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Synthase Gene (synthase + gene)
Terms modified by Synthase Gene Selected AbstractsA New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional ComplementationJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2006Zhi-Hua Liao Abstract Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a rate-limiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pI of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homology-based structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant. This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level. (Managing editor: Wei Wang) [source] Biosynthesis and Compositional Regulation of Poly[(3-hydroxybutyrate)- co -(3-hydroxyhexanoate)] in Recombinant Ralstonia eutropha Expressing Mutated Polyhydroxyalkanoate Synthase GenesMACROMOLECULAR BIOSCIENCE, Issue 3 2004Takeharu Tsuge Abstract Summary: A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB,4 harboring mutated PHA synthase genes (phaCAc) from Aeromona caviae was investigated. The strain harboring wild-type phaCAc gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB- co -3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaCAc gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0,5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes. Relationship between PHA synthase activity and 3HHx fraction in P(3HB- co -3HHx). [source] Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activityFEBS JOURNAL, Issue 15 2002Hannes E. Bülow In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11,-hydroxylase [Bülow, H.E.,Möbius, K., Bähr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304,312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11,-hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11,-hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals. [source] Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrolFEMS YEAST RESEARCH, Issue 1 2003John V.W. Becker Abstract The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p -coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p -coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p -coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following ,-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol ,-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol. [source] Cloning and expression of a geranylgeranyl diphosphate synthase gene: insights into the synthesis of termite defence secretionINSECT MOLECULAR BIOLOGY, Issue 1 2007Masaru Hojo Abstract In Nasutitermes takasagoensis, a termite in which soldiers perform specialized chemical defence, Nts19-1 gene is highly expressed exclusively in soldier head. In this study, two types of transcripts for this gene were obtained, and the full-length cDNAs were determined by rapid amplification of cDNA ends (RACE). These transcripts were putative homologues of the geranylgeranyl diphosphate (GGPP) synthase gene, involved in the condensation of dimethylallyl diphosphate with isopentenyl diphosphate in the isoprenoid biosynthetic pathway. The genes were thus termed NtGGPPS1. GGPP is a precursor of diterpenes in plants. In situ hybridization localized NtGGPPS1 expression to the epidermal secretory cells of the frontal gland reservoir where many kinds of diterpenes are produced, suggesting that NtGGPPS1 is involved in the biosynthesis of defence secretion. [source] An intron 4 VNTR polymorphism of the endothelial nitric oxide synthase gene is associated with early-onset colorectal cancerINTERNATIONAL JOURNAL OF CANCER, Issue 7 2009Chih-Ching Yeh Abstract Endothelial-derived nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), may play an important role in colorectal carcinogenesis. However, the putative contribution of common eNOS genetic polymorphisms to colorectal cancer risk remains unknown. We genotyped 3 polymorphisms of eNOS (T-786C, G894T, and intron4b/a) in 727 colorectal adenocarcinoma cases and 736 age- and sex-matched healthy controls in Taiwan. Genotypes of the T-786C and G894T polymorphisms were determined by fluorescence polarization assays and the 27-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 (intron4b/a) was analyzed by PCR. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Among younger participants (,60 yrs), the intron4a variant genotype was associated with a significantly increased risk of colorectal cancer, compared with the intron4bb genotype (OR = 1.60, 95% CI = 1.04,2.46). In addition, those young individuals bearing a greater number of high-risk genotypes (OR > 1, i.e., CT+TT for T-786C, ba+aa for intron4b/a, and GG for G894T) of eNOS had a higher colorectal cancer risk (ptrend = 0.039). Compared with younger individuals without any putative high-risk genotypes, those with 3 high-risk genotypes had a significantly greater cancer risk (OR = 1.89, 95% CI = 1.04,3.43). Our results suggest that the eNOS intron4b/a polymorphism may contribute to early-onset colorectal cancer risk in the Taiwanese population. © 2008 Wiley-Liss, Inc. [source] Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicolaJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2009X. Li Abstract Aims:, To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean (Phaseolus vulgaris L.). Methods and Results:, Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium, Erwinia and Pantoea. Conclusion:, A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria. Significance and Impact of the Study:, This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences. [source] Use of induction promoters to regulate hyaluronan synthase and UDP-glucose-6-dehydrogenase of Streptococcus zooepidemicus expression in Lactococcus lactis: a case study of the regulation mechanism of hyaluronic acid polymerJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009J.Z. Sheng Abstract Aims:, To determine the effects of the ratios of hyaluronan synthase expression level to precursor sugar UDP-GlcA biosynthesis ability on the molecular weight (MW) of hyaluronic acid (HA) in recombinant Lactococcus lactis. Methods and Results:, The genes szHasA (hyaluronan synthase gene) and szHasB (UDP-glucose-6-dehydrogenase gene) of Streptococcus zooepidemicus were introduced into L. lactis under the control of nisA promoter and lacA promoter respectively, resulting in a dual-plasmid controlled expression system. The effects of the ratios of hyaluronan synthase expression level to the precursor sugar UDP-GlcA biosynthesis ability under different induction concentration collocations with nisin and lactose on the MW of HA in recombinant L. lactis were determined. The results showed that the final weight-average molecular weight () of HA correlated with the relative ratios of HasA (hyaluronan synthase) expression level to the concentration of UDP-GlcA. Conclusions:, Regulating the relative ratios of HasA expression level to the precursor sugar biosynthesis ability was an efficient method to control the size of HA. Significance and Impact of the Study:, This study put forward a guide to establish an efficacious way to control the size of HA in fermentation. [source] Selective protection against oxidative damage in brain of mice with a targeted disruption of the neuronal nitric oxide synthase geneJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2007Juan Carlos Martínez-Lazcano Abstract Nitric oxide (NO) is an essential messenger molecule in brain, where it is produced in neurons mostly by the activity of the neuronal isoform of nitric oxide synthase (nNOS). To understand the participation of the different isoforms of NOS in physiological functioning and in pathological processes, mice with null mutations for each of the NOS isoforms have been generated. In the present paper, we report that there is a selective protection from oxidative damage in the brain of mice with a targeted disruption of the nNOS gene. The cerebellum of these mice shows reduced levels of lipid peroxidation (LP) at the different ages tested, compared with wild-type mice, and also a reduction in the formation of reactive oxygen species (ROS). We observed a decrease of LP in cortex, and no effect on either LP or ROS formation was observed in striatum of knockout mice compared with wild type. We also report increased spontaneous motor activity of knockout mice. The expression and activity of nNOS are crucial to maintain redox status in brain, and we consider that the alteration in oxidative damage may help us to explain the phenotypical characteristics of nNOS knockout mice and their differential susceptibility to brain insults. © 2007 Wiley-Liss, Inc. [source] Requirement of spermidine for developmental transitions in Aspergillus nidulansMOLECULAR MICROBIOLOGY, Issue 3 2002Yuan Jin Summary Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, ,spdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when ,spdA is grown on media supplemented with 0.05,0.10 mM exogenous spermidine. Supplementation of ,spdA with , 0.10 mM spermidine restores sterigmatocystin production and , 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. ,spdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in ,spdA strains grown in media lacking spermidine but could force both processes in ,spdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over-ride the spermidine requirement for the latter but not the former transition. [source] Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregationMOLECULAR MICROBIOLOGY, Issue 5 2002Vinod S. Dubey Summary Cell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosic acid synthase gene (mas) encodes the enzyme that produces one class of such acids. Seven mas -like genes (msls) were identified in the genome. One of them, msl3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl3 mutant does not produce mycolipanoic acids and mycolipenic (phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface-exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead-like growth morphology without affecting the overall growth rate. [source] The synergistic effects of sugar and abscisic acid on myo -inositol-1-phosphate synthase expressionPHYSIOLOGIA PLANTARUM, Issue 4 2002Kaoru T. Yoshida 1L- myo -inositol-1-phosphate [Ins(1)P1] synthase (EC 5.5.1.4) catalyses the formation of Ins(1)P1 from glucose-6-phosphate, the first step in the biosynthesis of myo -inositol. Ins(1)P1 is a precursor of phytin (inositol hexakisphosphate), a storage form of phosphate and cations in seeds. Since sucrose and abscisic acid (ABA) are known to affect synthesis of storage compounds in seeds, we investigated the effects of ABA and sucrose on Ins(1)P1 synthase gene (RINO1) expression in cultured cells derived from the scutellum of mature rice seeds. Higher levels of RINO1 transcript accumulation were evident after treatment with either sucrose (10,100 mM) or ABA (10,8M to 10,4M). Glucose was also effective in the upregulation, whereas mannitol was not, suggesting that sucrose and glucose acted as metabolizable sugars and not as osmotica. Treatment with ABA and sucrose together resulted in much higher levels of transcript accumulation, suggesting a synergistic induction of the Ins(1)P1 synthase gene. [source] Molecular control of ethylene production by cyanide in Arabidopsis thalianaPHYSIOLOGIA PLANTARUM, Issue 2 2000Jennifer McMahon Smith Although cyanide has long been recognized as a co-product of ethylene synthesis, little attention has been given to its potential physiological and molecular roles. In the present work, the long-term effects of cyanide on growth and development were observed in Arabidopsis thaliana. Two days after a single 20-min application of cyanide, plants demonstrated visible signs of stress. Long-term detrimental effects on growth and photosynthetic capabilities were noted, including low chlorophyll accumulation and stunted growth. Because of the relationship between cyanide and ethylene production, we chose to evaluate the results of cyanide treatment on genes encoding proteins involved in ethylene synthesis. We have found that only the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene, ACS6, is rapidly activated in response to cyanide treatment, while other ACS genes were unaffected. This same gene has previously been shown to be transcriptionally activated in response to touch and other environmental stimuli. Cyanide was capable of activating ACS6 transcription within 10 min of treatment, and the amount of transcript correlated positively with the cyanide dosage. Due to the toxic nature of cyanide, plant in vivo concentrations are generally maintained lower than 10 ,M, but can increase under certain stresses. In the present work, we observed that physiologically relevant concentrations as low as 1 ,M HCN, considered metabolically ,safe', were capable of initiating ACS6 transcription. ACS6 transcripts were not substantially reduced as a result of multiple cyanide treatments, which is in contrast with the effects of mechanical stimulation on transcription. Our results suggest a relationship between cyanide production during ethylene synthesis and the molecular control of ethylene synthesis. This work corresponds with earlier experiments that have demonstrated that ethylene and cyanide can elicit some similar physiological responses. It is possible that cyanide may play an active role in ethylene regulation under conditions where rapid cyanide accumulation occurs. Since cyanide can rapidly activate ethylene synthesis, it is possible that it is involved in the positive-feedback regulation of ethylene that occurs in some plant tissues. [source] Generation of stable ,low phytic acid' transgenic rice through antisense repression of the 1d - myo -inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoterPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2009Mio Kuwano Summary Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1d - myo -inositol 3-phosphate [Ins(3)P1] synthase (EC 5.5.1.4) catalyses the first step of myo -inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P1 synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong ,low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants. [source] Polymorphism of the promoter region of prostacyclin synthase gene in chronic thromboembolic pulmonary hypertensionRESPIROLOGY, Issue 2 2004Shinya AMANO Objective: Decreased expression of prostacyclin synthase (PGIS) is observed in the lung vasculature of patients with pulmonary arterial hypertension and the biosynthesis of prostacyclin (PGI2) may be impaired in chronic thromboembolic pulmonary hypertension (CTEPH). Whether it is genetically determined or develops as the disease progresses is unclear. A variable-number tandem repeat (VNTR) polymorphism has been detected in the 5,-upstream promoter region of the PGIS gene. It has been demonstrated that the alleles vary in size from three to seven repeats of nine base pairs, and transcriptional activity increased with the number of repeats. The purpose of the present study was to elucidate the association between the VNTR polymorphisms of the PGIS gene and CTEPH in Japanese subjects. Methodology: Ninety patients with CTEPH and 144 control subjects were investigated for the presence of VNTR polymorphisms. Sixty-two blood samples were obtained from CTEPH patients and the plasma concentrations of prostacyclin and thromboxane A2 metabolites were measured. Results: VNTR polymorphisms in the prostacyclin synthase gene were grouped into L alleles (five, six and seven repeats) and S alleles (three and four repeats). The overall distribution of the alleles and genotypes were not significantly different between CTEPH patients and the control subjects. The patients with the LL genotype had higher plasma concentrations of 6-keto-prostaglandin F1, than patients with the LS and SS genotypes. Conclusions: Our results suggested that the specific VNTR polymorphism in the 5,-upstream promoter region of the PGIS gene regulated prostacyclin production, but did not seem to be associated with the development of CTEPH in this patient population. [source] A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horsesANIMAL GENETICS, Issue 1 2009B. Herszberg Summary Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene (GBE1). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene (GYS1) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy. [source] Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiaeBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Mohammad A. Asadollahi Abstract The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over-expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over-expression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol. Biotechnol. Bioeng. 2010; 106: 86,96. © 2010 Wiley Periodicals, Inc. [source] Enhanced Hyaluronic Acid Production in Bacillussubtilis by Coexpressing Bacterial HemoglobinBIOTECHNOLOGY PROGRESS, Issue 5 2007Liang-Jung Chien Bacillus subtilis strains that can produce hyaluronic acid (HA) were constructed by integrating the HA synthase gene (hasA) and the UDP-glucose dehydrogenase gene of group C Streptococcus (hasB) or of B. subtilis itself (tauD) into the amyE locus of the B. subtilis chromosome. All of the inserted genes were under the control of a strong constitutive vegII promoter of B. subtilis. Although HA production could be achieved by expressing hasA alone, coexpressing hasB or tauD with hasA could enhance HA production at least 2-fold. To replenish the energy consumed for HA biosynthesis, Vitreoscilla hemoglobin (VHb) was coexpressed with the HA-expressing genes. With the expression of VHb, not only the cell concentration was enhanced 25%, but also HA production was further increased by 100%. About 1.8 g/L of HA was obtained by the recombinant strain B.subtilis carrying VHb, hasA, and tauD genes in the expression cassette after 30 h cultivation. [source] Human MIP synthase splice variants in bipolar disorderBIPOLAR DISORDERS, Issue 7 2007Alon Shamir Objectives:, Alternative splicing allows the production of multiple gene products with different functions from a given sequence, affecting cellular function control. Tissue-specific splicing is most prevalent in the brain. We therefore investigate whether splice variants contribute to complex psychiatric disorders. A database search suggested that the myo -inositol-1-phosphate (MIP) synthase gene, possibly involved in pathophysiology of bipolar disorder, has splice variants. Methods:, Human RNA was purified from lymphocytes and postmortem brain. MIP synthase alternative splice variants were amplified using reverse transcription-polymerase chain reaction. Results:, The bioinformatics finding was confirmed in both tissues. No difference in lymphocyte MIP synthase mRNA splice-variant levels was found between bipolar patients and controls. However, patients with family history of a major psychiatric disorder had significantly higher levels of the variant lacking exons 3 and 4 versus patients with no family history and controls. Conclusions:, As alternative splicing may be a mechanism by which the ,30,000 genes are amplified in mammalian brain, further studies with other candidate genes for psychiatric disorders are needed. [source] GM3 synthase gene is a novel biomarker for histological classification and drug sensitivity against epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancerCANCER SCIENCE, Issue 10 2007Mariko Noguchi Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT-I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT-I mRNA as well as GM3 in 40 tumor tissues surgically removed from non-small cell lung cancer patients. Adenocarcinoma tissues expressed SAT-I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT-I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT-I mRNA and epidermal growth factor receptor (EGFR)-tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR-TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC50 values for gefitinib and AG1478 decreased dramatically with increasing SAT-I mRNA levels (R2 = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR-TK inhibitors, the SAT-I gene was introduced stably into a GM3-negative clone of murine 3LL lung cancer cells to produce GM3-reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3-reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT-I mRNA in lung biopsy samples from non-small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR-TK inhibitors. (Cancer Sci 2007; 98: 1625,1632) [source] PKSP-dependent reduction of phagolysosome fusion and intracellular kill of Aspergillus fumigatus conidia by human monocyte-derived macrophagesCELLULAR MICROBIOLOGY, Issue 12 2002Bernhard Jahn Summary Previously, we described the isolation of an Aspergillus fumigatus mutant producing non-pigmented conidia, as a result of a defective polyketide synthase gene, pksP (polyketide synthase involved in pigment biosynthesis). The virulence of the pksP mutant was attenuated in a murine animal infection model and its conidia showed enhanced susceptibility towards damage by monocytes in vitro. Because macrophage-mediated killing is critical for host resistance to aspergillosis, the interaction of both grey-green wild-type conidia and white pksP mutant conidia with human monocyte-derived macrophages (MDM) was studied with respect to intracellular processing of ingested conidia. After phagocytosis, the percentage of wild-type conidia residing in an acidic environment was approximately fivefold lower than that observed for non-pigmented pksP mutant conidia. The phagolysosome formation, as assessed by co-localization of LAMP-1 and cathepsin D with ingested conidia, was significantly lower for wild-type conidia compared with pksP mutant conidia. Furthermore, the intracellular kill of pksP mutant conidia was significantly higher than of wild-type conidia, which was markedly increased by chloroquine, a known enhancer of phagolysosome fusion. Taken together, these findings suggest that the presence of a functional pksP gene in A. fumigatus conidia is associated with an inhibition of phagolysosome fusion in human MDM. These data show for the first time that a fungus has the capability to inhibit the fusion of the phagosome with the lysosome. This finding might help explain the attenuated virulence of pksP mutant strains in a murine animal model and provides a conceptual frame to understand the virulence of A. fumigatus. [source] Organisation of the Biosynthetic Gene Cluster and Tailoring Enzymes in the Biosynthesis of the Tetracyclic Quinone Glycoside Antibiotic PolyketomycinCHEMBIOCHEM, Issue 6 2009Martina Daum Abstract Surprising results regarding the function of methyltransferases and oxygenases: Investigations on oxygenase and methyltransferase genes that are located in the polyketomycin biosynthetic gene cluster of Streptomyces diastatochromogenes Tü6028 shed light into polyketide-modifying reactions. Polyketomycin is a tetracyclic quinone glycoside produced by Streptomyces diastatochromogenes Tü6028. It shows cytotoxic and antibiotic activity, in particular against Gram-positive multi-drug-resistant strains (for example, MRSA). The polyketomycin biosynthetic gene cluster has been sequenced and characterised. Its identity was proven by inactivation of a ,-ketoacyl synthase gene (pokP1) of the "minimal polyketide synthase II" system. In order to obtain valuable information about tailoring steps, we performed further gene-inactivation experiments. The generation of mutants with deletions in oxygenase genes (pokO1, pokO2, both in parallel and pokO4) and methyltransferase genes (pokMT1, pokMT2 and pokMT3) resulted in new polyketomycin derivatives, and provided information about the organisation of the biosynthetic pathway. [source] Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathwayCLINICAL GENETICS, Issue 1 2009R Do High levels of plasma low-density lipoprotein cholesterol (LDL-C) are a significant risk factor for heart disease. Statins (3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors) have been extensively used to treat high-plasma LDL-C levels and are effective in preventing heart disease. However, statins can be associated with adverse side effects in some patients and do not work effectively in others. As an alternative to statins, the development of cholesterol-lowering agents that directly inhibit squalene synthase have shown promise. Clinical studies have shown that squalene synthase inhibitors are effective in lowering plasma levels of total cholesterol and LDL-C. Squalene synthase plays an important role in the cholesterol biosynthesis pathway as it is responsible for the flow of metabolites into either the sterol or the non-sterol branches of the pathway. In addition, variants of the squalene synthase gene appear to modulate plasma cholesterol levels in human populations and therefore may be linked to cardiovascular disease. In this review, we examine squalene synthase and the gene that codes for it (farnesyldiphosphate farnesyltransferase 1). In particular, we investigate their role in the regulation of cellular and plasma cholesterol levels, including data that suggest that squalene synthase may be involved in the etiology of hypercholesterolemia. [source] ,Klotho: A new kid on the bile acid biosynthesis block,HEPATOLOGY, Issue 1 2006Marco Arrese We have generated a line of mutant mouse that lacks ,Klotho, a protein that structurally resembles Klotho. The synthesis and excretion of bile acids were found to be dramatically elevated in these mutants, and the expression of 2 key bile acid synthase genes, cholesterol 7,-hydroxylase (Cyp7a1) and sterol 12,- hydroxylase (Cyp8b1), was strongly upregulated. Nuclear receptor pathways and the enterohepatic circulation, which regulates bile acid synthesis, seemed to be largely intact; however, bile acid,dependent induction of the small heterodimer partner (SHP) NR0B2, a common negative regulator of Cyp7a1 and Cyp8b1, was significantly attenuated. The expression of Cyp7a1 and Cyp8b1 is known to be repressed by dietary bile acids via both SHP-dependent and -independent regulations. Interestingly, the suppression of Cyp7a1 expression by dietary bile acids was impaired, whereas that of Cyp8b1 expression was not substantially altered in ,klotho,/, mice. Therefore, ,Klotho may stand as a novel contributor to Cyp7a1 -selective regulation. Additionally, ,Klotho-knockout mice exhibit resistance to gallstone formation, which suggests the potential future clinical relevance of the ,Klotho system. [source] The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrixINSECT MOLECULAR BIOLOGY, Issue 5 2005Y. Arakane Abstract Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1- specific RNA interference (RNAi) disrupted all three types of moult (larval,larval, larval,pupal and pupal,adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2 -specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut. [source] Single nucleotide polymorphisms in succinate dehydrogenase subunits and citrate synthase genes: association results for impaired spermatogenesisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2007Sandra Bonache Abstract Evaluation of the possible implication of the SDHA, SDHB, SDHC, SDHD and CS genes in non-obstructive male infertility was performed on the basis that sperm concentration in the ejaculate has been previously correlated with nuclear-encoded mitochondrial enzyme activities (the four subunits of succinate dehydrogenase/complex II of the respiratory chain and citrate synthase). We performed an exhaustive analysis of the five genes for the presence of sequence variants that could be associated with impairment of sperm production. blastn searches in the genomic sequence NCBI database evidenced the presence of highly homologous sequences elsewhere on the genome that can interfere with polymerase chain reaction experiments. Therefore, a careful design of the analytical strategy to search for sequence variants was performed. In this report, we provide primer sequences that allowed selective amplification of coding and immediate flanking regions of the five genes. Fifty-five sequence variations in the five genes were identified in infertile and normozoospermic fertile individuals as controls and only one of them (SDHA c.456+32G>A) showed significant genotype association with impairment of sperm production. Moreover, new single nucleotide polymorphisms identified should be useful in future association studies for other human diseases related to nuclear-encoded genes, leading to mitochondrial respiratory chain activity impairment revealing the physiological role of these genes. [source] Simvastatin effects on portal-systemic collaterals of portal hypertensive ratsJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 8 2010Hui-Chun Huang Abstract Background and Aim:, Portal-systemic collateral vascular resistance and vasoconstrictor responsiveness are crucial in portal hypertension and variceal bleeding control. Statins enhance vasodilators production, but their influence on collaterals is unknown. This study aimed to survey the effect of simvastatin on collaterals. Methods:, Partially portal vein-ligated rats received oral simvastatin (20 mg/kg/day) or distilled water from ,2 to +7 day of ligation. After hemodynamic measurements on the eighth postoperative day, baseline perfusion pressure (i.e. an index of collateral vascular resistance) and arginine vasopressin (AVP, 0.1 nM,0.1 µM) responsiveness were evaluated with an in situ perfusion model for collateral vascular beds. RT-PCR of endothelial NO synthase (eNOS), inducible NOS (iNOS), cyclooxygenase-1 (COX-1), COX-2, thromboxane A2 synthase (TXA2 -S) and prostacyclin synthase genes was performed in parallel groups for splenorenal shunt (SRS), the most prominent intra-abdominal collateral vessel. To determine the acute effects of simvastatin, collateral AVP response was assessed with vehicle or simvastatin. SRS RT-PCR of eNOS, iNOS, COX-1, COX-2 and TXA2 -S, and measurements of perfusate nitrite/nitrate, 6-keto-PGF1, and TXB2 levels were performed in parallel groups without AVP. Results:, Acute simvastatin administration enhanced SRS eNOS expression and elevated perfusate nitrite/nitrate and 6-keto-PGF1, concentrations. Chronic simvastatin treatment reduced baseline collateral vascular resistance and portal pressure and enhanced SRS eNOS, COX-2 and TXA2 -S mRNA expression. Neither acute nor chronic simvastatin administration influenced collateral AVP responsiveness. Conclusion:, Simvastatin reduces portal-systemic collateral vascular resistance and portal pressure in portal hypertensive rats. This may be related to the enhanced portal-systemic collateral vascular NO and prostacyclin activities. [source] Cellulose structure and biosynthesis: What is in store for the 21st century?JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 3 2004R. Malcolm Brown Jr. Abstract This article briefly summarizes historical developments in fundamental research related to the structure and biosynthesis of cellulose. Major advances concerning the structure of cellulose include the discovery of a new suballomorph of cellulose I, the lattice imaging of glucan chains showing no fringe micelle structure, parallel chain orientation in cellulose I, and the discovery of nematic ordered cellulose. Major advances in biosynthesis include the discovery of the terminal synthesizing complex, the isolation and purification of cellulose synthase, the in vitro synthesis of cellulose I, and synthetic cellulose assembly. This article focuses on recent advances in molecular biology with cellulose, including the cloning and sequencing of cellulose synthase genes from bacteria, cyanobacteria, and vascular plants; proof of the terminal synthesizing complex as the site of the catalytic subunit of cellulose synthase; cellulose and callose synthase expression during growth and development; and phylogenetic aspects of cellulose synthase evolution. This article concludes with thoughts about future uses for the accumulating genetic information on cellulose biosynthesis for textiles and forest products and discusses possibilities of new global resources for cellulose production. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 487,495, 2004 [source] Biosynthesis and Compositional Regulation of Poly[(3-hydroxybutyrate)- co -(3-hydroxyhexanoate)] in Recombinant Ralstonia eutropha Expressing Mutated Polyhydroxyalkanoate Synthase GenesMACROMOLECULAR BIOSCIENCE, Issue 3 2004Takeharu Tsuge Abstract Summary: A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB,4 harboring mutated PHA synthase genes (phaCAc) from Aeromona caviae was investigated. The strain harboring wild-type phaCAc gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB- co -3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaCAc gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0,5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes. Relationship between PHA synthase activity and 3HHx fraction in P(3HB- co -3HHx). [source] MicroReview: LuxR-type quorum-sensing regulators that are detached from common scentsMOLECULAR MICROBIOLOGY, Issue 5 2010Ching-Sung Tsai Summary The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and at least some of them require AHLs for folding and protease resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. Complexes between some of these proteins and their DNA binding sites are disrupted by AHLs in vitro. All such proteins are fairly closely related within the larger LuxR family, indicating that they share a relatively recent common ancestor. The 3, ends of the genes encoding these receptors invariably overlap with the 3, ends of the cognate AHL synthase genes, suggesting additional antagonism at the level of mRNA synthesis, stability or translation. [source] | |||||||||||||||||||||||||||||||