose or fructose) levels were concomitant with higher HXK activities and protein levels. After the onset of ripening, we demonstrated a drastic reduction in HXK activity and protein levels accompanied by a rising hexose level. Therefore, our results revealed that HXK activity and protein levels had an inverse relationship with the endogenous glucose or fructose levels during grape berry development. A 51 kDa HXK protein band was detected throughout grape berry development. In addition, HXK located in the vacuoles, cytoplasm, nucleus, proplastid, chloroplast, and mitochondrion of the berry flesh cells. During grape berry development, HXK transcriptional level changed slightly, while cell wall invertase (CWINV) and sucrose synthase (SuSy) expression was enhanced after véraison stage. Intriguingly, when sliced grape berries were incubated in different glucose solutions, CWINV and SuSy expression was repressed by glucose, and the intensity of repression depended on glucose concentration and incubation time. After sliced, grape berries were treated with different glucose analogs, CWINV and SuSy expression analyses revealed that phosphorylation of hexoses by hexokinase was an essential component in the glucose-dependent CWINV and SuSy expression. In the meantime, mannoheptulose, a specific inhibitor of hexokinase, blocked the repression induced by glucose on CWINV and SuSy expression. It suggested that HXK played a major role in regulating CWINV and SuSy expression during grape berry development.

  9. Relationship of large and small invertases in Saccharomyces: mutant selectively deficient in small invertase.

    PubMed Central

    Abrams, B B; Hackel, R; Mizunaga, T; Lampen, J O

    1978-01-01

    A mutant strain of Saccharomyces cerevisiae (D10-ER1) has been isolated after a two-step mutagenesis of strain 4059-358D (SUC 1) using ethyl methane sulfonate. Cells of this new strain produced a level of total invertase equaling that of 4059 but contained only trace amounts of the small, internal, aglycan form of the enzyme (less than 0.1% of total in D10-ER1 compared with 6% in 4059). When D10.ER1 was crossed with an invertase-hyperproducing strain dgr3 (SUC3), progeny were isolated (HZ400-5A and HZ400-2C) in which levels of total invertase had at least quadrupled. The percentage of small invertase, however, remained insignificant. Levels of small invertase in strain HZ400-5A were determined by affinity chromatography on conconavalin A-Sepharose, gel permeation chromatography, and isopycnic centrifugation in CsCl. The large invertase of the SUC1 yeasts described here was found to contain a form apparently greater in size than the large invertase of the SUC2 strain FH4C; this probably reflects a higher content of carbohydrate. The overall results of this study do not support a direct structural relationship between large and small invertases. The implications on invertase biosynthesis and structure are discussed. PMID:357425

  10. Cloning and functional analysis of chicory root fructan1-exohydrolase I (1-FEH I): a vacuolar enzyme derivedfrom a cell-wall invertase ancestor? Mass fingerprint of the 1-FEH I enzyme.

    PubMed

    Van den Ende, W; Michiels, A; De Roover, J; Verhaert, P; Van Laere, A

    2000-11-01

    This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.

  11. Sucrolytic Enzyme Activities in Cotyledons of the Faba Bean (Developmental Changes and Purification of Alkaline Invertase).

    PubMed Central

    Ross, H. A.; McRae, D.; Davies, H. V.

    1996-01-01

    In terms of maximum extractable catalytic activity, sucrose synthase is the predominant sucrolytic enzyme in developing cotyledons of faba bean (Vicia faba L.). Although acid invertase activity is extremely low, there is significant activity of alkaline invertase, the majority of which is extractable only with high concentrations of NaCl. Calculations of potential activity in vivo indicate that alkaline invertase is the predominant sucrolytic enzyme from 50 days after anthesis onward. However, at almost all stages of cotyledon development analyzed, the maximum extractable catalytic activities of both enzymes is in excess of the actual rate of starch deposition. Two forms of alkaline invertase were identified in developing cotyledons. The major form has been purified to homogeneity, and antibodies have been raised against it. The native protein has a molecular mass of about 238 [plus or minus] 4.5 kD. It is apparently a homotetramer (subunit molecular mass 53.4 [plus or minus] 0.9 kD). The enzyme has a pH optimum of 7.4, an isoelectric point of 5.2, and a Km[sucrose] of 10 mM and is inhibited by Tris (50% inhibition at 5 mM) and fructose (30% inhibition at 10 mM). Bean alkaline invertase is a [beta]-fructofuranosidase with no significant activity against raffinose, stachyose, trehalose, maltose, or lactose. PMID:12226291

  12. Purification and characterization of neutral and alkaline invertase from carrot.

    PubMed Central

    Lee, H S; Sturm, A

    1996-01-01

    Neutral and alkaline invertase were identified in cells of a suspension culture of carrot (Daucus carota L.) and purified to electrophoretic homogeneity. Neutral invertase is an octamer with a molecular mass of 456 kD and subunits of 57 kD, whereas alkaline invertase is a tetramer with a molecular mass of 504 kD and subunits of 126 kD. Both enzymes had sharp pH profiles, with maximal activities at pH 6.8 for neutral invertase and pH 8.0 for alkaline invertase, and both hydrolyzed sucrose with typical hyperbolic kinetics and similar Km values of about 20 mM at pH 7.5. Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a beta-fructofuranosidase. In contrast, alkaline invertase was highly specific for sucrose. Fructose acted as a competitive inhibitor of both enzymes, with Ki values of about 15 mM. Glucose was a noncompetitive inhibitor of both neutral and alkaline invertase, with a Ki of about 30 mM. Neither enzyme was inhibited by HgCl2. Alkaline invertase was markedly inhibited by CaCl2, MgCl2, and MnCl2, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP. PMID:8972597

  13. 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

  14. 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

  15. Invertase from a strain of Rhodotorula glutinis.

    PubMed

    Rubio, María C; Runco, Rosa; Navarro, Antonio R

    2002-11-01

    An invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from Rhodotorula glutinis was purified by ammonium sulfate fractionation, gel filtration and anion exchange chromatography. Invertase molecular weight was estimated to be 100 kDa by analytical gel filtration and 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular mass determinations indicated that the native enzyme exists as a homodimer. It is a glycoprotein that contains 19% carbohydrate. The enzyme attacks beta-D-fructofuranoside (raffinose, stachyose and sucrose) from the fructose end. It has a K(m) of 0.227 M and a V(max) of 0.096 micromol/min with sucrose as a substrate. Invertase activity is stable between pH 2.6 and 5.5 for 30 min, maximum activity being observed at pH 4.5. The activation energy was 6520 cal/mol. The enzyme is stable between 20 and 60 degrees C. Mg(2+) and Ca(2+) ions stimulated invertase activity 3-fold, while Fe(2+), K(+), Co(2+), Na(+) and Cu(2+) increased activity about 2-fold. The transfructosylation reaction could not be observed. This enzyme is of particular interest since it appears to have a high hydrolytic activity in 1 M sucrose solution. This fact would make the enzymatic hydrolysis process economically efficient for syrup production using by-products with high salt and sugar contents such as sugar cane molasses.

  16. Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

    PubMed

    Schonberger, O; Knox, C; Bibi, E; Pines, O

    1996-09-01

    The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

  17. 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

  18. 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.

  19. Selective transcriptional down-regulation of anther invertases precedes the failure of pollen development in water-stressed wheat.

    PubMed

    Koonjul, P K; Minhas, J S; Nunes, C; Sheoran, I S; Saini, H S

    2005-01-01

    Water deficit during male meiosis in wheat (Triticum aestivum L.) causes pollen sterility. With a view to identifying the internal trigger for this failure, it was found that water stress specifically impairs the activities of vacuolar and cell-wall invertases in anthers prior to the arrest of pollen development. The enzymes are affected only when water deficit occurs around meiosis. Three invertase cDNAs, two encoding the cell-wall (Ivr1, Ivr3) and one the vacuolar (Ivr5) isoform, were isolated from an anther cDNA library. RNA gel-blot analysis using floral organs of well-watered plants revealed that these genes were expressed preferentially, though not exclusively, in anthers. Semi-quantitative RT-PCR demonstrated that transitory water deficit during meiosis selectively down-regulated the transcription of two of the three genes, one encoding the vacuolar (Ivr5) and the other a cell-wall (Ivr1) isoform, without affecting the Ivr3 message. Their expression did not recover upon resumption of watering. Another homologue of Ivr1 was also down-regulated, but only during the post-stress period. The stress effects on invertase transcripts were consistent with those on the developmental profiles of the corresponding enzyme activities. In situ hybridization revealed that the stress-sensitive invertase genes, unlike an insensitive one, were expressed within the microspores. No evidence for an invertase inhibitor under stress was found. Together the results show that the decline in invertase activity is probably regulated primarily at the transcriptional level in a gene- and cell-specific manner.

  20. Invertase-nanogold clusters decorated plant membranes for fluorescence-based sucrose sensor.

    PubMed

    Bagal-Kestwal, Dipali; Kestwal, Rakesh Mohan; Chiang, Been-Huang

    2015-04-12

    In the present study, invertase-mediated nanogold clusters were synthesized on onion membranes, and their application for sucrose biosensor fabrication was investigated. Transmission electron microscopy revealed free nanoparticles of various sizes (diameter ~5 to 50 nm) along with clusters of nanogold (~95 to 200 nm) on the surface of inner epidermal membranes of onions (Allium cepa L.). Most of the polydispersed nanoparticles were spherical, although some were square shaped, triangular, hexagonal or rod-shaped. Ultraviolet-visible spectrophotometric observations showed the characteristic peak for nanoparticles decorated invertase-onion membrane at approximately 301 nm. When excited at 320 nm in the presence of sucrose, the membranes exhibited a photoemission peak at 348 nm. The fluorescence lifetime of this nanogold modified onion membrane was 6.20 ns, compared to 2.47 ns for invertase-onion membrane without nanogold. Therefore, a sucrose detection scheme comprised of an invertase/nanogold decorated onion membrane was successfully developed. This fluorescent nanogold-embedded onion membrane drop-test sensor exhibited wide acidic to neutral working pH range (4.0-7.0) with a response time 30 seconds (<1 min). The fabricated quenching-based probe had a low detection limit (2x10(-9) M) with a linear dynamic range of 2.25x10(-9) to 4.25x10(-8) M for sensing sucrose. A microplate designed with an enzyme-nanomaterial-based sensor platform exhibited a high compliance, with acceptable percentage error for the detection of sucrose in green tea samples in comparison to a traditional method. With some further, modifications, this fabricated enzyme-nanogold onion membrane sensor probe could be used to estimate glucose concentrations for a variety of analytical samples. Graphical abstract Synthesis and characterization of invertase assisted nanogold clusters on onion membranes and their application for fluorescence-based sucrose sensor.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana.

    PubMed

    Xiang, Li; Le Roy, Katrien; Bolouri-Moghaddam, Mohammad-Reza; Vanhaecke, Mieke; Lammens, Willem; Rolland, Filip; Van den Ende, Wim

    2011-07-01

    Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.

  6. 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...

  7. 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.

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

    PubMed Central

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

    1995-01-01

    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

  9. 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

  10. 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

  11. 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

  12. 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).

  13. Invertase inhibitors from red beet, sugar beet, and sweet potato roots.

    PubMed

    Pressey, R

    1968-09-01

    Invertase inhibitors have been isolated and partially purified from red beets, sugar beets, and sweet potatoes. These inhibitors are thermolabile proteins with molecular weights of 18,000 to 23,000. They do not inhibit yeast and Neurospora invertases, but they are reactive with potato tuber invertase and other plant invertases with pH optima near 4.5. There are differences in reactivity of the inhibitors with some of the plant invertases, however. For most invertases, red beet and sugar beet inhibitors are most effective at pH 4.5 while sweet potato inhibitor is most effective at pH 5. PMID:16656933

  14. Invertase inhibitors from red beet, sugar beet, and sweet potato roots.

    PubMed

    Pressey, R

    1968-09-01

    Invertase inhibitors have been isolated and partially purified from red beets, sugar beets, and sweet potatoes. These inhibitors are thermolabile proteins with molecular weights of 18,000 to 23,000. They do not inhibit yeast and Neurospora invertases, but they are reactive with potato tuber invertase and other plant invertases with pH optima near 4.5. There are differences in reactivity of the inhibitors with some of the plant invertases, however. For most invertases, red beet and sugar beet inhibitors are most effective at pH 4.5 while sweet potato inhibitor is most effective at pH 5.

  15. 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

  16. Quantification of invertase activity in ants under field conditions.

    PubMed

    Heil, Martin; Büchler, Rita; Boland, Wilhelm

    2005-02-01

    Invertases (EC 3.2.1.26) are hydrolases that cleave sucrose into the monosacccharides, glucose, and fructose. They play a central role in carbohydrate metabolism of plants and animals. Methods presented so far to quantify invertase activity in ants or other animals have been hampered by the variability in both substrates and products of the enzymatic reaction in animals whose carbohydrate metabolism is highly active. Our method is based on a spectrophotometric quantification of the kinetics of glucose release. We first obtained an equilibrium state summarizing reactions of any carbohydrates and enzymes that are present in the extract. Sucrose was then added to quantify invertase activity as newly released glucose. Invertase activities differed significantly among species of ants. Variances were lowest among individuals from the same colony and highest among different species. When preparations were made from ants of the same species, invertase activity was linearly related to the number of ants used for extraction. Our method does not require ants to be kept on specific substrates prior to the experiment, or expensive or large equipment. It, thus, appears suitable for dealing with a broad range of physiological, ecological, and evolutionary questions.

  17. Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp.

    PubMed

    López-Igual, Rocío; Flores, Enrique; Herrero, Antonia

    2010-10-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N(2) fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO(2). The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

  18. Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions.

    PubMed

    Sonnewald, U; Brauer, M; von Schaewen, A; Stitt, M; Willmitzer, L

    1991-07-01

    In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis. PMID:1844880

  19. Invertase in immobilized cells of Papaver somniferum L.

    PubMed

    Stano, J; Nemec, P; Bezáková, L; Kovács, P; Kákoniova, D; Neubert, K; Lisková, D

    1997-03-01

    Papaver somniferum L., (opium poppy) cells were after permeabilization in Tween 80 immobilized by glutaraldehyde without any carrier. Cells immobilized by cross-linking performed the hydrolysis of sucrose. The immobilized cells were characterized by high invertase activity and appropriate physico-mechanical properties.

  20. Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase.

    PubMed

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

    2013-12-01

    The activity of vacuolar invertase (VI) is vital to potato cold-induced sweetening (CIS). A post-translational regulation of VI activity has been proposed which involves invertase inhibitor (VIH), but the mechanism for the interaction between VI and VIH has not been fully understood. To identify the potential partners of VI and VIH, two cDNA libraries were respectively constructed from CIS-resistant wild potato species Solanum berthaultii and CIS-sensitive potato cultivar AC035-01 for the yeast two-hybrid analysis. The StvacINV1 (one of the potato VIs) and StInvInh2B (one of the potato VIHs), previously identified to be associated with potato CIS, were used as baits to screen the two libraries. Through positive selection and sequencing, 27 potential target proteins of StvacINV1 and eight of StInvInh2B were clarified. The Kunitz-type protein inhibitors were captured by StvacINV1 in both libraries and the interaction between them was confirmed by bimolecular fluorescence complementation assay in tobacco cells, reinforcing a fundamental interaction between VI and VIH. Notably, a sucrose non-fermenting-1-related protein kinase 1 was captured by both the baits, suggesting that a protein complex could be necessary for fine turning of the invertase activity. The target proteins clarified in present research provide a route to elucidate the mechanism by which the VI activity can be subtly modulated.

  1. Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

    PubMed Central

    Bracho, Geracimo E.; Whitaker, John R.

    1990-01-01

    Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (Mr 30,000). The enzyme had a Km for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH4)2SO4 fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (Mr 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour. Images Figure 2 PMID:16667287

  2. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis.

    PubMed

    Chen, Su-Fen; Liang, Ke; Yin, Dong-Mei; Ni, Di-An; Zhang, Zhi-Guo; Ruan, Yong-Ling

    2016-12-01

    There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance.

  3. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis.

    PubMed

    Chen, Su-Fen; Liang, Ke; Yin, Dong-Mei; Ni, Di-An; Zhang, Zhi-Guo; Ruan, Yong-Ling

    2016-12-01

    There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance. PMID:26899912

  4. CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis.

    PubMed

    Ruhlmann, Jeffrey M; Kram, Brian W; Carter, Clay J

    2010-01-01

    To date, no genes have been reported to directly affect the de novo production of floral nectar. In an effort to identify genes involved in nectar production, the Affymetrix((R)) ATH1 GeneChip was previously used to examine global gene expression profiles in Arabidopsis thaliana nectaries. One of the genes displaying highly enriched expression in nectaries was CELL WALL INVERTASE 4 (AtCWINV4, At2g36190), which encodes an enzyme that putatively catalyses the hydrolysis of sucrose into glucose and fructose. RT-PCR was used to confirm the nectary-enriched expression of AtCWINV4, as well as an orthologue from Brassica rapa. To probe biological function, two independent Arabidopsis cwinv4 T-DNA mutants were isolated. Unlike wild-type plants, cwinv4 lines did not produce nectar. While overall nectary morphology appeared to be normal, cwinv4 flowers accumulated higher than normal levels of starch in the receptacle, but not within the nectaries themselves. Conversely, wild-type, but not cwinv4, nectarial stomata stained intensely for starch. Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar. Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production. In addition, CWINV4 is probably responsible for the hexose-rich composition observed for many Brassicaceae nectars.

  5. 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

  6. 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

  7. Gene Expressions for Signal Transduction under Acidic Conditions

    PubMed Central

    Fukamachi, Toshihiko; Ikeda, Syunsuke; Wang, Xin; Saito, Hiromi; Tagawa, Masatoshi; Kobayashi, Hiroshi

    2013-01-01

    Although it is now well known that some diseased areas, such as cancer nests, inflammation loci, and infarction areas, are acidified, little is known about cellular signal transduction, gene expression, and cellular functions under acidic conditions. Our group showed that different signal proteins were activated under acidic conditions compared with those observed in a typical medium of around pH 7.4 that has been used until now. Investigations of gene expression under acidic conditions may be crucial to our understanding of signal transduction in acidic diseased areas. In this study, we investigated gene expression in mesothelioma cells cultured at an acidic pH using a DNA microarray technique. After 24 h culture at pH 6.7, expressions of 379 genes were increased more than twofold compared with those in cells cultured at pH 7.5. Genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors numbered 35, 32, and 17 among the 379 genes, respectively. Since the functions of 78 genes are unknown, it can be argued that cells may have other genes for signaling under acidic conditions. The expressions of 37 of the 379 genes were observed to increase after as little as 2 h. After 24 h culture at pH 6.7, expressions of 412 genes were repressed more than twofold compared with those in cells cultured at pH 7.5, and the 412 genes contained 35, 76, and 7 genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors, respectively. These results suggest that the signal pathways in acidic diseased areas are different, at least in part, from those examined with cells cultured at a pH of around 7.4. PMID:24705103

  8. Expression patterns, activities and carbohydrate-metabolizing regulation of sucrose phosphate synthase, sucrose synthase and neutral invertase in pineapple fruit during development and ripening.

    PubMed

    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.

  9. Crystallization and preliminary X-ray diffraction analysis of the invertase from Saccharomyces cerevisiae

    PubMed Central

    Sainz-Polo, M. Angela; Lafraya, Alvaro; Polo, Aitana; Marín-Navarro, Julia; Polaina, Julio; Sanz-Aparicio, Julia

    2012-01-01

    Saccharomyces cerevisiae invertase (ScInv) is an enzyme encoded by the SUC2 gene that releases β-fructose from the nonreducing termini of various β-­d-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which presents a high degree of oligomerization, was crystallized by vapour-diffusion methods. The crystals belonged to space group P3121, with unit-cell parameters a = 268.6, b = 268.6, c = 224.4 Å. The crystals diffracted to 3.3 Å resolution and gave complete data sets using a synchrotron X-ray source. PMID:23192042

  10. Lipoic acid functionalized amino acids cationic lipids as gene vectors.

    PubMed

    Su, Rong-Chuan; Liu, Qiang; Yi, Wen-Jing; Zheng, Li-Ting; Zhao, Zhi-Gang

    2016-10-01

    A series of reducible cationic lipids 4a-4f with different amino acid polar-head groups were prepared. The novel lipid contains a hydrophobic lipoic acid (LA) moiety, which can be reduced under reductive conditions to release of the encapsulated plasmid DNA. The particle size, zeta potential and cellular uptake of lipoplexes formed with DNA, as well as the transfection efficacy (TE) were characterized. The TE of the cationic lipid based on arginine was especially high, and was 2.5times higher than that of a branched polyethylenimine in the presence of 10% serum.

  11. Most acid-tolerant chickpea mesorhizobia show induction of major chaperone genes upon acid shock.

    PubMed

    Brígido, Clarisse; Oliveira, Solange

    2013-01-01

    Our goals were to evaluate the tolerance of mesorhizobia to acid and alkaline conditions as well as to investigate whether acid tolerance is related to the species or the origin site of the isolates. In addition, to investigate the molecular basis of acid tolerance, the expression of chaperone genes groEL and dnaKJ was analyzed using acid-tolerant and sensitive mesorhizobia. Tolerance to pH 5 and 9 was evaluated in liquid medium for 98 Portuguese chickpea mesorhizobia belonging to four species clusters. All isolates showed high sensitivity to pH 9. In contrast, mesorhizobia revealed high diversity in terms of tolerance to acid stress: 35 % of the isolates were acid sensitive and 45 % were highly tolerant to pH 5 or moderately acidophilic. An association between mesorhizobia tolerance to acid conditions and the origin soil pH was found. Furthermore, significant differences between species clusters regarding tolerance to acidity were obtained. Ten isolates were used to investigate the expression levels of the chaperone genes by northern hybridization. Interestingly, most acid-tolerant isolates displayed induction of the dnaK and groESL genes upon acid shock while the sensitive ones showed repression. This study suggests that acid tolerance in mesorhizobia is related to the pH of the origin soil and to the species cluster of the isolates. Additionally, the transcriptional analysis suggests a relationship between induction of major chaperone genes and higher tolerance to acid pH in mesorhizobia. This is the first report on transcriptional analysis of the major chaperones genes in mesorhizobia under acidity, contributing to a better understanding of the molecular mechanisms of rhizobia acidity tolerance.

  12. Evaluation of invertase (B-fructo furanosidase) activity in irradiated Mazafaty dates during storage

    NASA Astrophysics Data System (ADS)

    Zare, Z.; Sohrabpour, M.; Fazeli, T. Z.; Kohan, K. G.

    2002-10-01

    Invertase activity of irradiated and non-irradiated Mazafaty dates during four months storage time has been studied. There are large differences in invertase activity in different dates cultivars. The soft and good quality dates usually have higher activity compared to dry or semi-dry varieties. Irradiated dates with doses 1-5 kGy, which could be used for decontamination and disinfestations of dates with a dose rate of 1.87 Gy/s were used. The samples were stored in two temperatures of 5°C and 25°C for four months. The activity of invertase enzyme was analysed at different time intervals. Inactivation study of invertase (B-fructo furanosidase) activity showed that the invertase is sensitive to temperature, storage time and also inactivation of enzyme occurred in dose range of 10-50 kGy.

  13. 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

  14. 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

  15. Extracellular Invertase Is an Essential Component of Cytokinin-Mediated Delay of SenescenceW⃞

    PubMed Central

    Balibrea Lara, Maria Encarnación; Gonzalez Garcia, Maria-Cruz; Fatima, Tahira; Ehneß, Rainer; Lee, Taek Kyun; Proels, Reinhard; Tanner, Widmar; Roitsch, Thomas

    2004-01-01

    Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the

  16. Yeast invertase polymorphism is correlated with variable states of oligosaccharide chain phosphorylation.

    PubMed Central

    Frevert, J; Ballou, C E

    1982-01-01

    Saccharomyces cerevisiae invertase (EC 3.2.1.26) isolated from wild-type strain X2180 can be resolved by isoelectric focusing into at least seven bands revealed by an activity stain. Most of this polymorphism is eliminated in mutants that are defective in phosphorylation of the mannoprotein carbohydrate chains (mnn4 and mnn6). In contrast to strain X2180, invertase from the mnn9 mutant, which makes mannoprotein lacking the outer portion of the polymannose chains, shows only two major bands on isoelectric focusing. Although mnn2 mannoprotein is though not to have any branches in its outer chain, the invertase of this mutant shows at least six bands on isoelectric focusing, and digestion of this invertase with an endo-alph aI leads to 6-mannanase that removes the unbranched outer chain produces an invertase with two bands that are similar to those from the mnn9 mutant. The invertase from mnn2 cells, grown with [32P]orthophosphate and precipitated with specific antiserum, gives at least five radioactive bands on isoelectric focusing, and after digestion with the endomannanase the radioactivity no longer migrates with the residual invertase. Mutants with shortened and unbranched outer chains (mnn2 mnn7, mnn2 mnn8, and mnn2 mnn10) give invertase patterns similar to mnn2. The results suggest that multiple states of outer chain phosphorylation lead to isoelectric polymorphism of S. cerevisiae external invertase and, because invertase has nine carbohydrate chains, no more than one phosphate group per chain would be required to account for this property. Images PMID:6755465

  17. Characterization of salicylic acid-induced genes in Chinese cabbage.

    PubMed

    Park, Y-S; Min, H-J; Ryang, S-H; Oh, K-J; Cha, J-S; Kim, H Y; Cho, T-J

    2003-06-01

    Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.

  18. 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.

  19. Ribonucleic acid interference induced gene knockdown

    PubMed Central

    Gottumukkala, Sruthima N. V. S.; Dwarakanath, C. D.; Sudarsan, Sabitha

    2013-01-01

    Despite major advances in periodontal regeneration over the past three decades, complete regeneration of the lost periodontium on a regular and predictable basis in humans has still remained elusive. The identification of stem cells in the periodontal ligament together with the growing concept of tissue engineering has opened new vistas in periodontal regenerative medicine. In this regard, ribonucleic acid interference (RNAi) opens a new gate way for a novel RNA based approach in periodontal management. This paper aims to summarize the current opinion on the mechanisms underlying RNAi, in vitro and in vivo existing applications in the dental research, which could lead to their future use in periodontal regeneration. PMID:24174717

  20. 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 (...

  1. Enhancement of invertase production by Aspergillus niger OZ-3 using low-intensity static magnetic fields.

    PubMed

    Taskin, Mesut; Esim, Nevzat; Genisel, Mucip; Ortucu, Serkan; Hasenekoglu, Ismet; Canli, Ozden; Erdal, Serkan

    2013-01-01

    The aim of this study is to investigate the effect of low-intensity static magnetic fields (SMFs) on invertase activity and growth on different newly identified molds. The most positive effect of SMFs on invertase activity and growth was observed for Aspergillus niger OZ-3. The submerged production of invertase was performed with the spores obtained at the different exposure times (120, 144, 168, and 196 hr) and magnetic field intensities (0.45, 3, 5, 7, and 9 mT). The normal magnetic field of the laboratory was assayed as 0.45 mT (control). Optimization of magnetic field intensity and exposure time significantly increased biomass production and invertase activity compared to 0.45 mT. The maximum invertase activity (51.14 U/mL) and biomass concentration (4.36 g/L) were achieved with the spores obtained at the 144 hr exposure time and 5 mT magnetic field intensity. The effect of low-intensity static magnetic fields (SMFs) on invertase activities of molds was investigated for the first time in the present study. As an additional contribution, a new hyper-invertase-producing mold strain was isolated.

  2. Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

    PubMed

    Dai, Zhimin; Guo, Xue; 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.

  3. 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

  4. 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.

  5. Control of Invertase Synthesis in Sugar Cane. Loci of Auxin and Glucose Effects

    PubMed Central

    Glasziou, K. T.; Waldron, J. C.; Bull, T. A.

    1966-01-01

    In tissue slices from rapidly expanding internodes of sugar cane the vacuolar invertase level is a function of the balance between synthesis and destruction. The enzyme is destroyed in the tissue at an approximately constant rate with a half time of 2 hours. Invertase synthesis is regulated by both auxin and glucose. From studies with inhibitors of protein and RNA synthesis we conclude that auxin alters the rate of synthesis and glucose increases the rate of destruction of messenger RNA required for the production of invertase. PMID:16656252

  6. 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.

  7. 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.

  8. 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.

  9. Dietary polyunsaturated fatty acid regulation of gene transcription.

    PubMed

    Clarke, S D; Jump, D B

    1994-01-01

    We have known for nearly 30 years that dietary polyenoic (n-6) and (n-3) fatty acids potentially inhibit hepatic fatty acid biosynthesis. The teleological explanation for this unique action of PUFAs resides in their ability to suppress the synthesis of (n-9) fatty acids. By inhibiting fatty acid biosynthesis, dietary PUFAs reduce the availability of substrate for delta 9 desaturase (7, 22, 34, 36) and in turn reduce the availability of (n-9) fatty acids for incorporation into plasma membranes. In this way, essential biological processes dependent on essential fatty acids (e.g. reproduction and trans-dermal water loss) continue to operate normally. Therefore, if essential fatty acid intake did not regulate (n-9) fatty acid synthesis, the survival of the organism would be threatened. During the past 20 years, we have gradually elucidated the cellular and molecular mechanisms by which dietary PUFAs modulate fatty acid biosynthesis and (n-9) fatty acid availability. Central to this mechanism has been our ability to determine that dietary PUFAs regulate the transcription of genes coding for lipogenic enzymes (12, 40). The potential mechanisms by which PUFAs govern gene transcription are numerous, and it is unlikely that any one mechanism can fully elucidate the nuclear actions of PUFA. The difficulty in providing a unifying hypothesis at this time stems from: (a) the many metabolic routes taken by PUFAs upon entering the hepatocyte (Figure 1); and (b) the lack of identity of a specific PUFA-regulated trans-acting factor. However, the studies described above indicate that macronutrients, like PUFA, are not only utilized as fuel and structural components of cells, but also serve as important mediators of gene expression (12, 14, 40). As regulators of gene expression, PUFAs (or metabolites) are thought to affect the activity of transcription factors, which in turn target key cis-linked elements associated with specific genes. Whether this targeting involves DNA

  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. Thymine, adenine and lipoamino acid based gene delivery systems.

    PubMed

    Skwarczynski, Mariusz; Ziora, Zyta M; Coles, Daniel J; Lin, I-Chun; Toth, Istvan

    2010-05-14

    A novel class of thymine, adenine and lipoamino acid based non-viral carriers for gene delivery has been developed. Their ability to bind to DNA by hydrogen bonding was confirmed by NMR diffusion, isothermal titration calorimetry and transmission electron microscopy experiments.

  12. Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

    PubMed

    Tauzin, Alexandra S; Sulzenbacher, Gerlind; Lafond, Mickael; Desseaux, Véronique; Reca, Ida Barbara; Perrier, Josette; Bellincampi, Daniela; Fourquet, Patrick; Lévêque, Christian; Giardina, Thierry

    2014-06-01

    Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.

  13. 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

  14. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    PubMed

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  15. Amino acid recognition and gene regulation by riboswitches

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2012-01-01

    Riboswitches specifically control expression of genes predominantly involved in biosynthesis, catabolism and transport of various cellular metabolites in organisms from all three kingdoms of life. Amongst many classes of identified riboswitches, two riboswitches respond to amino acids lysine and glycine to date. Though these riboswitches recognize small compounds, they both belong to the largest riboswitches and have unique structural and functional characteristics. In this review, we attempt to characterize molecular recognition principles employed by amino acid-responsive riboswitches to selectively bind their cognate ligands and to effectively perform a gene regulation function. We summarize up-to-date biochemical and genetic data available for the lysine and glycine riboswitches and correlate these results with recent high-resolution structural information obtained for the lysine riboswitch. We also discuss the contribution of lysine riboswitches to antibiotic resistance and outline potential applications of riboswitches in biotechnology and medicine. PMID:19619684

  16. Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.

    PubMed

    Cruz, Tiago M D; Carvalho, Raquel F; Richardson, Dale N; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  17. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    PubMed Central

    Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  18. 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

  19. 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.

  20. The pea gene NA encodes ent-kaurenoic acid oxidase.

    PubMed

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  1. Large-scale analysis of membrane transport in yeast using invertase reporters.

    PubMed

    Dalton, Lauren; Davey, Michael; Conibear, Elizabeth

    2015-01-01

    Transport of membrane proteins between cellular organelles requires the concerted action of many regulatory factors, which aid in cargo recognition and vesicle formation, targeting, and fusion. The yeast Saccharomyces cerevisiae is a useful model system for studying such regulators, due to the availability of genome-wide mutant collections and reporter proteins that provide sensitive biochemical readouts of individual transport pathways. Here, we describe an enzymatic invertase assay for evaluating endocytic recycling using a chimeric GFP-Snc1-Suc2 reporter. Cell surface levels of this reporter can be measured by a colorimetric assay that monitors sucrose hydrolysis at the plasma membrane, using two different methods. The first is a semiquantitative agar overlay assay followed by image densitometry that is suitable for high-throughput screening of arrayed yeast colonies. In the second, more quantitative assay, an enzymatic solution is added to yeast cultures in a multi-well plate and the absorbance is assessed by a plate reader. Furthermore, the modular nature of the chimeric reporter allows alternate transport signals to be introduced, thereby expanding the range of transport pathways that can be evaluated by this method. Together these techniques can be used to explore the function of genes involved in a variety of cellular trafficking pathways.

  2. Inducible gene expression and environmentally regulated genes in lactic acid bacteria.

    PubMed

    Kok, J

    1996-10-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transcription was studied and found to be regulatable. Examples are the lactose operon, the operon for nisin production, and genes in the proteolytic pathway of Lactococcus lactis, as well as xylose metabolism in Lactobacillus pentosus. Some other operons were specifically targetted with the aim to compare their mode of regulation with known regulatory mechanisms in other well-studied bacteria. These studies, dealing with the biosynthesis of histidine, tryptophan, and of the branched chain amino acids in L. lactis, have given new insights in gene regulation and in the occurrence of auxotrophy in these bacteria. Also, nucleotide sequence analyses of a number of lactococcal bacteriophages was recently initiated to, among other things, specifically learn more about regulation of the phage life cycle. Yet another approach in the analysis of regulated genes is the 'random' selection of genetic elements that respond to environmental stimuli and the first of such sequences from lactic acid bacteria have been identified and characterized. The potential of these regulatory elements in fundamental research and practical (industrial) applications will be discussed.

  3. Interplay of sugar, light and gibberellins in expression of Rosa hybrida vacuolar invertase 1 regulation.

    PubMed

    Rabot, Amélie; Portemer, Virginie; Péron, Thomas; Mortreau, Eric; Leduc, Nathalie; Hamama, Latifa; Coutos-Thévenot, Pierre; Atanassova, Rossitza; Sakr, Soulaiman; Le Gourrierec, José

    2014-10-01

    Our previous findings showed that the expression of the Rosa hybrida vacuolar invertase 1 gene (RhVI1) was tightly correlated with the ability of buds to grow out and was under sugar, gibberellin and light control. Here, we aimed to provide an insight into the mechanistic basis of this regulation. In situ hybridization showed that RhVI1 expression was localized in epidermal cells of young leaves of bursting buds. We then isolated a 895 bp fragment of the promoter of RhVI1. In silico analysis identified putative cis-elements involved in the response to sugars, light and gibberellins on its proximal part (595 bp). To carry out functional analysis of the RhVI1 promoter in a homologous system, we developed a direct method for stable transformation of rose cells. 5' deletions of the proximal promoter fused to the uidA reporter gene were inserted into the rose cell genome to study the cell's response to exogenous and endogenous stimuli. Deletion analysis revealed that the 468 bp promoter fragment is sufficient to trigger reporter gene activity in response to light, sugars and gibberellins. This region confers sucrose- and fructose-, but not glucose-, responsive activation in the dark. Inversely, the -595 to -468 bp region that carries the sugar-repressive element (SRE) is required to down-regulate the RhVI1 promoter in response to sucrose and fructose in the dark. We also demonstrate that sugar/light and gibberellin/light act synergistically to up-regulate β-glucuronidase (GUS) activity sharply under the control of the 595 bp pRhVI1 region. These results reveal that the 127 bp promoter fragment located between -595 and -468 bp is critical for light and sugar and light and gibberellins to act synergistically.

  4. 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

  5. Docosahexaenoic Acid (DHA) and Hepatic Gene Transcription1,3

    PubMed Central

    Jump, Donald B.; Botolin, Daniela; Wang, Yun; Xu, Jinghua; Demeure, Olivier; Christian, Barbara

    2008-01-01

    The type and quantity of dietary fat ingested contributes to the onset and progression of chronic diseases, like diabetes and atherosclerosis. The liver plays a central role in whole body lipid metabolism and responds rapidly to changes in dietary fat composition. Polyunsaturated fatty acids (PUFA) play a key role in membrane composition and function, metabolism and the control of gene expression. Certain PUFA, like the n-3 PUFA, enhance hepatic fatty acid oxidation and inhibit fatty acid synthesis and VLDL secretion, in part, by regulating gene expression. Our studies have established that key transcription factors, like PPARα, SREBP-1, ChREBP and MLX, are regulated by n-3 PUFA, which in turn control levels of proteins involved in lipid and carbohydrate metabolism. Of the n-3 PUFA, 22:6,n-3 has recently been established as a key controller of hepatic lipid synthesis. 22:6,n-3 controls the 26S proteasomal degradation of the nuclear form of SREBP-1. SREBP-1 is a major transcription factor that controls the expression of multiple genes involved fatty acid synthesis and desaturation. 22:6,n-3 suppresses nuclear SREBP-1 which, in turn suppresses lipogenesis. This mechanism is achieved, in part, through control of the phosphorylation status of protein kinases. This review will examine both the general features of PUFA-regulated hepatic gene transcription and highlight the unique mechanisms by which 22:6,n-3 impacts gene expression. The outcome of this analysis will reveal that changes in hepatic 22:6,n-3 content has a major impact on hepatic lipid and carbohydrate metabolism. Moreover, the mechanisms involve 22:6,n-3 control of several well-known signaling pathways, such as Akt, Erk1/2, Gsk3β and PKC (novel or atypical). 22:6,n-3 control of these same signaling pathways in non-hepatic tissues may help explain the diverse actions of n-3 PUFA on such complex physiological processes as visual acuity and learning. PMID:18343222

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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'.

  11. Fatty acid-gene interactions, adipokines and obesity.

    PubMed

    Stryjecki, C; Mutch, D M

    2011-03-01

    It is now recognized that the low-grade inflammation observed with obesity is associated with the development of a wide range of downstream complications. As such, there is considerable interest in elucidating the regulatory mechanisms underlying the production of inflammatory molecules to improve the prevention and treatment of obesity and its co-morbidities. White adipose tissue is no longer considered a passive reservoir for storing lipids, but rather an important organ influencing energy metabolism, insulin sensitivity and inflammation by the secretion of proteins, commonly referred to as adipokines. Dysregulation of several adipokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and adiponectin, contributes to the low-grade inflammation that is a hallmark of obesity. Evidence now suggests that fatty acids represent a class of molecules that can modulate adipokine production, thereby influencing inflammatory status. Although the precise molecular mechanisms by which dietary fats regulate adipokine production remain unclear, recent findings indicate that diet-gene interactions may have an important role in the transcriptional and secretory regulation of adipokines. Single-nucleotide polymorphisms in the genes encoding TNF-α, IL-6 and adiponectin can modify circulating levels of these adipokines and, subsequently, obesity-related phenotypes. This genetic variation can also alter the influence of dietary fatty acids on adipokine production. Therefore, the current review will show that it is paramount to consider both genetic information and dietary fat intake to unravel the inter-individual variability in inflammatory response observed in intervention protocols targeting obesity.

  12. 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.

  13. 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

  14. 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

  15. [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.

  16. [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

  17. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

    PubMed Central

    Duester, G; Shean, M L; McBride, M S; Stewart, M J

    1991-01-01

    Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis. Images PMID:1996113

  18. Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA.

    PubMed

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

    2014-05-01

    The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.

  19. 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.

  20. 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

  1. 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.

  2. 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...

  3. 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

  4. 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.

  5. Highly efficient production of inverted syrup in an analytical column with immobilized invertase.

    PubMed

    Upadhyay, Lata Sheo Bachan; Verma, Nishant

    2014-12-01

    This paper presents a procedure by which a simple and economical analytical column containing immobilized invertase was developed. This column has high efficiency of converting sucrose into inverted syrup rapidly. Gelatine beads were used for the immobilization of invertase. The enzyme was entrapped efficiently and was found to be stable and retained its activity over a period of 3 months. Immobilization parameters for maximum enzyme activity were estimated as temperature optima at 60 °C, pH optima 7.0 and 30 mg/mL enzyme concentration was found to give maximum immobilization (72 %). The reusability of the gelatine immobilized invertase was found to be seven times with a time interval of 24 h. The immobilized invertase presented a KM of 51.28 mM and Vmax of 0.334 mM/min. The time required to hydrolyse 50 % sucrose solution by a column of length 10 cm and diameter of 1.5 cm was found to be 15 min at room temperature. The column was found effective for inversion of biological samples like sugar cane juice.

  6. 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 %.

  7. [Invertase Overproduction May Provide for Inulin Fermentation by Selection Strains of Saccharomyces cerevisiae].

    PubMed

    Naumov, G I; Naumova, E S

    2015-01-01

    In some recent publications, the ability of selection strains of Saccharomyces cerevisiae to ferment inulin was attributed to inulinase activity. The review summarizes the literature data indicating that overproduction of invertase, an enzyme common to S. cerevisiae, may be responsible for this phenomenon. PMID:26263621

  8. Highly efficient production of inverted syrup in an analytical column with immobilized invertase.

    PubMed

    Upadhyay, Lata Sheo Bachan; Verma, Nishant

    2014-12-01

    This paper presents a procedure by which a simple and economical analytical column containing immobilized invertase was developed. This column has high efficiency of converting sucrose into inverted syrup rapidly. Gelatine beads were used for the immobilization of invertase. The enzyme was entrapped efficiently and was found to be stable and retained its activity over a period of 3 months. Immobilization parameters for maximum enzyme activity were estimated as temperature optima at 60 °C, pH optima 7.0 and 30 mg/mL enzyme concentration was found to give maximum immobilization (72 %). The reusability of the gelatine immobilized invertase was found to be seven times with a time interval of 24 h. The immobilized invertase presented a KM of 51.28 mM and Vmax of 0.334 mM/min. The time required to hydrolyse 50 % sucrose solution by a column of length 10 cm and diameter of 1.5 cm was found to be 15 min at room temperature. The column was found effective for inversion of biological samples like sugar cane juice. PMID:25477691

  9. Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

    PubMed

    An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

    2015-03-01

    Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid.

  10. 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

    2014-06-05

    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.

  11. 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.

  12. Agrobacterium tumefaciens exoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer, and virulence gene expression.

    PubMed

    Heckel, Brynn C; Tomlinson, Amelia D; Morton, Elise R; Choi, Jeong-Hyeon; Fuqua, Clay

    2014-09-01

    Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression. PMID:24982308

  13. Molecular evolution of monotreme and marsupial whey acidic protein genes.

    PubMed

    Sharp, Julie A; Lefèvre, Christophe; Nicholas, Kevin R

    2007-01-01

    Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

  14. Biotransformation of pineapple juice sugars into dietetic derivatives by using a cell free oxidoreductase from Zymomonas mobilis together with commercial invertase.

    PubMed

    Aziz, M G; Michlmayr, H; Kulbe, K D; Del Hierro, A M

    2011-01-01

    An easy procedure for cell free biotransformation of pineapple juice sugars into dietetic derivatives was accomplished using a commercial invertase and an oxidoreductase from Zymomonas mobilis. First, pineapple juice sucrose was quantitatively converted into glucose and fructose by invertase, thus increasing the concentration of each monosaccharide in the original juice to almost twice. In a second step, glucose-fructose oxidoreductase (GFOR) transformed glucose into gluconolactone, and fructose into the low calorie sweetener sorbitol. The advantage of using GFOR is simultaneous reduction of fructose and oxidation of glucose, allowing the continuous regeneration of the essential coenzyme NADP(H), that is tightly bound to the enzyme. The yield of GFOR catalyzed sugar conversion depends on initial pH and control of pH during the reaction. At optimal conditions (pH control at 6.2) a maximum of 80% (w/v) sugar conversion was obtained. Without pH control, GFOR is inactivated rapidly due to gluconic acid formation. Therefore, conversion yields are relatively low at the natural pH of pineapple juice. The application of this process might be more advantageous on juices of other tropical fruits (papaya, jackfruit, mango) due to their naturally given higher pH. PMID:22112775

  15. The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn4451.

    PubMed Central

    Crellin, P K; Rood, J I

    1997-01-01

    Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostridium perfringens and is found on the conjugative plasmid pIP401. The element undergoes spontaneous excision from multicopy plasmids in Escherichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is probably the transposition intermediate. Excision of Tn4451 is dependent upon the site-specific recombinase TnpX, which contains potential motifs associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid residues within these domains was used to show that the resolvase/invertase domain was essential for TnpX-mediated excision of Tn4451 from multicopy plasmids in E. coli. An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity. The Tn4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanked by directly repeated GA dinucleotides, and there was also a GA at the junction of the circular form, where the left and right termini of Tn4451 were fused. We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp staggered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives with altered GA dinucleotides provided experimental evidence to support the model. PMID:9260958

  16. Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

    PubMed Central

    Li, Rongfeng; Khaleeli, Nusrat; Townsend, Craig A.

    2000-01-01

    Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed. PMID:10869089

  17. 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

  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. 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

  20. Cloning and characterization of the ferulic acid catabolic genes of Sphingomonas paucimobilis SYK-6.

    PubMed

    Masai, Eiji; Harada, Kyo; Peng, Xue; Kitayama, Hirotaka; Katayama, Yoshihiro; Fukuda, Masao

    2002-09-01

    Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells. A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders. On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of E. coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively. p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5'-dehydrodivanillic acid. The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase

  1. 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...

  2. 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

    2015-12-16

    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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Association of an ACSL1 gene variant with polyunsaturated fatty acids in bovine skeletal muscle

    PubMed Central

    2011-01-01

    Background The intramuscular fat deposition and the fatty acid profiles of beef affect meat quality. High proportions of unsaturated fatty acids are related to beef flavor and are beneficial for the nutritional value of meat. Moreover, a variety of clinical and epidemiologic studies showed that particularly long-chain omega-3 fatty acids from animal sources have a positive impact on human health and disease. Results To screen for genetic factors affecting fatty acid profiles in beef, we initially performed a microsatellite-based genome scan in a F2 Charolais × German Holstein resource population and identified a quantitative trait locus (QTL) for fatty acid composition in a region on bovine chromosome 27 where previously QTL affecting marbling score had been detected in beef cattle populations. The long-chain acyl-CoA synthetase 1 (ACSL1) gene was identified as the most plausible functional and positional candidate gene in the QTL interval due to its direct impact on fatty acid metabolism and its position in the QTL interval. ACSL1 is necessary for synthesis of long-chain acyl-CoA esters, fatty acid degradation and phospholipid remodeling. We validated the genomic annotation of the bovine ACSL1 gene by in silico comparative sequence analysis and experimental verification. Re-sequencing of the complete coding, exon-flanking intronic sequences, 3' untranslated region (3'UTR) and partial promoter region of the ACSL1 gene revealed three synonymous mutations in exons 6, 7, and 20, six noncoding intronic gene variants, six polymorphisms in the promoter region, and four variants in the 3' UTR region. The association analysis identified the gene variant in intron 5 of the ACSL1 gene (c.481-233A>G) to be significantly associated with the relative content of distinct fractions and ratios of fatty acids (e.g., n-3 fatty acids, polyunsaturated, n-3 long-chain polyunsaturated fatty acids, trans vaccenic acid) in skeletal muscle. A tentative association of the ACSL1 gene

  8. 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

  9. Phytanic acid and docosahexaenoic acid increase the metabolism of all-trans-retinoic acid and CYP26 gene expression in intestinal cells.

    PubMed

    Lampen, A; Meyer, S; Nau, H

    2001-10-31

    Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all-trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRalpha) and peroxisome proliferator activating receptor (PPARalpha) activator, also activated PPARdelta, and to a lesser degree PPARgamma, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all-trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all-trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all-trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all-trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all-trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all-trans-RA in intestinal cells.

  10. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    PubMed

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  11. Neutral invertase, hexokinase and mitochondrial ROS homeostasis: emerging links between sugar metabolism, sugar signaling and ascorbate synthesis.

    PubMed

    Xiang, Li; Li, Yi; Rolland, Filip; Van den Ende, Wim

    2011-10-01

    Alkaline/neutral invertases (A/N-Invs) are unique to plants and photosynthetic bacteria. Although considerable advances have been made in our understanding of sucrose metabolic enzymes in plants, the function of A/N-Invs remained puzzling. In a recent study, we have analyzed the subcellullar localization of a cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) Arabidopsis A/N-Inv. Unexpectedly, At-A/N-InvA knockout plants showed a more severe growth defect than At-A/N-InvG knockout plants and a link between the two A/N-Invs and oxidative stress defence was found. Overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts reduced the activity of the ascorbate peroxidase 2 (APX2) promoter, that was stimulated by hydrogen peroxide and abscisic acid. It is discussed here how sugars and ascorbate might contribute to mitochondrial reactive oxygen species homeostasis. We hypothesize that both mitochondrial and cytosolic A/N-Invs and mitochondria-associated hexokinases are key mediators, integrating metabolic and sugar signalling processes. 

  12. Amino Acid Supplementation Affects Imprinted Gene Transcription Patterns in Parthenogenetic Porcine Blastocysts

    PubMed Central

    Park, Chi-Hun; Jeong, Young-Hee; Jeong, Yeun-Ik; Kwon, Jeong-Woo; Shin, Taeyoung; Hyun, Sang-Hwan; Jeung, Eui-Bae; Kim, Nam-Hyung; Seo, Sang-Kyo; Lee, Chang-Kyu; Hwang, Woo-Suk

    2014-01-01

    To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine

  13. Nucleic acid binding property of the gene products of rice stripe virus.

    PubMed

    Liang, Delin; Ma, Xiangqiang; Qu, Zhicai; Hull, Roger

    2005-10-01

    GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function. PMID:16025246

  14. Column chromatography of human small-intestinal maltase, isomaltase and invertase activities

    PubMed Central

    Dahlqvist, A.; Telenius, U.

    1969-01-01

    1. The maltase, isomaltase and invertase (sucrase) activities of solubilized mucosal preparations from human jejunum and ileum were studied with column chromatography on anion-exchange (diethylaminoethyl- and triethylaminoethyl-)cellulose and Sephadex G-200 gel. 2. On ion-exchange cellulose columns both kinds of enzyme preparations yielded two major disaccharidase peaks. The first peak contained maltase Ia (=isomaltase) and maltase Ib (=invertase). The second peak contained maltase II and maltase III. 3. On Sephadex G-200 gel columns jejunal preparations yielded the corresponding peaks as on ion-exchange columns, but the peaks appeared in the reverse order in the effluent. The ileal preparation studied yielded a single peak on gel columns, containing all the activities studied and eluted with the `void volume'. 4. Precipitation with ethanol did not affect the behaviour of the enzymes during ion-exchange chromatography. When gel filtration was performed after ethanol precipitation of the enzymes, however, two peaks were obtained also with the ileal preparation, and subfractionation of the invertase was obtained with both kinds of preparations. 5. The second peak from ion-exchange chromatograms, containing maltase II and maltase III, on concentration was found to have very weak isomaltase activity, probably exerted by these enzymes as such. This activity accounts for only about 1% of the total isomaltase activity of the mucosa. 6. The results support the concept of the specificity of the human small-intestinal disaccharidases previously described after heat-inactivation experiments. The subfractionation of the invertase that under certain conditions is seen on Sephadex G-200 columns appears most likely to be an artifact. Consequently the nomenclature for the human maltose-, isomaltose- and sucrose-splitting enzymes proposed by another research group after gel-filtration chromatography studies should be abandoned. It seems more logical to keep the nomenclature based

  15. Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

    PubMed Central

    2010-01-01

    Background Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are

  16. Cloning of genes responsible for acetic acid resistance in Acetobacter aceti.

    PubMed

    Fukaya, M; Takemura, H; Okumura, H; Kawamura, Y; Horinouchi, S; Beppu, T

    1990-04-01

    Five acetic acid-sensitive mutants of Acetobacter aceti subsp. aceti no. 1023 were isolated by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Three recombinant plasmids that complemented the mutations were isolated from a gene bank of the chromosome DNA of the parental strain constructed in Escherichia coli by using cosmid vector pMVC1. One of these plasmids (pAR1611), carrying about a 30-kilobase-pair (kb) fragment that conferred acetic acid resistance to all five mutants, was further analyzed. Subcloning experiments indicated that a 8.3-kb fragment was sufficient to complement all five mutations. To identify the mutation loci and genes involved in acetic acid resistance, insertional inactivation was performed by insertion of the kanamycin resistance gene derived from E. coli plasmid pACYC177 into the cloned 8.3-kb fragment and successive integration into the chromosome of the parental strain. The results suggested that three genes, designated aarA, aarB, and aarC, were responsible for expression of acetic acid resistance. Gene products of these genes were detected by means of overproduction in E. coli by use of the lac promoter. The amino acid sequence of the aarA gene product deduced from the nucleotide sequence was significantly similar to those of the citrate synthases (CSs) of E. coli and other bacteria. The A. aceti mutants defective in the aarA gene were found to lack CS activity, which was restored by introduction of a plasmid containing the aarA gene. A mutation in the CS gene of E. coli was also complemented by the aarA gene. These results indicate that aarA is the CS gene.

  17. Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death1[OPEN

    PubMed Central

    2016-01-01

    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

  18. Acidity-Activated Shielding Strategies of Cationic Gene Delivery for Cancer Therapy.

    PubMed

    Xia, Jialiang; Feng, Zongcai; Yang, Hongyan; Lin, Sanqing; Han, Bing

    2016-01-01

    Cationic gene vectors increased attractive for gene therapy. However, unstable systemic circulation due to the interaction of gene delivery system with blood cells limited the further application. Therefore, pH sensitive shielding systems were exploited, by which, the positive surface charge density of polyplexes was reduced, circulation time was improved and pH-triggered targeting delivery was promised. This mini review mainly focuses on the development of solid tumors pH environment activated shielding systems for cationic gene vectors. This shielding strategy shows great potential for enhancing efficient gene transporting and achieving better therapeutic effects in acidic tumor treatment.

  19. 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

  20. Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense.

    PubMed

    Chen, R D; Yu, L X; Greer, A F; Cheriti, H; Tabaeizadeh, Z

    1994-10-28

    We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24,943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress. PMID:7816027

  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.

  2. 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...

  3. 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...

  4. 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...

  5. 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%.

  6. [Gene therapy: nucleic acids as drugs. Action mechanisms and delivery into the cell].

    PubMed

    Cavagnari, Brian M

    2011-06-01

    Gene therapy involves the transference of new genetic material to the cell in order to obtain a therapeutic benefit, offering a new option for the treatment of various diseases. In this article, some of these nucleic acid-based drugs, such as plasmids, aptamers, oligonucleotides, ribozymes and small interfering ribonucleic acid, are presented. Their mechanism and level of action is commented and several delivery systems, such as liposomes, cationic polymers, direct nucleic acid transfer and viral vectors, are also discussed.

  7. Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1.

    PubMed Central

    Vimr, E R; Aaronson, W; Silver, R P

    1989-01-01

    The kps gene cluster of Escherichia coli K1 encodes functions for sialic acid synthesis, activation, polymerization, and possibly translocation of polymer to the cell surface. The size and complexity of this membrane polysaccharide biosynthetic cluster have hindered genetic mapping and functional descriptions of the kps genes. To begin a detailed investigation of the polysialic acid synthetic mechanism, acapsular mutants were characterized to determine their probable defects in polymer synthesis. The mutants were tested for complementation with kps fragments subcloned from two separately isolated, functionally intact kps gene clusters. Complementation was assayed by immunological and biochemical methods and by sensitivity to the K1-specific bacteriophage K1F. The kps cluster consisted of a central 5.8-kilobase region that contained at least two genes coding for sialic acid synthetic enzymes, a gene encoding the sialic acid-activating enzyme, and a gene encoding the sialic acid polymerase. This biosynthetic region is flanked on one side by an approximately 2.8-kilobase region that contains a potential regulatory locus and at least one structural gene for a polypeptide that appears to function in polysialic acid assembly. Flanking the biosynthetic region on the opposite side is a 6- to 8.4-kilobase region that codes for at least three proteins which may also function in polymer assembly and possibly in translocating polymer to the outer cell surface. Results of transduction crosses supported these conclusions and indicated that some of the kps genes flanking the central biosynthetic region may not function directly in transporting polymer to the cell surface. The results also demonstrate that the map position and probable function of most of the kps cluster genes have been identified. Images PMID:2644224

  8. Characterization of a salicylic acid- and pathogen-induced lipase-like gene in Chinese cabbage.

    PubMed

    Lee, Kyung-Ah; Cho, Tae-Ju

    2003-09-30

    A cDNA clone for a salicylic acid-induced gene in Chinese cabbage (Brassica rapa subsp. pekinensis) was isolated and characterized. The cabbage gene, designated Br-sil1 (for Brassica rapa salicylate-induced lipase-like 1 gene), encodes a putative lipase that has the family II lipase motif GDSxxDxG around the active site serine. A database search showed that plant genomes have a large number of genes that contain the family II lipase motif. The lipase-like proteins include a myrosinase-associated protein, an anther-specific proline-rich protein APG, a pollen coat protein EXL, and an early nodule-specific protein. The Br-sil1 gene is strongly induced by salicylic acid and a nonhost pathogen, Pseudomonas syringae pv. tomato, that elicits a hypersensitive response in Chinese cabbage. Treatment of the cabbage leaves with BTH, methyl jasmonate, or ethephon showed that the Br-sil1 gene expression is induced by BTH, but not by methyl jasmonate or ethylene. This indicates that the cabbage gene is activated via a salicylic acid-dependent signaling pathway. An examination of the tissue-specific expression revealed that the induction of the Br-sil1 gene expression by BTH occurs in leaves and stems, but not in roots and flowers. Without the BTH treatment, however, the Br-sil1 gene is not expressed in any of the tissues that were examined.

  9. Five Additional Genes Are Involved in Clavulanic Acid Biosynthesis in Streptomyces clavuligerus

    PubMed Central

    Jensen, S. E.; Paradkar, A. S.; Mosher, R. H.; Anders, C.; Beatty, P. H.; Brumlik, M. J.; Griffin, A.; Barton, B.

    2004-01-01

    An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for β-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster. PMID:14693539

  10. The relationship between dietary fatty acids and inflammatory genes on the obese phenotype and serum lipids.

    PubMed

    Joffe, Yael T; Collins, Malcolm; Goedecke, Julia H

    2013-05-21

    Obesity, a chronic low-grade inflammatory condition is associated with the development of many comorbidities including dyslipidemia. This review examines interactions between single nucleotide polymorphisms (SNP) in the inflammatory genes tumor necrosis alpha (TNFA) and interleukin-6 (IL-6) and dietary fatty acids, and their relationship with obesity and serum lipid levels. In summary, dietary fatty acids, in particular saturated fatty acids and the omega-3 and omega-6 polyunsaturated fatty acids, impact the expression of the cytokine genes TNFA and IL-6, and alter TNFα and IL-6 production. In addition, sequence variants in these genes have also been shown to alter their gene expression and plasma levels, and are associated with obesity, measures of adiposity and serum lipid concentrations. When interactions between dietary fatty acids and TNFA and IL-6 SNPs on obesity and serum lipid were analyzed, both the quantity and quality of dietary fatty acids modulated the relationship between TNFA and IL-6 SNPs on obesity and serum lipid profiles, thereby impacting the association between phenotype and genotype. Researching these diet-gene interactions more extensively, and understanding the role of ethnicity as a confounder in these relationships, may contribute to a better understanding of the inter-individual variability in the obese phenotype.

  11. Chlorogenic acid protects MSCs against oxidative stress by altering FOXO family genes and activating intrinsic pathway.

    PubMed

    Li, Shiyong; Bian, Hetao; Liu, Zhe; Wang, Ye; Dai, Jianghua; He, Wenfeng; Liao, Xingen; Liu, Rongrong; Luo, Jun

    2012-01-15

    Chlorogenic acid as an antioxidant exists widely in edible and medicinal plants, and can protect cell against apoptosis induced by oxidative stress. However, its molecular mechanisms remain largely unknown. Here, we showed that Chlorogenic acid suppressed reactive oxygen species increase by activation of Akt phosphorylation,and increased FOXO family genes and anti-apoptotic protein Bcl-2 expression in MSCs culturing under oxidative stress. In addition, PI-3Kinase Inhibitor (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, LY294002) could suppress the Chlorogenic acid-induced: (1) the cellular protective role, (2) the increase of the FOXO family genes expression, (3) increased expression of Bcl-2. These results suggested that Chlorogenic acid protected MSCs against apoptosis via PI3K/AKT signal and FOXO family genes.

  12. 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

  13. [Control of gene expression by antisense nucleic acids].

    PubMed

    Lebleu, B; Clarenc, J P; Degols, G; Leonetti, J P; Milhaud, P

    1992-01-01

    The use of antisense RNA or of antisense oligonucleotides for the specific control of viral or cellular genes expression has undergone rapid developments recently; their respective advantages and drawbacks will be discussed. Progresses in oligonucleotides chemistry have lead to the synthesis of analogs with improved pharmacological properties. Besides the antisense approach, which usually targets translation initiation or splicing sites, it is possible to interfere specifically with gene expression through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense approach). A major problem encountered in the use of synthetic oligonucleotides is their delivery to their nuclear or cytoplasmic targets after cell uptake by an endocytic pathway; our own work in this field will be discussed. Finally, we will describe the strategies followed by our group to improve the bioavailability of antisense oligonucleotides, as for instance conjugation to poly (L-lysine) or encapsidation in antibody-targeted liposomes.

  14. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

    PubMed

    Harashima, S; Hinnebusch, A G

    1986-11-01

    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

  15. Deletion of the pyc Gene Blocks Clavulanic Acid Biosynthesis Except in Glycerol-Containing Medium: Evidence for Two Different Genes in Formation of the C3 Unit

    PubMed Central

    Pérez-Redondo, Rosario; Rodríguez-García, Antonio; Martín, Juan F.; Liras, Paloma

    1999-01-01

    The β-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the β-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing d-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin. PMID:10559157

  16. Gene-Enzyme Relationships of Aromatic Amino Acid Biosynthesis in Higher Plants

    SciTech Connect

    2002-08-12

    Inhibition studies of amino acids in Nicotiana silvestris suspension cells gave clues to the difficulties for obtaining mutants deficient in post prephenate pathway proteins of aromatic amino acid biosynthesis (prephenate aminotransferase, arogenate dehydrogenase and arogenate dehydratase). Such mutants, if successfully obtained, would allow gene-enzyme relationships of aromatic amino acid proteins to be studied. We found that amino acids were inhibitory toward plant cell growth, and thus were unable to rescue analog resistant mutants. Toxicity of all amino acids toward exponentially dividing Nicotiana silvestris suspension cultured cells was monitored by following growth rates. Except for L-glutamine, all 19 protein amino acids inhibited cell growth. Inhibition of growth progressed to cell deterioration. Electron microscopy showed that amino acids triggered a state of cell shrinkage that eventually degenerated to total cellular disorganization. L-glutamine was not only an effective agent for prevention of amino acid toxicity, but enhanced the final growth yield. L-glutamine also was able to completely reverse inhibition effects in cells that had been in the slowed exponential phase. Two types of inhibition occurred and we have proposed that any amino acid inhibition that can be completely antagonized by L-glutamine be called ''general amino acid inhibition''. ''Specific amino acid inhibition'' resulting from particular pathway imbalances caused by certain exogenous amino acids, can be recognized and studied in the presence of L-glutamine which can abolishes the complication effects of general amino acid inhibition.

  17. Differential Gene Expression in GPR40-Overexpressing Pancreatic β-cells Treated with Linoleic Acid

    PubMed Central

    Kim, In-Su; Yang, So-Young; Han, Joo-Hui; Jung, Sang-Hyuk; Park, Hyun-Soo

    2015-01-01

    "G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic β-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with 30 µM linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus. PMID:25729276

  18. Isolation and Molecular Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase Genes in Hevea brasiliensis

    PubMed Central

    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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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...

  4. [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

  5. 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

  6. Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus▿ §

    PubMed Central

    Zelyas, Nathan J.; Cai, Hui; Kwong, Thomas; Jensen, Susan E.

    2008-01-01

    Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway. PMID:18931110

  7. Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases.

    PubMed Central

    Kim, S J; Lafyatis, R; Kim, K Y; Angel, P; Fujiki, H; Karin, M; Sporn, M B; Roberts, A B

    1990-01-01

    Human collagenase gene expression is regulated transcriptionally and is inducible by various mitogens in many cell types. To investigate the molecular mechanisms of this response, we examined the effects on collagenase gene expression of okadaic acid, a non-12-O-tetradecanoyl-phorbol-13-acetate (TPA)-type tumor promoter, which induces apparent "activation" of protein kinases by inhibition of protein phosphatases. Steady state levels of collagenase mRNA were markedly increased by okadaic acid treatment. We show that the AP-1 consensus sequence in the collagenase promoter is required for the induction of collagenase gene expression by okadaic acid, even though sequences upstream of the AP-1 consensus site have an additive effect. We also examined the regulation by okadaic acid of expression of the components of the AP-1 complex, c-fos and c-jun. c-fos expression is dramatically stimulated by okadaic acid, whereas c-jun expression is stimulated to a lesser extent. Induction of c-fos gene mRNA occurs through a region known to contain multiple regulatory elements. These results suggest that phosphorylation regulates collagenase gene expression mediated by an AP-1 binding site. Images PMID:1966042

  8. 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.

  9. 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

  10. 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.

  11. Sialic acid catabolism and transport gene clusters are lineage specific in Vibrio vulnificus.

    PubMed

    Lubin, Jean-Bernard; Kingston, Joseph J; Chowdhury, Nityananda; Boyd, E Fidelma

    2012-05-01

    Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several Vibrio species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In Vibrio cholerae on chromosome I, these genes are carried on the Vibrio pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced Vibrio vulnificus clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within V. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of V. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in V. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in siaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus and that the TRAP transporter is essential for sialic acid uptake.

  12. [Effect of acid rain, copper, and atrazine on soil hydrolase activity].

    PubMed

    Liu, Guangshen; Xu, Dongmei; Li, Kebin; Liu, Weiping

    2004-01-01

    The effects of acid rain, Cu2+ and atrazine on the activities of soil urease, invertase and acid phosphatase were studied by means of orthogonal test. The results showed that the inhibition rate was H+ > Cu2+, and atrazine had no significant influence on urease and intertase. Interaction analysis revealed that Cu x atrazine exhibited synergism on soil acid phosphatase activity, Cu x H had antagonism on soil invertase and urease, but atrazine x H had no interaction within the investigated concentration range. Among the three enzymes, soil acid phosphatase was the most sensitive one to the contaminations.

  13. Dynamic changes in prefrontal cortex gene expression following lysergic acid diethylamide administration.

    PubMed

    Nichols, Charles D; Garcia, Efrain E; Sanders-Bush, Elaine

    2003-03-17

    Lysergic acid diethylamide (LSD) is a psychoactive drug that transiently alters human perception, behavior, and mood at extremely low doses. Certain aspects of the behavior elicited by acute doses of LSD closely resemble symptoms of mental disorders such as schizophrenia. Characterizing gene expression profiles after LSD will be important for understanding how it alters behavior, and will lead to novel insights into disorders, such as schizophrenia, whose behavioral symptoms resemble the temporary effects of hallucinogenic drugs. We previously identified a small collection of genes within the rat prefrontal cortex that respond to LSD. Many of the products of these genes are involved in the process of synaptic plasticity. In the current report, we present a detailed analysis of the expression of these genes within the brain using RNase protection analysis. We find that the gene response to LSD is quite dynamic. The expression of some genes increases rapidly and decreases rapidly, while other genes change more gradually. Dose-response studies show two classes of expression; gene expression maximally stimulated at lower doses, versus gene expression that continues to rise at the higher doses. The role of the 5-HT(1A) and 5-HT(2A) receptor in mediating the increases in gene expression was examined in a series of experiments using receptor specific antagonists. Most expression increases were due to activation of the 5-HT(2A) receptor, however expression of two genes had neither a 5-HT(1A) nor a 5-HT(2A) receptor component.

  14. Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel "cell-wall invertase-like" specific 6-FEH from sugar beet (Beta vulgaris L.).

    PubMed

    Van den Ende, Wim; De Coninck, Barbara; Clerens, Stefan; Vergauwen, Rudy; Van Laere, André

    2003-12-01

    About 15% of flowering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans. The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levan-type fructans (beta-2,6) are the best substrates for the enzyme, while inulin-type fructans (beta-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and beta-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.

  15. 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

  16. Genome-wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus.

    PubMed

    Medema, Marnix H; Alam, Mohammad T; Heijne, Wilbert H M; van den Berg, Marco A; Müller, Ulrike; Trefzer, Axel; Bovenberg, Roel A L; Breitling, Rainer; Takano, Eriko

    2011-03-01

    To increase production of the important pharmaceutical compound clavulanic acid, a β-lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome-wide gene expression of an industrial S. clavuligerus strain, obtained through iterative mutagenesis, with that of the wild-type strain. Intriguingly, we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters. A few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid. In general, the observed changes largely coincide with genes that have been targeted by rational engineering in recent years, yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology.

  17. Genome‐wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus

    PubMed Central

    Medema, Marnix H.; Alam, Mohammad T.; Heijne, Wilbert H. M.; van den Berg, Marco A.; Müller, Ulrike; Trefzer, Axel; Bovenberg, Roel A. L.; Breitling, Rainer; Takano, Eriko

    2011-01-01

    Summary To increase production of the important pharmaceutical compound clavulanic acid, a β‐lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome‐wide gene expression of an industrial S. clavuligerus strain, obtained through iterative mutagenesis, with that of the wild‐type strain. Intriguingly, we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters. A few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid. In general, the observed changes largely coincide with genes that have been targeted by rational engineering in recent years, yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology. PMID:21342474

  18. 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

  19. 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

  20. 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.

  1. Fatty acid composition of beef is associated with exonic nucleotide variants of the gene encoding FASN.

    PubMed

    Oh, Dongyep; Lee, Yoonseok; La, Boomi; Yeo, Jungsou; Chung, Euiryong; Kim, Younyoung; Lee, Chaeyoung

    2012-04-01

    Genetic associations of fatty acid composition with exonic single nucleotide polymorphisms (SNPs) in the gene encoding fatty acid synthase (FASN) were examined using 513 Korean cattle. All five individual SNPs of g.12870 T>C, g.13126 T>C, g.15532 C>A, g.16907 T>C and g.17924 G>A were associated with a variety of fatty acid compositions and further with marbling score (P < 0.05). Their genotypes of CC, TT, AA, TT, and GG were associated with increased monounsaturated fatty acids and with decreased saturated fatty acids (P < 0.05). The genotypes at all the SNPs also increased marbling score (P < 0.05). Further genetic associations with fatty acid composition suggested that homozygous genotype with the haplotype of ATG at g.15532, g.16907, and g.17924 in a linkage disequilibrium block increased monounsaturated fatty acids and marbling score (P < 0.05). We concluded that the five exonic SNPs of g.12870, g.13126, g.15532, g.16907, and g.17924 in the FASN gene could change fatty acid contents. Their genotypes of CC, TT, AA, TT, and GG and haplotype of ATG at g.15532, g.16907, and g.17924 were recommended for genetic improvement of beef quality.

  2. A role for Lon protease in the control of the acid resistance genes of Escherichia coli.

    PubMed

    Heuveling, Johanna; Possling, Alexandra; Hengge, Regine

    2008-07-01

    Lon protease is a major protease in cellular protein quality control, but also plays an important regulatory role by degrading various naturally unstable regulators. Here, we traced additional such regulators by identifying regulons with co-ordinately altered expression in a lon mutant by genome-wide transcriptional profiling. Besides many members of the RcsA regulon (which validates our approach as RcsA is a known Lon substrate), many genes of the sigmaS-dependent general stress response were upregulated in the lon mutant. However, the lon mutation did not affect sigmaS levels nor sigmaS activity in general, suggesting specific effects of Lon on secondary regulators involved in the control of subsets of sigmaS-controlled genes. Lon-affected genes also included the major acid resistance genes (gadA, gadBC, gadE, hdeAB and hdeD), which led to the discovery that the essential acid resistance regulator GadE (whose expression is sigmaS-controlled) is degraded in vivo in a Lon-dependent manner. GadE proteolysis is constitutive as it was observed even under conditions that induce the system (i.e. at low pH or during entry into stationary phase). GadE degradation was found to rapidly terminate the acid resistance response upon shift back to neutral pH and to avoid overexpression of acid resistance genes in stationary phase.

  3. 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

  4. 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

  5. vir-Gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens.

    PubMed

    Berthelot, K; Buret, D; Guerin, B; Delay, D; Negrel, J; Delmotte, F M

    1998-11-20

    Expression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds. Several alkylsyringamides have recently been shown to be powerful inducers of these vir-genes. These synthetic amides, and especially ethylsyringamide, are much stronger inducers than syringic acid. In this work, four alkylamides derived from ferulic or sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to induce vir-gene expression on A. tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids. Their effectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occurring amide in plants. Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or beta-alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria of the respective acid at high concentration and thereby increased the vir-inducing potential. However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-gene induction, with the exception of ethylsyringamide at concentrations above 1mM. When tested on Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more efficient than the corresponding phenolic acids but only above 100 microM. PMID:11711062

  6. 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.

  7. Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

    PubMed

    Campos, Laura; Granell, Pablo; Tárraga, Susana; López-Gresa, Pilar; Conejero, Vicente; Bellés, José María; Rodrigo, Ismael; Lisón, Purificación

    2014-04-01

    We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

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

    PubMed

    Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Zhu, Junwei J; Zhuang, Xiaofeng; Liu, Wusheng; Pantalone, Vincent R; Arelli, Prakash R; Stewart, Charles N; Chen, Feng

    2013-12-01

    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 soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

  9. 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

  10. 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

  11. 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.

  12. Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

    PubMed Central

    Thill, G P; Kramer, R A; Turner, K J; Bostian, K A

    1983-01-01

    The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion. Images PMID:6343840

  13. Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

    PubMed Central

    Paradkar, A. S.; Mosher, R. H.; Anders, C.; Griffin, A.; Griffin, J.; Hughes, C.; Greaves, P.; Barton, B.; Jensen, S. E.

    2001-01-01

    Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine ɛ-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine. PMID:11319114

  14. Folic acid supplementation dysregulates gene expression in lymphoblastoid cells--implications in nutrition.

    PubMed

    Junaid, Mohammed A; Kuizon, Salomon; Cardona, Juan; Azher, Tayaba; Murakami, Noriko; Pullarkat, Raju K; Brown, W Ted

    2011-09-01

    For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ≥ four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics. PMID:21867686

  15. 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

  16. 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…

  17. 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…

  18. 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

  19. 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

  20. Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

    PubMed

    Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Rowland, Gordon; Booker, Helen; You, Frank M; Cloutier, Sylvie

    2013-10-01

    Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  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. 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

  10. Changes in oleic Acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.

    PubMed

    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.

  11. 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

  12. 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.

  13. Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.

    PubMed

    Wang, G; Xu, X; Pace, B; Dean, D A; Glazer, P M; Chan, P; Goodman, S R; Shokolenko, I

    1999-07-01

    Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

  14. Regulation of Cell Wall-Bound Invertase in Pepper Leaves by Xanthomonas campestris pv. vesicatoria Type Three Effectors

    PubMed Central

    Sonnewald, Sophia; Priller, Johannes P. R.; Schuster, Julia; Glickmann, Eric; Hajirezaei, Mohammed-Reza; Siebig, Stefan; Mudgett, Mary Beth; Sonnewald, Uwe

    2012-01-01

    Xanthomonas campestris pv. vesicatoria (Xcv) possess a type 3 secretion system (T3SS) to deliver effector proteins into its Solanaceous host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that Xcv may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient Xcv strain showed a higher level of cw-Inv mRNA and enzyme activity relative to Xcv wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the pathogenesis-related gene PRQ. These results suggest that Xcv suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify Xcv T3Es that regulate cw-Inv activity, a screen was performed with eighteen Xcv strains, each deficient in an individual T3E. Seven Xcv T3E deletion strains caused a significant change in cw-Inv activity compared to Xcv wild type. Among them, Xcv lacking the xopB gene (Xcv ΔxopB) caused the most prominent increase in cw-Inv activity. Deletion of xopB increased the mRNA abundance of PRQ in Xcv ΔxopB-infected pepper leaves, but not of Pti5 and Acre31, two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited Xcv-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity in planta to suppress sugar

  15. 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.

  16. 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...

  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. 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.

  19. 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

  20. 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

  1. 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

  2. Enhanced succinic acid productivity by expression of mgtCB gene in Escherichia coli mutant.

    PubMed

    Wang, Jing; Yang, Le; Wang, Dan; Dong, Lichun; Chen, Rachel

    2016-04-01

    In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg(2+) concentration and mgtB to enhance the transport of Mg(2+) into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)2 and NH3·H2O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97 g L(-1) h(-1)) compared with that by using the engineering strain with the overexpression of mgtA gene. PMID:26711444

  3. 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

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

    PubMed

    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.

  5. Mutations in the 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria.

    PubMed

    Tomoeda, K; Awata, H; Matsuura, T; Matsuda, I; Ploechl, E; Milovac, T; Boneh, A; Scott, C R; Danks, D M; Endo, F

    2000-11-01

    The enzyme 4-hydroxyphenylpyruvic acid dioxygenase (HPD) catalyzes the reaction of 4-hydroxyphenylpyruvic acid to homogentisic acid in the tyrosine catabolism pathway. A deficiency in the catalytic activity of HPD may lead to tyrosinemia type III, an autosomal recessive disorder characterized by elevated levels of blood tyrosine and massive excretion of tyrosine derivatives into urine. It has been postulated that hawkinsinuria, an autosomal dominant disorder characterized by the excretion of 'hawkinsin,' may also be a result of HPD deficiency. Hawkinsin is a sulfur amino acid identified as (2-l-cystein-S-yl, 4-dihydroxycyclohex-5-en-1-yl)acetic acid. Patients with hawkinsinuria excrete this metabolite in their urine throughout their life, although symptoms of metabolic acidosis and tyrosinemia improve in the first year of life. We performed analyses of the HPD gene in a patient with tyrosinemia type III and two unrelated patients with hawkinsinuria. A homozygous missense mutation predicting an Ala to Val change at codon 268 (A268V) in the HPD gene was found in the patient with tyrosinemia type III. A heterozygous missense mutation predicting an Ala to Thr change at codon 33 (A33T) was found in the same HPD gene in the two patients with hawkinsinuria. These findings support the hypothesis that alterations in the structure and activity of HPD are causally related to two different metabolic disorders, tyrosinemia type III and hawkinsinuria.

  6. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    PubMed

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  7. 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

  8. 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.

  9. Relative gene expression in acid-adapted Escherichia coli O157:H7 during lactoperoxidase and lactic acid challenge in Tryptone Soy Broth.

    PubMed

    Parry-Hanson, Angela A; Jooste, Piet J; Buys, Elna M

    2010-09-20

    Cross-protection of acid-adapted Escherichia coli O157:H7 against inimical stresses is mediated by the glucose-repressed sigma factor RpoS. However, many food systems in which E. coli O157:H7 occurs are complex and contain glucose. This study was aimed at investigating the contribution of acid and lactoperoxidase (LP)-inducible genes to cross-protection of E. coli O157:H7 against LP system and lactic acid (LA) in Tryptone Soy Broth (TSB). Acid-adapted and non-adapted E. coli O157:H7 were challenged to activated LP and LA at pH 4.0 and 5.0 in TSB for 6h at 25°C followed by expression of acid and LP-inducible genes. Acid-adapted E. coli showed cross-protection against activated LP and LA. All the acid-inducible genes tested were repressed at pH 4.0 with or without activated LP system. At pH 7.4, gadA, ompC and ompF were induced in acid-adapted cells. Induction of corA occurred in non-adapted cells but was repressed in acid-adapted cells. Although acid-inducible genes were repressed at pH 4.0, high resistance of acid-adapted cells indicates that expression of acid-inducible genes occurred during acid adaptation and not the actual challenge. Repression of rpoS indicates that RpoS-independent systems contribute to cross-protection in acid-adapted E. coli O157:H7.

  10. Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

    PubMed Central

    Li, Mimi; Peng, Bin; Guo, Haisong; Yan, Qinqin; Hang, Yueyu

    2013-01-01

    The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants. PMID:24358289

  11. Caproic acid grafted chitosan cationic nanocomplexes for enhanced gene delivery: effect of degree of substitution.

    PubMed

    Layek, Buddhadev; Singh, Jagdish

    2013-04-15

    This work was designed to investigate the effect of the degree of substitutions of caproic acid on plasmid DNA (pDNA) binding, cellular uptake, biocompatibility, and transfection efficiency of caproic acid grafted chitosan (CGC). The CGC with three substitution degrees (CGC-5, CGC-15, and CGC-25) were synthesized by coupling caproic acid with chitosan. Chemical characterization of graft polymers was performed using FTIR, NMR, and elemental analysis. The CGC polymers showed good pDNA condensing capacity and efficient protection of pDNA from DNase I. The nanosized CGC/pDNA polyplexes exhibited well-defined spherical shapes and stability in serum. Isothermal titration calorimetry demonstrated reduction in CGC-pDNA binding constant with increase in the degree of caproic acid substitution. Caproic acid substitution resulted in 2-7-fold higher cellular uptake in HEK 293 cells mainly via the clathrin-mediated pathway without affecting biocompatibility. In vitro transfection study suggested a dependence of transfection efficiency on the variability of caproic acid substitution. The CGC-15 polymer exhibited 31-fold and 1.33-fold higher gene expression compared to chitosan and the marketed non-viral vector FuGENEHD, respectively. These finding suggests that the CGC-15 graft polymer is a promising non-viral gene delivery vector due to its superior transfection efficiency and biocompatibility.

  12. Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor.

    PubMed

    Bastías, Adriana; López-Climent, María; Valcárcel, Mercedes; Rosello, Salvador; Gómez-Cadenas, Aurelio; Casaretto, José A

    2011-03-01

    Growing evidence suggests that the phytohormone abscisic acid (ABA) plays a role in fruit development. ABA signaling components of developmental programs and responses to stress conditions include the group of basic leucine zipper transcriptional activators known as ABA-response element binding factors (AREBs/ABFs). AREB transcription factors mediate ABA-regulated gene expression involved in desiccation tolerance and are expressed mainly in seeds and in vegetative tissues under stress; however, they are also expressed in some fruits such as tomato. In order to get an insight into the role of ABA signaling in fruit development, the expression of two AREB-like factors were investigated during different developmental stages. In addition, tomato transgenic lines that overexpress and downregulate one AREB-like transcription factor, SlAREB1, were used to determine its effect on the levels of some metabolites determining fruit quality. Higher levels of citric acid, malic acid, glutamic acid, glucose and fructose were observed in SlAREB1-overexpressing lines compared with those in antisense suppression lines in red mature fruit pericarp. The higher hexose concentration correlated with increased expression of genes encoding a vacuolar invertase (EC 3.2.1.26) and a sucrose synthase (EC 2.4.1.13). No significant changes were found in ethylene content which agrees with the normal ripening phenotype observed in transgenic fruits. These results suggest that an AREB-mediated ABA signal affects the metabolism of these compounds during the fruit developmental program.

  13. Effect of repeated applications of buprofezin and acephate on soil cellulases, amylase, and invertase.

    PubMed

    Raju, M Naga; Venkateswarlu, K

    2014-10-01

    The impact of repeated applications of buprofezin and acephate, at concentrations ranging from 0.25 to 1.0 kg ha(-1), on activities of cellulases, amylase, and invertase in unamended and nitrogen, phosphorous, and potassium (NPK) fertilizer-amended soil planted with cotton was studied. The nontarget effect of selected insecticides, when applied once, twice, or thrice on soil enzyme activities, was dose-dependent; the activities decreased with increasing concentrations of insecticides. However, there was a rapid decline in activities of enzymes after three repeated applications of insecticides in unamended or NPK-amended soil. Our data clearly suggest that insecticides must be applied judiciously in pest management in order to protect the enzymes largely implicated in soil fertility. PMID:24869954

  14. [Constructing recombinant plasmid pSH-CUP and knockout of acid trehalase gene in baker's yeast].

    PubMed

    He, Dongqin; Xiao, Dongguang; Lv, Ye

    2008-02-01

    The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(delta ATH1, G418(r)), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kan(r) gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.

  15. 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.

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

    PubMed

    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

    2015-04-24

    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.

  17. Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

    PubMed

    Benoit, Isabelle; Asther, Michèle; Bourne, Yves; Navarro, David; Canaan, Stéphane; Lesage-Meessen, Laurence; Herweijer, Marga; Coutinho, Pedro M; Asther, Marcel; Record, Eric

    2007-09-01

    The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.

  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. 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...

  1. Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery.

    PubMed

    Lei, Yuguo; Rahim, Maha; Ng, Quinn; Segura, Tatiana

    2011-08-10

    Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.

  2. Dynamic and combinatorial control of gene expression by nuclear retinoic acid receptors (RARs)

    PubMed Central

    Rochette-Egly, Cécile; Germain, Pierre

    2009-01-01

    Nuclear retinoic acid receptors (RARs) are transcriptional regulators controlling the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on transcription of cognate target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, new roles for the N-terminal domain and the ubiquitin-proteasome system recently emerged. Moreover, the functions of RARs are not limited to the regulation of cognate target genes, as they can transrepress other gene pathways. Finally, RARs are also involved in nongenomic biological activities such as the activation of translation and of kinase cascades. Here we will review these mechanisms, focusing on how kinase signaling and the proteasome pathway cooperate to influence the dynamics of RAR transcriptional activity. PMID:19471584

  3. 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

  4. 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

  5. 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.

  6. Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

    PubMed

    Allam, Uday Sankar; Krishna, M Gopala; Sen, Minakshi; Thomas, Rony; Lahiri, Amit; Gnanadhas, Divya Prakash; Chakravortty, Dipshikha

    2012-01-01

    During the course of infection, Salmonella has to face several potentially lethal environmental conditions, one such being acidic pH. The ability to sense and respond to the acidic pH is crucial for the survival and replication of Salmonella. The physiological role of one gene (STM1485) involved in this response, which is upregulated inside the host cells (by 90- to 113-fold) is functionally characterized in Salmonella pathogenesis. In vitro, the ΔSTM1485 neither exhibited any growth defect at pH 4.5 nor any difference in the acid tolerance response. The ΔSTM1485 was compromised in its capacity to proliferate inside the host cells and complementation with STM1485 gene restored its virulence. We further demonstrate that the surface translocation of Salmonella pathogenicity island-2 (SPI-2) encoded translocon proteins, SseB and SseD were reduced in the ΔSTM1485. The increase in co-localization of this mutant with lysosomes was also observed. In addition, the ΔSTM1485 displayed significantly reduced competitive indices (CI) in spleen, liver and mesenteric lymph nodes in murine typhoid model when infected by intra-gastric route. Based on these results, we conclude that the acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

  7. Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

    PubMed Central

    Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.

    2012-01-01

    Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment. PMID:22247133

  8. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    PubMed

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P < 0.05). We identified one SNP in the genomic sequence of the gene and found that this SNP was associated significantly with body length (P < 0.05), but not with resistance to S. agalactiae. The results of this study suggest that the LAO gene plays an important role in innate immune responses to the bacterial pathogen in tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene. PMID:26546307

  9. Transcriptome analysis and identification of genes associated with omega-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), an omega-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 cD...

  10. 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

  11. Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

    PubMed

    Simonson, Oscar E; Svahn, Mathias G; Törnquist, Elisabeth; Lundin, Karin E; Smith, C I E

    2005-01-01

    Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

  12. Metazoan remaining genes for essential amino acid biosynthesis: sequence conservation and evolutionary analyses.

    PubMed

    Costa, Igor R; Thompson, Julie D; Ortega, José Miguel; Prosdocimi, Francisco

    2014-12-24

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  13. 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

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

    PubMed Central

    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

  15. The Saccharomyces Cerevisiae Spt7 Gene Encodes a Very Acidic Protein Important for Transcription in Vivo

    PubMed Central

    Gansheroff, L. J.; Dollard, C.; Tan, P.; Winston, F.

    1995-01-01

    Mutations in the SPT7 gene of Saccharomyces cerevisiae originally were identified as suppressors of Ty and {delta small} insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Other genes that have been identified in mutant hunts of this type have been shown to play a role in transcription. In this work we show that SPT7 is also important for proper transcription in vivo. We have cloned and sequenced the SPT7 gene and have shown that it encodes a large, acidic protein that is localized to the nucleus. The SPT7 protein contains a bromodomain sequence; a deletion that removes the bromodomain from the SPT7 protein causes no detectable mutant phenotype. Strains that contain an spt7 null mutation are viable but grow very slowly and have transcriptional defects at many loci including insertion mutations, Ty elements, the INO1 gene and the MFA1 gene. These transcriptional defects and other mutant phenotypes are similar to those caused by certain mutations in SPT15, which encodes the TATA binding protein (TBP). The similarity of the phenotypes of spt7 and spt15 mutants, including effects of spt7 mutations on the transcription start site of certain genes, suggests that SPT7 plays an important role in transcription initiation in vivo. PMID:7713415

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

    PubMed Central

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

    1990-01-01

    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. Images PMID:2342468

  17. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

    PubMed

    Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J; Ivison, David; Généreux, Catherine; Dubus, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J; Lloyd, Adrian J; Roper, David I; Schofield, Christopher J; Andersson, Inger; McDonough, Michael A

    2013-08-01

    Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

  18. 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

  19. Deletion of genes encoding fatty acid desaturases leads to alterations in stress sensitivity in Pichia pastoris.

    PubMed

    Zhang, Meng; Liu, Zhe; Yu, Qilin; Mao, Jiwei; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2015-06-01

    Unsaturated fatty acids (UFAs) are key compounds which have important roles in maintaining cell membrane physiological functions and the adaption to tough conditions. Defects of fatty acid desaturases will change cellular UFA constitution. Pichia pastoris GS115 has four fatty acid desaturase genes, namely FAD9A, FAD9B, FAD12 and FAD15. Their products catalyze the synthesis of three kinds of UFAs, oleic acid (catalyzed by Fad9A and Fad9B), linoleic acid (catalyzed by Fad12) and α-linolenic acid (catalyzed by Fad15), respectively. In this study, we found that deletion of FAD12 led to increased resistance to oxidative stress. Cellular lipid peroxidation levels declined in the fad12Δ mutant upon H2O2 treatment. Cellular fatty acids compositions were changed with the increased expression of FAD9A. On the other hand, deletion of FAD9A resulted in increased tolerance to the plasma membrane (PM) damage agent SDS, and PM deformation was not detected in the fad9AΔ mutant under this stress. Our results showed that UFAs are related to cell adaption to adverse environmental changes.

  20. 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.

  1. Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in Streptomyces clavuligerus

    PubMed Central

    Jensen, Susan E.; Elder, Kenneth J.; Aidoo, Kwamena A.; Paradkar, Ashish S.

    2000-01-01

    Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a “supercluster” in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway. PMID:10681345

  2. 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.

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

    DOE PAGES

    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

  4. Gene-nutrient interactions: importance of folic acid and vitamin B12 during early embryogenesis.

    PubMed

    Finnell, Richard H; Shaw, Gary M; Lammer, Edward J; Rosenquist, Thomas H

    2008-06-01

    The role that nutritional factors play in mammalian development has received renewed attention over the past two decades as the scientific literature has exploded with reports that folic acid supplementation in the periconceptional period can protect embryos from a number of highly significant malformations. As is often the case, the relationship between B vitamin supplementation and improved pregnancy outcomes is more complicated than initially perceived, as the interaction between nutritional factors and selected genes must be considered. In this review, we attempt to summarize the complex clinical and experimental literature on nutritional factors, their biological transport mechanisms, and interactions with genetic polymorphisms that impact early embryogenesis. While not exhaustive, our goal was to provide an overview of important gene-nutrient interactions, focusing on folic acid and vitamin B12, to serve as a framework for understanding the multiple roles they play in early embryogenesis.

  5. Metabolism of Very Long-Chain Fatty Acids: Genes and Pathophysiology

    PubMed Central

    Sassa, Takayuki; Kihara, Akio

    2014-01-01

    Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology. PMID:24753812

  6. 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

  7. The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae.

    PubMed

    Zhou, Yanrong; Lin, Yanli; Wu, Xiaojie; Feng, Chong; Long, Chuan; Xiong, Fuyin; Wang, Ning; Pan, Dengke; Chen, Hongxing

    2014-02-01

    Livestock meat is generally low in n-3 polyunsaturated fatty acids (PUFAs), which are beneficial to human health. An alternative approach to increasing the levels of n-3 PUFAs in meat is to generate transgenic livestock animals. In this study, we describe the generation of cloned pigs that express the cbr-fat-1 gene from Caenorhabditis briggsae, encoding an n-3 fatty acid desaturase. Analysis of fatty acids demonstrated that the cbr-fat-1 transgenic pigs produced high levels of n-3 fatty acids from n-6 analogs; consequently, a significantly reduced ratio of n-6/n-3 fatty acids was observed. We demonstrated that the n-3 desaturase gene from C. briggsae was functionally expressed, and had a significant effect on the fatty acid composition of the transgenic pigs, which may allow the production of pork enriched in n-3 PUFAs.

  8. A co-expression gene network associated with developmental regulation of apple fruit acidity.

    PubMed

    Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Xu, Kenong

    2015-08-01

    Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant (P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly (P FDR < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03-WAF08) and late (WAF16-WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in 'Golden Delicious'.

  9. The oct3 gene, a gene for an embryonic transcription factor, is controlled by a retinoic acid repressible enhancer.

    PubMed Central

    Okazawa, H; Okamoto, K; Ishino, F; Ishino-Kaneko, T; Takeda, S; Toyoda, Y; Muramatsu, M; Hamada, H

    1991-01-01

    Oct3 is an embryonic octamer-binding transcription factor, whose expression is rapidly repressed by retinoic acid (RA). In this report, we have determined the transcriptional control region of the oct3 gene and studied the mechanism of the RA-mediated repression. The chromosomal oct3 gene consists of five exons. Three subdomains of the POU region and transactivating domain are located in separate exons. Transcription initiates at multiple sites in the GC-rich region lacking a typical TATA box. The upstream 2 kb region can confer the cell type-specific expression and RA-mediated repression. Analysis of the upstream region by deletion mutagenesis locates a cis element (RARE1) which functions as a stem cell-specific, yet RA-repressible, enhancer. Footprint and gel-retardation assays show that RARE1 is composed of two domains, each of which is recognized by distinct factors. Microinjection of oct3-lacZ constructs into fertilized eggs indicates that RARE1 can function in early embryos. We suggest that RARE1 is a critical cis element for oct3 gene expression in embryonic stem cells and for the RA-mediated repression. Images PMID:1915274

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. Comprehensive analysis of amino acid and nucleotide composition in eukaryotic genomes, comparing genes and pseudogenes.

    PubMed

    Echols, Nathaniel; Harrison, Paul; Balasubramanian, Suganthi; Luscombe, Nicholas M; Bertone, Paul; Zhang, Zhaolei; Gerstein, Mark

    2002-06-01

    Based on searches for disabled homologs to known proteins, we have identified a large population of pseudogenes in four sequenced eukaryotic genomes-the worm, yeast, fly and human (chromosomes 21 and 22 only). Each of our nearly 2500 pseudogenes is characterized by one or more disablements mid-domain, such as premature stops and frameshifts. Here, we perform a comprehensive survey of the amino acid and nucleotide composition of these pseudogenes in comparison to that of functional genes and intergenic DNA. We show that pseudogenes invariably have an amino acid composition intermediate between genes and translated intergenic DNA. Although the degree of intermediacy varies among the four organisms, in all cases, it is most evident for amino acid types that differ most in occurrence between genes and intergenic regions. The same intermediacy also applies to codon frequencies, especially in the worm and human. Moreover, the intermediate composition of pseudogenes applies even though the composition of the genes in the four organisms is markedly different, showing a strong correlation with the overall A/T content of the genomic sequence. Pseudogenes can be divided into 'ancient' and 'modern' subsets, based on the level of sequence identity with their closest matching homolog (within the same genome). Modern pseudogenes usually have a much closer sequence composition to genes than ancient pseudogenes. Collectively, our results indicate that the composition of pseudogenes that are under no selective constraints progressively drifts from that of coding DNA towards non-coding DNA. Therefore, we propose that the degree to which pseudogenes approach a random sequence composition may be useful in dating different sets of pseudogenes, as well as to assess the rate at which intergenic DNA accumulates mutations. Our compositional analyses with the interactive viewer are available over the web at http://genecensus.org/pseudogene.

  15. Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour.

    PubMed

    Armas, Pablo; Cachero, Sebastián; Lombardo, Verónica A; Weiner, Andrea; Allende, Miguel L; Calcaterra, Nora B

    2004-08-01

    Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.

  16. Characterisation of the FAD2 gene family from Hiptage benghalensis: a ricinoleic acid accumulating plant.

    PubMed

    Zhou, Xue-Rong; Singh, Surinder P; Green, Allan G

    2013-08-01

    We have characterised the FAD2 gene family from Hiptage benghalensis, a tropical plant that accumulates high levels of ricinoleic acid in its seeds. Functional characterisation of six FAD2 gene family members showed that two of them were capable of functioning as Δ12-hydroxylases while the other FAD2 members were confirmed to be Δ12-desaturases. The Δ12-hydroxylation function of these two genes was confirmed in yeast cells, using C16:1(Δ9) and C18:1(Δ9) monounsaturated fatty acids as substrates. These Δ12-hydroxylases, like the other Δ12-hydroxylases previously cloned from plants Ricinus communis (castor), Physaria fendleri and fungus Claviceps purpurea, also showed some Δ12-desaturase activity. The hydroxylation activity of the two Hiptage hydroxylases was further confirmed by their expression in the Arabidopsis fad2/fae1 double mutant where they were able to produce equivalent or higher levels hydroxylated fatty acids in the seed oil when compared with the other known hydroxylases.

  17. Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes.

    PubMed Central

    Tomic, M; Jiang, C K; Epstein, H S; Freedberg, I M; Samuels, H H; Blumenberg, M

    1990-01-01

    In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters. Images PMID:1712634

  18. Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants.

    PubMed

    Pla, M; Gómez, J; Goday, A; Pagès, M

    1991-12-01

    We have isolated a new maize gene, rab28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction. rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vp1 mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vp1 contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.

  19. A systems biology approach for the identification of target genes for the improvement of itaconic acid production in Aspergillus species

    PubMed Central

    2013-01-01

    Background In this paper, a clone based transcriptome analysis towards the identification of genes related to itaconic acid production in Aspergillus terreus was carried out as an extension of a previously published a clone-based transcriptome analysis from a set of batch fermentation experiments. Also a publically available A. niger transcriptome dataset from cultures similar to those of the A. terreus data set was analyzed to evaluate the specificity of the approach followed for A. terreus. Results Besides the itaconic acid gene cluster (cis-aconitate decarboxylase, mitochondrial tri-carboxylic acid transporter and major facilitator superfamily transporter) discovered previously, additional genes of interest were identified in the A. terreus transcriptome data correlating to itaconic acid production, including 6 genes encoding enzymes in glycolysis and the pentose phosphate pathway, 4 genes functioning in vitamins synthesis, and a gene encoding a copper transporter. Only three of the 83 low pH specific genes identified from the A. niger dataset corresponded to high itaconic acid / low pH expressed genes identified from the A. terreus data set. However, in all three cases, the regulation of pH dependent gene expression was completely different between the two species. Conclusions An extended clone based transcriptome analysis using a clone based transcription array to identify genes correlating with itaconic acid production revealed novel genes both in the central metabolism and in other more secondary pathways such as vitamin biosynthesis and Cu2+ transport, providing targets for further metabolic and process engineering to optimize itaconic acid production. PMID:24304666

  20. [Soil Microbial Respiration Under Different Soil Temperature Conditions and Its Relationship to Soil Dissolved Organic Carbon and Invertase].

    PubMed

    Wu, Jing; Chen, Shu-tao; Hu, Zheng-hua; Zhang, Xu

    2015-04-01

    In order to investigate the soil microbial respiration under different temperature conditions and its relationship to soil dissolved organic carbon ( DOC) and invertase, an indoor incubation experiment was performed. The soil samples used for the experiment were taken from Laoshan, Zijinshan, and Baohuashan. The responses of soil microbial respiration to the increasing temperature were studied. The soil DOC content and invertase activity were also measured at the end of incubation. Results showed that relationships between cumulative microbial respiration of different soils and soil temperature could be explained by exponential functions, which had P values lower than 0.001. The coefficient of temperature sensitivity (Q10 value) varied from 1.762 to 1.895. The Q10 value of cumulative microbial respiration decreased with the increase of soil temperature for all soils. The Q10 value of microbial respiration on 27 days after incubation was close to that of 1 day after incubation, indicating that the temperature sensitivity of recalcitrant organic carbon may be similar to that of labile organic carbon. For all soils, a highly significant ( P = 0.003 ) linear relationship between cumulative soil microbial respiration and soil DOC content could be observed. Soil DOC content could explain 31.6% variances of cumulative soil microbial respiration. For the individual soil and all soils, the relationship between cumulative soil microbial respiration and invertase activity could be explained by a highly significant (P < 0.01) linear regression function, which suggested that invertase was a good indicator of the magnitude of soil microbial respiration.

  1. Expression, purification and immobilization of the intracellular invertase INVA, from Zymomonas mobilis on crystalline cellulose and Nylon-6.

    PubMed

    de Los Angeles Calixto-Romo, María; Santiago-Hernández, José Alejandro; Vallejo-Becerra, Vanessa; Amaya-Delgado, Lorena; del Carmen Montes-Horcasitas, María; Hidalgo-Lara, María Eugenia

    2008-11-01

    This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBDCex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 degrees C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. Km and Vmax were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 degrees C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to Vmax increased up to 5.7-fold, following immobilization, whereas Km increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.

  2. [Soil Microbial Respiration Under Different Soil Temperature Conditions and Its Relationship to Soil Dissolved Organic Carbon and Invertase].

    PubMed

    Wu, Jing; Chen, Shu-tao; Hu, Zheng-hua; Zhang, Xu

    2015-04-01

    In order to investigate the soil microbial respiration under different temperature conditions and its relationship to soil dissolved organic carbon ( DOC) and invertase, an indoor incubation experiment was performed. The soil samples used for the experiment were taken from Laoshan, Zijinshan, and Baohuashan. The responses of soil microbial respiration to the increasing temperature were studied. The soil DOC content and invertase activity were also measured at the end of incubation. Results showed that relationships between cumulative microbial respiration of different soils and soil temperature could be explained by exponential functions, which had P values lower than 0.001. The coefficient of temperature sensitivity (Q10 value) varied from 1.762 to 1.895. The Q10 value of cumulative microbial respiration decreased with the increase of soil temperature for all soils. The Q10 value of microbial respiration on 27 days after incubation was close to that of 1 day after incubation, indicating that the temperature sensitivity of recalcitrant organic carbon may be similar to that of labile organic carbon. For all soils, a highly significant ( P = 0.003 ) linear relationship between cumulative soil microbial respiration and soil DOC content could be observed. Soil DOC content could explain 31.6% variances of cumulative soil microbial respiration. For the individual soil and all soils, the relationship between cumulative soil microbial respiration and invertase activity could be explained by a highly significant (P < 0.01) linear regression function, which suggested that invertase was a good indicator of the magnitude of soil microbial respiration. PMID:26164932

  3. 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

    2016-01-29

    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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.

    PubMed Central

    Collart, F R; Huberman, E

    1987-01-01

    The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies. Images PMID:2890098

  11. 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.

  12. 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.

  13. Plastidial fatty acid levels regulate resistance gene-dependent defense signaling in Arabidopsis.

    PubMed

    Chandra-Shekara, A C; Venugopal, Srivathsa C; Barman, Subhankar Roy; Kachroo, Aardra; Kachroo, Pradeep

    2007-04-24

    In Arabidopsis, resistance to Turnip Crinkle Virus (TCV) depends on the resistance (R) gene, HRT, and the recessive locus rrt. Resistance also depends on salicylic acid (SA), EDS1, and PAD4. Exogenous application of SA confers resistance in RRT-containing plants by increasing HRT transcript levels in a PAD4-dependent manner. Here we report that reduction of oleic acid (18:1) can also induce HRT gene expression and confer resistance to TCV. However, the 18:1-regulated pathway is independent of SA, rrt, EDS1, and PAD4. Reducing the levels of 18:1, via a mutation in the SSI2-encoded stearoyl-acyl carrier protein-desaturase, or by exogenous application of glycerol, increased transcript levels of HRT as well as several other R genes. Second-site mutations in the ACT1-encoded glycerol-3-phosphate acyltransferase or GLY1-encoded glycerol-3-phosphate dehydrogenase restored 18:1 levels in HRT ssi2 plants and reestablished a dependence on rrt. Resistance to TCV and HRT gene expression in HRT act1 plants was inducible by SA but not by glycerol, whereas that in HRT pad4 plants was inducible by glycerol but not by SA. The low 18:1-mediated induction of R gene expression was also dependent on ACT1 but independent of EDS1, PAD4, and RAR1. Intriguingly, TCV inoculation did not activate this 18:1-regulated pathway in HRT plants, but instead resulted in the induction of several genes that encode 18:1-synthesizing isozymes. These results suggest that the 18:1-regulated pathway may be specifically targeted during pathogen infection and that altering 18:1 levels may serve as a unique strategy for promoting disease resistance.

  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.

  16. QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

    PubMed Central

    2010-01-01

    Background Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes. Results The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

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

    PubMed

    Akbas, F; Aydin, Z

    2012-04-03

    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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Urease gene-containing Archaea dominate autotrophic ammonia oxidation in two acid soils.

    PubMed

    Lu, Lu; Jia, Zhongjun

    2013-06-01

    The metabolic traits of ammonia-oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea-amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water-amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time-course incubations indicated that archaeal amoA genes were increasingly labelled by (13) CO2 in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the (13) C-DNA, and acetylene inhibition suggests that autotrophic growth of urease-containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a-associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.

  12. Polymorphisms in fatty-acid-metabolism-related genes are associated with colorectal cancer risk.

    PubMed

    Hoeft, Birgit; Linseisen, Jakob; Beckmann, Lars; Müller-Decker, Karin; Canzian, Federico; Hüsing, Anika; Kaaks, Rudolf; Vogel, Ulla; Jakobsen, Marianne U; Overvad, Kim; Hansen, Rikke D; Knüppel, Sven; Boeing, Heiner; Trichopoulou, Antonia; Koumantaki, Yvoni; Trichopoulos, Dimitrios; Berrino, Franco; Palli, Domenico; Panico, Salvatore; Tumino, Rosario; Bueno-de-Mesquita, H B; van Duijnhoven, Fränzel J B; van Gils, Carla H; Peeters, Petra H; Dumeaux, Vanessa; Lund, Eiliv; Huerta Castaño, José M; Muñoz, Xavier; Rodriguez, Laudina; Barricarte, Aurelio; Manjer, Jonas; Jirström, Karin; Van Guelpen, Bethany; Hallmans, Göran; Spencer, Elizabeth A; Crowe, Francesca L; Khaw, Kay-Tee; Wareham, Nick; Morois, Sophie; Boutron-Ruault, Marie-Christine; Clavel-Chapelon, Françoise; Chajes, Veronique; Jenab, Mazda; Boffetta, Paolo; Vineis, Paolo; Mouw, Traci; Norat, Teresa; Riboli, Elio; Nieters, Alexandra

    2010-03-01

    Colorectal cancer (CRC) is the third most common malignant tumor and the fourth leading cause of cancer death worldwide. The crucial role of fatty acids for a number of important biological processes suggests a more in-depth analysis of inter-individual differences in fatty acid metabolizing genes as contributing factor to colon carcinogenesis. We examined the association between genetic variability in 43 fatty acid metabolism-related genes and colorectal risk in 1225 CRC cases and 2032 controls participating in the European Prospective Investigation into Cancer and Nutrition study. Three hundred and ninety two single-nucleotide polymorphisms were selected using pairwise tagging with an r(2) cutoff of 0.8 and a minor allele frequency of >5%. Conditional logistic regression models were used to estimate odds ratios and corresponding 95% confidence intervals. Haplotype analysis was performed using a generalized linear model framework. On the genotype level, hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD), phospholipase A2 group VI (PLA2G6) and transient receptor potential vanilloid 3 were associated with higher risk for CRC, whereas prostaglandin E receptor 2 (PTGER2) was associated with lower CRC risk. A significant inverse association (P < 0.006) was found for PTGER2 GGG haplotype, whereas HPGD AGGAG and PLA2G3 CT haplotypes were significantly (P < 0.001 and P = 0.003, respectively) associated with higher risk of CRC. Based on these data, we present for the first time the association of HPGD variants with CRC risk. Our results support the key role of prostanoid signaling in colon carcinogenesis and suggest a relevance of genetic variation in fatty acid metabolism-related genes and CRC risk. PMID:20042636

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Use of Peptide Nucleic Acids to Manipulate Gene Expression in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Naik, Shankar; Yavin, Eylon; Dzikowski, Ron

    2014-01-01

    One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents. PMID:24466246

  18. Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.

    PubMed

    Bockstahler, L E; Li, Z; Nguyen, N Y; Van Houten, K A; Brennan, M J; Langone, J J; Morris, S L

    2002-03-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h. PMID:11926172

  19. Expression and retinoic acid regulation of the zebrafish nr2f orphan nuclear receptor genes

    PubMed Central

    Love, Crystal E.; Prince, Victoria E.

    2012-01-01

    Background The vertebrate nuclear receptor subfamily 2, group f (nr2f) genes encode orphan receptors that have the capacity to act as negative regulators of retinoic acid (RA) signaling. Results We describe embryonic and larval expression of four of the six zebrafish nr2f genes, nr2f1a, nr2f1b, nr2f2 and nr2f5. These genes show highly regulated patterns of expression within the CNS, including in the developing hindbrain, as well as in the mesoderm and endoderm. We also investigated the role of RA and Fgf signaling in regulating early nr2f gene expression. RA is not required for nr2f expression in the hindbrain; however, exogenous RA can repress this expression. Conversely, we find that RA positively regulates nr2f1a expression in trunk endoderm and mesoderm. Fgf signaling is not required for nr2f expression onset in the hindbrain; however, it may play a role in maintaining rhombomere-specific expression. Conclusions We report detailed expression analysis of four nr2f genes in all three germ layers. The onset of nr2f expression in the hindbrain does not require RA or Fgf signals. Our finding that RA positively regulates nr2f1a expression in the trunk supports the possibility that Nr2fs function in a negative feedback loop to modulate RA signaling in this region. PMID:22836912

  20. Polymorphisms in lipogenic genes and milk fatty acid composition in Holstein dairy cattle.

    PubMed

    Nafikov, Rafael A; Schoonmaker, Jon P; Korn, Kathleen T; Noack, Kristin; Garrick, Dorian J; Koehler, Kenneth J; Minick-Bormann, Jennifer; Reecy, James M; Spurlock, Diane E; Beitz, Donald C

    2014-12-01

    Changing bovine milk fatty acid (FA) composition through selection can decrease saturated FA (SFA) consumption, improve human health and provide a means for manipulating processing properties of milk. Our study determined associations between milk FA composition and genes from triacylglycerol (TAG) biosynthesis pathway. The GC dinucleotide allele of diacylglycerol O-acyltransferase 1:g.10433-10434AA >GC was associated with lower palmitic acid (16:0) concentration but higher oleic (18:1 cis-9), linoleic (18:2 cis-9, cis-12) acid concentrations, and elongation index. Accordingly, the GC dinucleotide allele was associated with lower milk fat percentage and SFA concentrations but higher monounsaturated FA and polyunsaturated FA (PUFA) concentrations. The glycerol-3-phosphate acyltransferase, mitochondrial haplotypes were associated with higher myristoleic acid (14:1 cis-9) concentration and C14 desaturation index. The 1-acylglycerol-3-phosphate acyltransferase 1 haplotypes were associated with higher PUFA and linoleic acid concentrations. The results of this study provide information for developing genetic tools to modify milk FA composition in dairy cattle.

  1. 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

  2. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.

    PubMed

    Gojgini, Shiva; Tokatlian, Talar; Segura, Tatiana

    2011-10-01

    The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

  3. 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.

  4. The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

    PubMed Central

    Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

    1999-01-01

    A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-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 β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-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-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-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

  5. Association between SLC2A9 transporter gene variants and uric acid phenotypes in African American and white families

    PubMed Central

    de Andrade, Mariza; Matsumoto, Martha; Mosley, Tom H.; Kardia, Sharon; Turner, Stephen T.

    2011-01-01

    Objectives. SLC2A9 gene variants associate with serum uric acid in white populations, but little is known about African American populations. Since SLC2A9 is a transporter, gene variants may be expected to associate more closely with the fractional excretion of urate, a measure of renal tubular transport, than with serum uric acid, which is influenced by production and extrarenal clearance. Methods. Genotypes of single nucleotide polymorphisms (SNPs) distributed across the SLC2A9 gene were obtained in the Genetic Epidemiology Network of Arteriopathy cohorts. The associations of SNPs with serum uric acid, fractional excretion of urate and urine urate-to-creatinine ratio were assessed with adjustments for age, sex, diuretic use, BMI, homocysteine and triglycerides. Results. We identified SLC2A9 gene variants that were associated with serum uric acid in 1155 African American subjects (53 SNPs) and 1132 white subjects (63 SNPs). The most statistically significant SNPs in African American subjects (rs13113918) and white subjects (rs11723439) were in the latter half of the gene and explained 2.7 and 2.8% of the variation in serum uric acid, respectively. After adjustment for this SNP in African Americans, 0.9% of the variation in serum uric acid was explained by an SNP (rs1568318) in the first half of the gene. Unexpectedly, SLC2A9 gene variants had stronger associations with serum uric acid than with fractional excretion of urate. Conclusions. These findings support two different loci by which SLC2A9 variants affect uric acid levels in African Americans and suggest SLC2A9 variants affect serum uric acid level via renal and extrarenal clearance. PMID:21186168

  6. 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.

  7. Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage▿ †

    PubMed Central

    Mirete, Salvador; de Figueras, Carolina G.; González-Pastor, Jose E.

    2007-01-01

    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

  8. 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

  9. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis.

    PubMed Central

    Xiang, C; Oliver, D J

    1998-01-01

    Glutathione plays a pivotal role in protecting plants from environmental stresses, oxidative stress, xenobiotics, and some heavy metals. Arabidopsis plants treated with cadmium or copper responded by increasing transcription of the genes for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione reductase. The response was specific for those metals whose toxicity is thought to be mitigated through phytochelatins, and other toxic and nontoxic metals did not alter mRNA levels. Feeding experiments suggested that neither oxidative stress, as results from exposure to H2O2, nor oxidized or reduced glutathione levels were responsible for activating transcription of these genes. Jasmonic acid also activated the same suite of genes, which suggests that it might be involved in the signal transduction pathway for copper and cadmium. Jasmonic acid treatment increased mRNA levels and the capacity for glutathione synthesis but did not alter the glutathione content in unstressed plants, which supports the idea that the glutathione concentration is controlled at multiple levels. PMID:9724699

  10. 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

  11. 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

  12. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids

    PubMed Central

    Santa Maria, John P.; Sadaka, Ama; Moussa, Samir H.; Brown, Stephanie; Zhang, Yanjia J.; Rubin, Eric J.; Gilmore, Michael S.; Walker, Suzanne

    2014-01-01

    Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine–tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks. PMID:25104751

  13. Mutations at the lysosomal acid cholesteryl ester hydrolase gene locus in Wolman disease.

    PubMed Central

    Anderson, R A; Byrum, R S; Coates, P M; Sando, G N

    1994-01-01

    The genomic sequences encoding the human lysosomal acid lipase/cholesteryl esterase (sterol esterase; EC 3.1.1.13) have been isolated and sequenced, and the information has been used to identify mutations in both alleles of the gene from a patient with Wolman disease, an autosomal recessive lysosomal lipid storage disorder. The genomic locus consists of 10 exons spread over 36 kb. The 5' flanking region is G+C-rich and has characteristics of a "housekeeping" gene promoter. One of the identified mutations involves the insertion of a T residue after position 634, resulting in the appearance of an in-frame translation stop signal 13 codons downstream. The second mutation is a T-to-C transition at nucleotide 638. This results in a leucine-to-proline substitution at amino acid 179 and is predicted to lead to the disruption of the alpha-helical structure in a highly conserved region of the protein. These mutations are each capable of completely disrupting the catalytic function of the lysosomal acid cholesteryl ester hydrolase; their presence can account for the extreme phenotype of the lysosomal lipid storage disorder manifested in members of this patient's family. Images PMID:8146180

  14. Folic Acid supplementary reduce the incidence of adenocarcinoma in a mouse model of colorectal cancer: microarray gene expression profile

    PubMed Central

    2011-01-01

    Background Whether Folic acid is a potential drug that may prevent the progression of colorectal carcinoma and when to use are important healthy issues we focus on. Our study is to examine the effect of folic acid on the development of the CRC and the optimal time folic acid should be provided in a mouse-ICR model induced by 1, 2-Dimethylhydrazine. Also, we investigated the gene expression profile of this model related to folic acid. Method Female ICR mouse (n = 130) were divided into 7 groups either with the treatment of 1, 2-Dimethylhydrazine (20 mg/kg bodyweight) weekly or folic acid (8 mg/kg bodyweight) twice a week for 12 or 24 weeks. Using a 4 × 44 K Agilent whole genome oligo microarray assay, different gene expression among groups (NS, DMH, FA2, FA3) were identified and selected genes were validated by real-time polymerase chain reaction. Results Animals with a supplementary of folic acid showed a significant decrease in the incidence, the maximum diameter and multiplicity of adenocarcinomas (P < 0.05). Furthermore, there were fewer adenomas or adenocarcinomas developed in the group of folic acid supplementation in pre-adenoma stage compared to group of post-adenoma stage. Meanwhile, about 1070 genes that were changed by 1, 2-Dimethylhydrazine can be reversed by folic acid and 172 differentially genes were identified between the groups of pre- and post- adenoma stage using microarray gene expression analysis. Conclusion Our study demonstrated that folic acid supplementary was significantly associated with the decrease risk of CRC. And the subgroup of providing folic acid without precancerous lesions was more effective than that with precancerous lesions. PMID:22206623

  15. Cell-wall invertases, key enzymes in the modulation of plant metabolism during defence responses.

    PubMed

    Proels, Reinhard Korbinian; Hückelhoven, Ralph

    2014-10-01

    Most plant-pathogen interactions do not result in pathogenesis because of pre-formed defensive plant barriers or pathogen-triggered activation of effective plant immune responses. The mounting of defence reactions is accompanied by a profound modulation of plant metabolism. Common metabolic changes are the repression of photosynthesis, the increase in heterotrophic metabolism and the synthesis of secondary metabolites. This enhanced metabolic activity is accompanied by the reduced export of sucrose or enhanced import of hexoses at the site of infection, which is mediated by an induced activity of cell-wall invertase (Cw-Inv). Cw-Inv cleaves sucrose, the major transport sugar in plants, irreversibly yielding glucose and fructose, which can be taken up by plant cells via hexose transporters. These hexose sugars not only function in metabolism, but also act as signalling molecules. The picture of Cw-Inv regulation in plant-pathogen interactions has recently been broadened and is discussed in this review. An interesting emerging feature is the link between Cw-Inv and the circadian clock and new modes of Cw-Inv regulation at the post-translational level.

  16. Synthesis of fructooligosaccharides in banana 'prata' and its relation to invertase activity and sucrose accumulation.

    PubMed

    Der Agopian, Roberta Ghedini; Purgatto, Eduardo; Cordenunsi, Beatriz Rosana; Lajolo, Franco Maria

    2009-11-25

    Levels of sucrose and total fructooligosaccharides (FOS) were quantified in different phases of banana 'Prata' ripening during storage at ambient (approximately 19 degrees C) and low (approximately 10 degrees C) temperature. Total FOS levels were detected in the first days after harvest, whereas 1-kestose remained undetectable until the sucrose levels reached approximately 200 mg/g (dry weight) in both groups. Sucrose levels increased slowly but constantly at low temperature, but they elevated rapidly when the temperature was raised to 19 degrees C. Total FOS and sucrose levels were higher in bananas stored at low temperature than in the control group. In both samples, total FOS levels were higher than those of 1-kestose. The carbohydrate profiles obtained by HPLC and TLC suggest the presence of neokestose, 6-kestose, and bifurcose. The enzymes putatively involved in banana fructosyltransferase activity were also evaluated. Results obtained indicate that the banana enzyme responsible for the synthesis of FOS by transfructosylation is an invertase rather than a sucrose-sucrosyl transferase-like enzyme. PMID:19860446

  17. 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

  18. Identification of genes expressed in response to acid stress in Synechocystis sp. PCC 6803 using DNA microarrays.

    PubMed

    Ohta, Hisataka; Shibata, Yousuke; Haseyama, Youhei; Yoshino, Yuka; Suzuki, Takehiro; Kagasawa, Tsuyoshi; Kamei, Ayako; Ikeuchi, Masahiko; Enami, Isao

    2005-06-01

    Plant cells are always exposed to various environmental stresses such as high light, low temperature and acid rain, and thus have to respond in order to survive these stresses. Although some mechanisms of responses to high light and low temperature etc., have been clarified, there is little information about the acclimation process to acid stress. In this study, the gene expression changes of Synechocystis sp. PCC 6803 in response to acid stress were examined using DNA microarrays (CyanoCHIP). We compared gene expression profiles of the cells treated at pH 8 (control) and pH 3 for 0.5, 1, 2 or 4 h. As a result, we found that 32 genes were upregulated by more than 3-fold, and 29 genes were downregulated by at least 3-fold after the acid treatment. Among these upregulated genes, expressions of slr0967 and sll0939 kept-increasing until 4 h under the acid stress and increased by 7 to 16-fold after the 4 h treatment. This suggests that the products of these two genes play important roles in the acid acclimation process.

  19. 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

  20. 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

  1. Gene-gene interactions of fatty acid synthase (FASN) using multifactor-dimensionality reduction method in Korean cattle.

    PubMed

    Lee, Jeayoung; Jin, Mehyun; Lee, Yoonseok; Ha, Jaejung; Yeo, Jungsou; Oh, Dongyep

    2014-01-01

    We examined the gene-gene interactions of five exonic single nucleotide polymorphisms (SNPs) in the gene encoding fatty acid synthase using 513 Korean cattle and using the model free and the non-parametrical multifactor dimensionality reduction method for the analysis. The five SNPs of g.12870 T>C, g.13126 T>C, g.15532 C>A, g.16907 T>C and g.17924 G>A associated with a variety of fatty acid compositions and marbling score were used in this study. The two-factor interaction between g.13126 T>C and g.15532 C>A had the highest training-balanced among the five-factor models and a testing-balanced accuracy at 70.18 % on C18:1 with a cross-validation consistency of 10 out of 10. Also, the two-factor interaction between g.13126 T>C and g.15532 C>A had the highest testing-balanced accuracy at 68.59 % with a 10 out of 10 cross-validation consistency, than any other models on MUFA. In MS, a single SNP g.15532 C>A had the best accuracy at 58.85 % and the two-factor interaction model g.12870 T>C and g.15532 C>A had the highest testing-balanced accuracy at 64.00 %. The three-factor interaction model g.12870 T>C, g.13126 T>C and g.15532 C>A was recorded as having a high testing-balanced accuracy of 63.24 %, but it was lower than the two-factor interaction model. We used likelihood ratio tests for interaction, and Chi square tests to validate our results, with all tests showing statistical significance. We also compared this with mean scores between the high-risk trait group and low-risk trait group. The genotypes of TTCA, TTAA and TCAA at g.15532 and g.13126 on C18:1, genotypes TTCC, TTCA, TTAA, TCAA CCAA at g.15532 and g.13126 on MUFA and genotypes CCCC, TCCA, CCCA, TTAA, TCAA and CCAA at g.15532 and g.12870 on MS were recommended for the genetic improvement of beef quality.

  2. Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders.

    PubMed

    Baelum, Jacob; Jacobsen, Carsten S; Holben, William E

    2010-03-01

    31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.

  3. 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.

  4. 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.

  5. Biodegradable DNA-Brush Block Copolymer Spherical Nucleic Acids Enable Transfection Agent-Free Intracellular Gene Regulation.

    PubMed

    Zhang, Chuan; Hao, Liangliang; Calabrese, Colin M; Zhou, Yu; Choi, Chung Hang J; Xing, Hang; Mirkin, Chad A

    2015-10-28

    By grafting multiple DNA strands onto one terminus of a polyester chain, a DNA-brush block copolymer that can assemble into micelle structure is constructed. These micelle spherical nucleic acids have a density of nucleic acids that is substantively higher than linear DNA block copolymer structures, which makes them effective cellular transfection and intracellular gene regulation agents.

  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.

  7. Obstructive heart defects associated with candidate genes, maternal obesity, and folic acid supplementation.

    PubMed

    Tang, Xinyu; Cleves, Mario A; Nick, Todd G; Li, Ming; MacLeod, Stewart L; Erickson, Stephen W; Li, Jingyun; Shaw, Gary M; Mosley, Bridget S; Hobbs, Charlotte A

    2015-06-01

    Right-sided and left-sided obstructive heart defects (OHDs) are subtypes of congenital heart defects, in which the heart valves, arteries, or veins are abnormally narrow or blocked. Previous studies have suggested that the development of OHDs involved a complex interplay between genetic variants and maternal factors. Using the data from 569 OHD case families and 1,644 control families enrolled in the National Birth Defects Prevention Study (NBDPS) between 1997 and 2008, we conducted an analysis to investigate the genetic effects of 877 single nucleotide polymorphisms (SNPs) in 60 candidate genes for association with the risk of OHDs, and their interactions with maternal use of folic acid supplements, and pre-pregnancy obesity. Applying log-linear models based on the hybrid design, we identified a SNP in methylenetetrahydrofolate reductase (MTHFR) gene (C677T polymorphism) with a main genetic effect on the occurrence of OHDs. In addition, multiple SNPs in betaine-homocysteine methyltransferase (BHMT and BHMT2) were also identified to be associated with the occurrence of OHDs through significant main infant genetic effects and interaction effects with maternal use of folic acid supplements. We also identified multiple SNPs in glutamate-cysteine ligase, catalytic subunit (GCLC) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) that were associated with elevated risk of OHDs among obese women. Our findings suggested that the risk of OHDs was closely related to a combined effect of variations in genes in the folate, homocysteine, or glutathione/transsulfuration pathways, maternal use of folic acid supplements and pre-pregnancy obesity. PMID:25846410

  8. Obstructive Heart Defects Associated with Candidate Genes, Maternal Obesity, and Folic Acid Supplementation

    PubMed Central

    Tang, Xinyu; Cleves, Mario A.; Nick, Todd G.; Li, Ming; MacLeod, Stewart L.; Erickson, Stephen W.; Li, Jingyun; Shaw, Gary M.; Mosley, Bridget S.; Hobbs, Charlotte A.

    2015-01-01

    Right-sided and left-sided obstructive heart defects (OHDs) are subtypes of congenital heart defects, in which the heart valves, arteries, or veins are abnormally narrow or blocked. Previous studies have suggested that the development of OHDs involved a complex interplay between genetic variants and maternal factors. Using the data from 569 OHD case families and 1644 control families enrolled in the National Birth Defects Prevention Study (NBDPS) between 1997 and 2008, we conducted an analysis to investigate the genetic effects of 877 single nucleotide polymorphisms (SNPs) in 60 candidate genes for association with the risk of OHDs, and their interactions with maternal use of folic acid supplements, and pre-pregnancy obesity. Applying log-linear models based on the hybrid design, we identified a SNP in methylenetetrahydrofolate reductase (MTHFR) gene (C677T polymorphism) with a main genetic effect on the occurrence of OHDs. In addition, multiple SNPs in betaine-homocysteine methyltransferase (BHMT and BHMT2) were also identified to be associated with the occurrence of OHDs through significant main infant genetic effects and interaction effects with maternal use of folic acid supplements. We also identified multiple SNPs in glutamate-cysteine ligase, catalytic subunit (GCLC) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) that were associated with elevated risk of OHDs among obese women. Our findings suggested that the risk of OHDs was closely related to a combined effect of variations in genes in the folate, homocysteine, or glutathione/transsulfuration pathways, maternal use of folic acid supplements and pre-pregnancy obesity. PMID:25846410

  9. Obstructive heart defects associated with candidate genes, maternal obesity, and folic acid supplementation.

    PubMed

    Tang, Xinyu; Cleves, Mario A; Nick, Todd G; Li, Ming; MacLeod, Stewart L; Erickson, Stephen W; Li, Jingyun; Shaw, Gary M; Mosley, Bridget S; Hobbs, Charlotte A

    2015-06-01

    Right-sided and left-sided obstructive heart defects (OHDs) are subtypes of congenital heart defects, in which the heart valves, arteries, or veins are abnormally narrow or blocked. Previous studies have suggested that the development of OHDs involved a complex interplay between genetic variants and maternal factors. Using the data from 569 OHD case families and 1,644 control families enrolled in the National Birth Defects Prevention Study (NBDPS) between 1997 and 2008, we conducted an analysis to investigate the genetic effects of 877 single nucleotide polymorphisms (SNPs) in 60 candidate genes for association with the risk of OHDs, and their interactions with maternal use of folic acid supplements, and pre-pregnancy obesity. Applying log-linear models based on the hybrid design, we identified a SNP in methylenetetrahydrofolate reductase (MTHFR) gene (C677T polymorphism) with a main genetic effect on the occurrence of OHDs. In addition, multiple SNPs in betaine-homocysteine methyltransferase (BHMT and BHMT2) were also identified to be associated with the occurrence of OHDs through significant main infant genetic effects and interaction effects with maternal use of folic acid supplements. We also identified multiple SNPs in glutamate-cysteine ligase, catalytic subunit (GCLC) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) that were associated with elevated risk of OHDs among obese women. Our findings suggested that the risk of OHDs was closely related to a combined effect of variations in genes in the folate, homocysteine, or glutathione/transsulfuration pathways, maternal use of folic acid supplements and pre-pregnancy obesity.

  10. 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

  11. 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

  12. An acute dose of gamma-hydroxybutyric acid alters gene expression in multiple mouse brain regions.

    PubMed

    Schnackenberg, B J; Saini, U T; Robinson, B L; Ali, S F; Patterson, T A

    2010-10-13

    Gamma-hydroxybutyric acid (GHB) is normally found in the brain in low concentrations and may function as a neurotransmitter, although the mechanism of action has not been completely elucidated. GHB has been used as a general anesthetic and is currently used to treat narcolepsy and alcoholism. Recreational use of GHB is primarily as a "club drug" and a "date rape drug," due to its amnesic effects. For this study, the hypothesis was that behavioral and neurochemical alterations may parallel gene expression changes in the brain after GHB administration. Adult male C57/B6N mice (n=5/group) were administered a single dose of 500 mg/kg GHB (i.p.) and were sacrificed 1, 2 and 4 h after treatment. Control mice were administered saline. Brains were removed and regionally dissected on ice. Total RNA from the hippocampus, cortex and striatum was extracted, amplified and labeled. Gene expression was evaluated using Agilent whole mouse genome 4x44K oligonucleotide microarrays. Microarray data were analyzed by ArrayTrack and differentially expressed genes (DEGs) were identified using P < or = 0.01 and a fold change > or = 1.7 as the criteria for significance. Principal component analysis (PCA) and Hierarchical Cluster Analysis (HCA) showed that samples from each time point clustered into distinct treatment groups with respect to sacrifice time. Ingenuity pathways analysis (IPA) was used to identify involved pathways. The results show that GHB induces gene expression alterations in hundreds of genes in the hippocampus, cortex and striatum, and the number of affected genes increases throughout a 4-h time course. Many of these DEGs are involved in neurological disease, apoptosis, and oxidative stress.

  13. Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens.

    PubMed

    Winstel, Volker; Liang, Chunguang; Sanchez-Carballo, Patricia; Steglich, Matthias; Munar, Marta; Bröker, Barbara M; Penadés, Jose R; Nübel, Ulrich; Holst, Otto; Dandekar, Thomas; Peschel, Andreas; Xia, Guoqing

    2013-01-01

    Mobile genetic elements (MGEs) encoding virulence and resistance genes are widespread in bacterial pathogens, but it has remained unclear how they occasionally jump to new host species. Staphylococcus aureus clones exchange MGEs such as S. aureus pathogenicity islands (SaPIs) with high frequency via helper phages. Here we report that the S. aureus ST395 lineage is refractory to horizontal gene transfer (HGT) with typical S. aureus but exchanges SaPIs with other species and genera including Staphylococcus epidermidis and Listeria monocytogenes. ST395 produces an unusual wall teichoic acid (WTA) resembling that of its HGT partner species. Notably, distantly related bacterial species and genera undergo efficient HGT with typical S. aureus upon ectopic expression of S. aureus WTA. Combined with genomic analyses, these results indicate that a 'glycocode' of WTA structures and WTA-binding helper phages permits HGT even across long phylogenetic distances thereby shaping the evolution of Gram-positive pathogens.

  14. A single dose of lysergic acid diethylamide influences gene expression patterns within the mammalian brain.

    PubMed

    Nichols, Charles D; Sanders-Bush, Elaine

    2002-05-01

    Hallucinogenic drugs such as lysergic acid diethylamide (LSD) have profound effects on humans including hallucinations and detachment from reality. These remarkable behavioral effects have many similarities to the debilitating symptoms of neuropsychiatric disorders such as schizophrenia. The effects of hallucinogens are thought to be mediated by serotonin receptor activation; however, how these drugs elicit the unusual behavioral effects remains largely a mystery, despite much research. We have undertaken the first comprehensive analysis of gene expression influenced by acute LSD administration in the mammalian brain. These studies represent a novel approach to elucidate the mechanism of action of this class of drugs. We have identified a number of genes that are predicted to be involved in the processes of synaptic plasticity, glutamatergic signaling and cytoskeletal architecture. Understanding these molecular events will lead to new insights into the etiology of disorders whose behavioral symptoms resemble the temporary effects of hallucinogenic drugs, and also may ultimately result in new therapies.

  15. 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

  16. Adenovirus carrying gene encoding Haliotis discus discus sialic acid binding lectin induces cancer cell apoptosis.

    PubMed

    Yang, Xinyan; Wu, Liqin; Duan, Xuemei; Cui, Lianzhen; Luo, Jingjing; Li, Gongchu

    2014-06-30

    Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.

  17. WinGene/WinPep: user-friendly software for the analysis of amino acid sequences.

    PubMed

    Hennig, L

    1999-06-01

    WinGene1.0/WinPep1.2 is a pair of Microsoft Windows programs designed to read nucleotide or amino acid sequence data. These versatile programs have the following capabilities: (i) searches for open reading frames and their translation, (ii) assisting the design of primers for PCR and (iii) calculation of molecular weight, isoelectric point and molar absorbtion coefficients of polypeptides. Furthermore, hydropathic plots and helical wheel displays are easily produced. The programs run with an intuitive Windows interface, contain a comprehensive help file and enable data exchange with other applications by means of the Copy&Paste command. The software is free for academic and noncommercial users.

  18. Improvement of clavulanic acid production in Streptomyces clavuligerus by genetic manipulation of structural biosynthesis genes.

    PubMed

    Jnawali, Hum Nath; Yoo, Jin Cheol; Sohng, Jae Kyung

    2011-06-01

    To enhance clavulanic acid production, four structural clavulanic acid biosynthesis genes, carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2) and proclavaminate amidinohydrolase (pah2), were amplified from Streptomyces clavuligerus genomic DNA. They were cloned in the pSET152 integration and pIBR25 expression vectors containing the strong ermE* promoter to generate pHN18 and pHN19, respectively, and both plasmids were introduced into S. clavuligerus by protoplast transformation. Clavulanic acid production was increased by 8.7-fold (to ~310 mg/l) in integrative pHN18 transformants and by 5.1-fold in pHN19 transformants compared to controls. Transcriptional analyses showed that the expression levels of ceas2, bls2, cas2 and pah2 were markedly increased in both transformants as compared with wild-type. The elevation of the ceas2, bls2, cas2 and pah2 transcripts was consistent with the enhanced production of clavulanic acid.

  19. A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity.

    PubMed

    Rossi-Alva, Juan Carlos; Rocha-Leão, Maria Helena Miguez

    2003-08-01

    Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.

  20. 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

  1. Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines.

    PubMed

    Niranjan, S K; Goyal, S; Dubey, P K; Kumari, N; Mishra, S K; Mukesh, M; Kataria, R S

    2016-01-10

    Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines

  2. 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.

  3. 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

  4. (+)-Abscisic Acid Metabolism, 3-Ketoacyl-Coenzyme A Synthase Gene Expression, and Very-Long-Chain Monounsaturated Fatty Acid Biosynthesis in Brassica napus Embryos1

    PubMed Central

    Qi, Qungang; Rose, Patricia A.; Abrams, Garth D.; Taylor, David C.; Abrams, Suzanne R.; Cutler, Adrian J.

    1998-01-01

    Microspore-derived embryos of Brassica napus cv Reston were used to examine the effects of exogenous (+)-abscisic acid (ABA) and related compounds on the accumulation of very-long-chain monounsaturated fatty acids (VLCMFAs), VLCMFA elongase complex activity, and induction of the 3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing enzyme of the VLCMFA elongation system. Of the concentrations tested, (+)-ABA at 10 μm showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 μm (+)-ABA treatment, was 60% higher than that of the untreated embryos at 24 h. The transcript of the KCS gene was induced by 10 μm (+)-ABA within 1 h and further increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with (+)-ABA for 72 h. Also, (+)-8′-methylene ABA, which is metabolized more slowly than ABA, had a stronger ABA-like effect on the KCS gene transcription, elongase complex activity (28% higher), and level of VLCMFAs (25–30% higher) than ABA. After 24 h approximately 60% of the added (+)-[3H]ABA (10 μm) was metabolized, yielding labeled phaseic and dihydrophaseic acid. This study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via increased expression of the KCS gene and that reducing ABA catabolism would increase VLCMFAs in microspore-derived embryos. PMID:9662540

  5. RNAi targeting putative genes in phosphatidylcholine turnover results in significant change in fatty acid composition in Crambe abyssinica seed oil.

    PubMed

    Guan, Rui; Li, Xueyuan; Hofvander, Per; Zhou, Xue-Rong; Wang, Danni; Stymne, Sten; Zhu, Li-Hua

    2015-04-01

    The aim of this study was to evaluate the importance of three enzymes, LPCAT, PDCT and PDAT, involved in acyl turnover in phosphatidylcholine in order to explore the possibility of further increasing erucic acid (22:1) content in Crambe seed oil. The complete coding sequences of LPCAT1-1 and LPCAT1-2 encoding lysophosphatidylcholine acyltransferase (LPCAT), PDCT1 and PDCT2 encoding phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), and PDAT encoding phospholipid:diacylglycerol acyltransferase (PDAT) were cloned from developing Crambe seeds. The alignment of deduced amino acid sequences displayed a high similarity to the Arabidopsis homologs. Transgenic lines expressing RNA interference (RNAi) targeting either single or double genes showed significant changes in the fatty acid composition of seed oil. An increase in oleic acid (18:1) was observed, to varying degrees, in all of the transgenic lines, and a cumulative effect of increased 18:1 was shown in the LPCAT-PDCT double-gene RNAi. However, LPCAT single-gene RNAi led to a decrease in 22:1 accumulation, while PDCT or PDAT single-gene RNAi had no obvious effect on the level of 22:1. In agreement with the abovementioned oil phenotypes, the transcript levels of the target genes in these transgenic lines were generally reduced compared to wild-type levels. In this paper, we discuss the potential to further increase the 22:1 content in Crambe seed oil through downregulation of these genes in combination with fatty acid elongase and desaturases.

  6. Efficient Gene Silencing by Self-Assembled Complexes of siRNA and Symmetrical Fatty Acid Amides of Spermine

    PubMed Central

    Metwally, Abdelkader A.; Pourzand, Charareh; Blagbrough, Ian S.

    2011-01-01

    Gene silencing by siRNA (synthetic dsRNA of 21-25 nucleotides) is a well established biological tool in gene expression studies and has a promising therapeutic potential for difficult-to-treat diseases. Five fatty acids of various chain length and oxidation state (C12:0, C18:0, C18:1, C18:2, C22:1) were conjugated to the naturally occurring polyamine, spermine, and evaluated for siRNA delivery and gene knock-down. siRNA delivery could not be related directly to gene silencing efficiency as N4,N9-dierucoyl spermine resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine. GFP silencing in HeLa cells showed that the unsaturated fatty acid amides are more efficient than saturated fatty acid amides, with N4,N9-dioleoyl spermine resulting in the most efficient gene silencing in the presence of serum. The alamarBlue cell viability assay showed that fatty acid amides of spermine have good viability (75%–85% compared to control) except N4,N9-dilauroyl spermine which resulted in low cell viability. These results prove that unsaturated fatty acid amides of spermine are efficient, non-toxic, non-viral vectors for siRNA mediated gene silencing. PMID:24310492

  7. 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

  8. Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

    PubMed

    Christiaens, H; Leer, R J; Pouwels, P H; Verstraete, W

    1992-12-01

    The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

  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.

  10. Promoter variants determine γ-aminobutyric acid homeostasis-related gene transcription in human epileptic hippocampi.

    PubMed

    Pernhorst, Katharina; Raabe, Anna; Niehusmann, Pitt; van Loo, Karen M J; Grote, Alexander; Hoffmann, Per; Cichon, Sven; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2011-12-01

    The functional consequences of single nucleotide polymorphisms associated with episodic brain disorders such as epilepsy and depression are unclear. Allelic associations with generalized epilepsies have been reported for single nucleotide polymorphisms rs1883415 (ALDH5A1; succinic semialdehyde dehydrogenase) and rs4906902 (GABRB3; GABAA β3), both of which are present in the 5' regulatory region of genes involved in γ-aminobutyric acid (GABA) homeostasis. To address their allelic association with episodic brain disorders and allele-specific impact on the transcriptional regulation of these genes in human brain tissue, DNA and messenger RNA (mRNA) isolated from hippocampi were obtained at epilepsy surgery of 146 pharmacoresistant mesial temporal lobe epilepsy (mTLE) patients and from 651 healthy controls. We found that the C allele of rs1883415 is accumulated to a greater extentin mTLE versus controls. By real-time quantitative reverse transcription-polymerase chain reaction analyses, individuals homozygous for the C allele showed higher ALDH5A1 mRNA expression. The rs4906902 G allele of the GABRB3 gene was overrepresented in mTLE patients with depression; individuals homozygous for the G allele showed reduced GABRB3 mRNA expression. Bioinformatic analyses suggest that rs1883415 and rs4906902 alter the DNA binding affinity of the transcription factors Egr-3 in ALDH5A1 and MEF-2 in GABRB3 promoters, respectively. Using in vitro luciferase transfection assays, we observed that, in both cases, the transcription factors regulate gene expression depending on the allelic variant in the same direction as in the human hippocampi. Our data suggest that distinct promoter variants may sensitize individuals for differential, potentially stimulus-induced alterations of GABA homeostasis-relevant gene expression. This might contribute to the episodic onset of symptoms and point to new targets for pharmacotherapies.

  11. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed Central

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-01-01

    A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses. Images PMID:3025846

  12. Mutations of lysophosphatidic acid receptor-1 gene during progression of lung tumors in rats

    SciTech Connect

    Yamada, Takanori; Obo, Yumi; Furukawa, Mami; Hotta, Mayuko; Yamasaki, Ayako; Honoki, Kanya; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2009-01-16

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2-4 were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 out of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung carcinogenesis induced in rats by BHP.

  13. Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

    PubMed

    Venugopal, Srivathsa C; Jeong, Rae-Dong; Mandal, Mihir K; Zhu, Shifeng; Chandra-Shekara, A C; Xia, Ye; Hersh, Matthew; Stromberg, Arnold J; Navarre, DuRoy; Kachroo, Aardra; Kachroo, Pradeep

    2009-07-01

    Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

  14. 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.

  15. Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells.

    PubMed

    Tran, C P; Familari, M; Parker, L M; Whitehead, R H; Giraud, A S

    1998-07-01

    Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.

  16. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-12-01

    A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses.

  17. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-01

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  18. Identification of Campylobacter jejuni Genes Involved in the Response to Acidic pH and Stomach Transit▿

    PubMed Central

    Reid, Anne N.; Pandey, Reenu; Palyada, Kiran; Naikare, Hemant; Stintzi, Alain

    2008-01-01

    Campylobacter jejuni causes food- and waterborne gastroenteritis, and as such it must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after the organism was exposed to in vitro and in vivo (piglet stomach) acid shock. The observed down-regulation of genes encoding ribosomal proteins likely reflects the need to reshuffle energy toward the expression of components required for survival. Acid shock also caused C. jejuni to up-regulate genes involved in stress responses. These included heat shock genes as well as genes involved in the response to oxidative and nitrosative stress. A role for the chaperone clpB in acid resistance was confirmed in vitro. Some genes showed expression patterns that were markedly different in vivo and in vitro, which likely reflects the complexity of the in vivo environment. For instance, transit through the stomach was characterized by up-regulation of genes that encode products that are involved in the use of nitrite as a terminal electron acceptor and down-regulation of genes that are involved in capsular polysaccharide expression. In conclusion, this study has enabled us to understand how C. jejuni modulates gene expression in response to acid shock in vitro and to correlate this with gene expression profiles of C. jejuni as it transits through the host stomach. PMID:18192414

  19. 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

  20. 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

  1. 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

  2. Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development.

    PubMed

    Leskow, Carla Coluccio; Kamenetzky, Laura; Dominguez, Pia Guadalupe; Díaz Zirpolo, José Antonio; Obata, Toshihiro; Costa, Hernán; Martí, Marcelo; Taboga, Oscar; Keurentjes, Joost; Sulpice, Ronan; Ishihara, Hirofumi; Stitt, Mark; Fernie, Alisdair Robert; Carrari, Fernando

    2016-07-01

    Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level. PMID:27194734

  3. 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 ...

  4. A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway.

    PubMed

    Zou, Xiaolu; Seemann, Jeffrey R; Neuman, Dawn; Shen, Qingxi J

    2004-12-31

    The creosote bush (Larrea tridentata) is a xerophytic evergreen C3 shrub thriving in vast arid areas of North America. As the first step toward understanding the molecular mechanisms controlling the drought tolerance of this desert plant, we have isolated a dozen genes encoding transcription factors, including LtWRKY21 that encodes a protein of 314 amino acid residues. Transient expression studies with the GFP-LtWRKY21 fusion construct indicate that the LtWRKY21 protein is localized in the nucleus and is able to activate the promoter of an abscisic acid (ABA)-inducible gene, HVA22, in a dosage-dependent manner. The transactivating activity of LtWRKY21 relies on the C-terminal sequence containing the WRKY domain and a N-terminal motif that is essential for the repression activity of some regulators in ethylene signaling. LtWRKY21 interacts synergistically with ABA and transcriptional activators VP1 and ABI5 to control the expression of the HVA22 promoter. Co-expression of VP1, ABI5, and LtWRKY21 leads to a much higher expression of the HVA22 promoter than does the ABA treatment alone. In contrast, the Lt-WRKY21-mediated transactivation is inhibited by two known negative regulators of ABA signaling: 1-butanol, an inhibitor of phospholipase D, and abi1-1, a dominant negative mutant protein phosphatase. Interestingly, abi1-1 does not block the synergistic effect of LtWRKY21, VP1, and ABI5 co-expression, indicating that LtWRKY21, VP1, and ABI5 may form a complex that functions downstream of ABI1 to control ABA-regulated expression of genes.

  5. Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species.

    PubMed

    Wu, YuHua; Xiao, Ling; Wu, Gang; Lu, ChangMing

    2007-06-01

    The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhongshuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at position 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and between the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.

  6. 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

  7. Effective antitumor gene therapy delivered by polyethylenimine-conjugated stearic acid-g-chitosan oligosaccharide micelles.

    PubMed

    Hu, F-Q; Chen, W-W; Zhao, M-D; Yuan, H; Du, Y-Z

    2013-06-01

    Non-viral vesicle composing of low-molecular weight polyethylenimine-conjugated stearic acid-g-chitosan oligosaccharide (CSOSA-g-PEI) was synthesized for gene delivery and therapy. The synthesized CSOSA-g-PEI had good ion-buffer capabilities and DNA-binding capacity, which could form positively charged nano-sized particles (100-150 nm) with plasmid DNA; in vitro gene transfection tests demonstrated that CSOSA-g-PEI presented much lower cytotoxicity and corresponding transfection efficiency in comparison with Lipofectamine 2000 in both human cancer cells (Hela and MCF-7). The gene transfection of CSOSA-g-PEI/pDNA could be further enhanced in the presence of serum or by adding arginine during incubation of CSOSA-g-PEI micelles with plasmid DNA. The biodistribution experiments demonstrated CSOSA-g-PEI conjugate highly localized in the tumor tissue and indicated a persistently increased accumulation. In vivo antitumor activity results showed that CSOSA-g-PEI/plasmid pigment epithelium-derived factor formulation could effectively suppress the tumor growth (above 60% tumor inhibition) without systematic toxicity against animal body after intravenous injection.

  8. 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.

  9. 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

  10. 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

  11. Influence of Different Levels of Lipoic Acid Synthase Gene Expression on Diabetic Nephropathy

    PubMed Central

    Xu, Longquan; Hiller, Sylvia; Simington, Stephen; Nickeleit, Volker; Maeda, Nobuyo; James, Leighton R.; Yi, Xianwen

    2016-01-01

    Oxidative stress is implicated in the pathogenesis of diabetic nephropathy (DN) but outcomes of many clinical trials are controversial. To define the role of antioxidants in kidney protection during the development of diabetic nephropathy, we have generated a novel genetic antioxidant mouse model with over- or under-expression of lipoic acid synthase gene (Lias). These models have been mated with Ins2Akita/+ mice, a type I diabetic mouse model. We compare the major pathologic changes and oxidative stress status in two new strains of the mice with controls. Our results show that Ins2Akita/+ mice with under-expressed Lias gene, exhibit higher oxidative stress and more severe DN features (albuminuria, glomerular basement membrane thickening and mesangial matrix expansion). In contrast, Ins2Akita/+ mice with highly-expressed Lias gene display lower oxidative stress and less DN pathologic changes. Our study demonstrates that strengthening endogenous antioxidant capacity could be an effective strategy for prevention and treatment of DN. PMID:27706190

  12. Genetic Variants in the FADS Gene: Implications for Dietary Recommendations for Fatty Acid Intake

    PubMed Central

    Mathias, Rasika A.; Pani, Vrindarani; Chilton, Floyd H.

    2014-01-01

    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

  13. Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii.

    PubMed

    Wang, Q H; Jiao, Y; Du, X D; Zhao, X X; Huang, R L; Deng, Y W; Yan, F

    2015-01-01

    Purple acid phosphatases (PAPs), also known as type 5 acid phosphatases, are widely present in animals, plants, and fungi. In mammal, PAP was reported to participate in immune defense and bone resorption. In this study, the characteristics and potential functions of a PAP gene from pearl oyster Pinctada martensii (pm-PAP) were examined. The Pm-PAP cDNA was found to be 2777 base pairs, containing a 1581-base pair open reading fragment encoding for 526 amino acids with an estimated molecular mass of 60.1 kDa and theoretical isoelectric point of 5.82. One signal peptide and five conserved motifs [GDXX/GDXXY/GNH(D/E)/XXXH/(A/G)HXH] were present in the entire sequence. Tissue expression profile analysis showed that pm-PAP mRNA was constitutively expressed in all tissues studied with abundant mRNA found in mollusk defense system, including hepatopancreas, gill, and hemocytes. After lipopolysaccharide stimulation, the expression of pm-PAP mRNA in hemocytes was dramatically upregulated at 2 h and achieved the highest level at 36 h. Additionally, pm-PAP mRNA expression was significantly increased and achieved the highest level at 2 days after the surgical implantation during pearl production. These results suggest that pm-PAP is a constitutive and inducible protein that may be involved in the immune defense of pearl oyster. PMID:25729991

  14. Development of a SCAR (sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum.