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Sample records for phosphoenolpyruvate-dependent phosphotransferase genes

  1. Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: Functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides

    SciTech Connect

    Lai, Xiaokuang; Davis, F.C.; Ingram, L.O.; Hespell, R.B.

    1997-02-01

    Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-{beta}-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-{beta}-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation. 63 refs., 4 figs., 4 tabs.

  2. Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose▿†

    PubMed Central

    Xiao, Han; Gu, Yang; Ning, Yuanyuan; Yang, Yunliu; Mitchell, Wilfrid J.; Jiang, Weihong; Yang, Sheng

    2011-01-01

    Efficient cofermentation of d-glucose, d-xylose, and l-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the d-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved d-xylose and l-arabinose consumption in the presence of d-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented d-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the d-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the d-xylose proton-symporter (cac1345), d-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of d-glucose, d-xylose, and l-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels. PMID:21926197

  3. Phosphoenolpyruvate-dependent phosphorylation of sucrose by Clostridium tyrobutyricum ZJU 8235: evidence for the phosphotransferase transport system.

    PubMed

    Jiang, Ling; Cai, Jin; Wang, Jufang; Liang, Shizhong; Xu, Zhinan; Yang, Shang-Tian

    2010-01-01

    The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the first definitive evidence for phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity in butyric acid-producing bacteria. Glucose was utilized preferentially to sucrose when both substrates were present in the medium. The PEP-dependent sucrose: PTS was induced by growing C. tyrobutyricum on sucrose (but not glucose) as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that both soluble and membrane components were required for bioactivity. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose-grown cultures. Based on these findings, a pathway of sucrose metabolism in this organism was proposed that includes the forming of sucrose-6-phosphate via the PTS and its further degradation into glucose-6-phosphate and fructose-6-phosphate. PMID:19726178

  4. Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by sup 15 N NMR spectroscopy

    SciTech Connect

    van Dijk, A.A.; de Lange, L.C.M.; Robillard, G.T. ); Bachovchin, W.W. )

    1990-09-04

    The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with {sup 15}N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with {sup 15}N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N{delta}1 and N{epsilon}2 positions or at only the N{delta}1 position. {sup 15}N NMR spectra of two synthesized model compound, phosphoimidazole and phosphomethylimidazole, were also recorded. The authors show that, prior to phosphorylation, the protonated His15 N{epsilon}2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N{delta}1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N{delta}1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.

  5. Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by 15N NMR spectroscopy.

    PubMed

    van Dijk, A A; de Lange, L C; Bachovchin, W W; Robillard, G T

    1990-09-01

    The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with 15N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with 15N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N delta 1 and N epsilon 2 positions or at only the N delta 1 position. 15N NMR spectra of two synthesized model compounds, phosphoimidazole and phosphomethylimidazole, were also recorded. We show that, prior to phosphorylation, the protonated His15 N epsilon 2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N delta 1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N delta 1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed. PMID:2261470

  6. Complete Sucrose Metabolism Requires Fructose Phosphotransferase Activity in Corynebacterium glutamicum To Ensure Phosphorylation of Liberated Fructose

    PubMed Central

    Dominguez, H.; Lindley, N. D.

    1996-01-01

    Sucrose uptake by Corynebacterium glutamicum involves a phosphoenolpyruvate-dependent sucrose phosphotransferase (PTS), but in the absence of fructokinase, further metabolism of the liberated fructose requires efflux of the fructose and reassimilation via the fructose PTS. Mutant strains lacking detectable fructose-transporting PTS activity accumulated fructose extracellularly but consumed sucrose at rates comparable to those of the wild-type strain. PMID:16535429

  7. Mapping of the gene specifying aminoglycoside 3'-phosphotransferase II on the Pseudomonas aeruginosa chromosome.

    PubMed Central

    Okii, M; Iyobe, S; Mitsuhashi, S

    1983-01-01

    We examined the aminoglycoside inactivation enzymes in Pseudomonas aeruginosa strains, seven clinical isolates and seven laboratory strains without plasmids. All strains were found to possess the enzyme aminoglycoside 3'-phosphotransferase II [APH(3')-II]. We isolated an APH(3')-II-deficient mutant from a PAO strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. By plasmid (FP5 or R68.45)-mediated conjugation, we determined the locus of the gene specifying the APH(3')-II between trp-6 and pro-82 on the PAO chromosome and designated this gene aphA. It was concluded that the intrinsic resistance of P. aeruginosa to kanamycins, neomycins, paromomycins, ribostamycin, and butirosins was due to this newly determined gene. PMID:6307974

  8. Genome-wide analysis suggests divergent evolution of lipid phosphotases/phosphotransferase genes in plants.

    PubMed

    Wang, Peng; Chen, Zhenxi; Kasimu, Rena; Chen, Yinhua; Zhang, Xiaoxiao; Gai, Jiangtao

    2016-08-01

    Genes of the LPPT (lipid phosphatase/phosphotransferase) family play important roles in lipid phosphorous transfer and triacylglycerol accumulation in plants. To provide overviews of the plant LPPT family and their overall relationships, here we carried out genome-wide identifications and analyses of plant LPPT family members. A total of 643 putative LPPT genes were identified from 48 sequenced plant genomes, among which 205 genes from 14 plants were chosen for further analyses. Plant LPPT genes belonged to three distinctive groups, namely the LPT (lipid phosphotransfease), LPP (lipid phosphatase), and pLPP (plastidic lipid phosphotransfease) groups. Genes of the LPT group could be further partitioned into three groups, two of which were only identified in terrestrial plants. Genes in the LPP and pLPP groups experienced duplications in early stages of plant evolution. Among 17 Zea mays LPPT genes, divergence of temporal-spatial expression patterns was revealed based on microarray data analysis. Peptide sequences of plant LPPT genes harbored different conserved motifs. A test of Branch Model versus One-ratio Model did not support significant selective pressures acting on different groups of LPPT genes, although quite different nonsynonymous evolutionary rates and selective pressures were observed. The complete picture of the plant LPPT family provided here should facilitate further investigations of plant LPPT genes and offer a better understanding of lipid biosynthesis in plants. PMID:27501416

  9. Efficient transformation and regeneration of transgenic cassava using the neomycin phosphotransferase gene as aminoglycoside resistance marker gene.

    PubMed

    Niklaus, Michael; Gruissem, Wilhelm; Vanderschuren, Hervé

    2011-01-01

    Cassava is one of the most important crops in the tropics. Its industrial use for starch and biofuel production is also increasing its importance for agricultural production in tropical countries. In the last decade cassava biotechnology has emerged as a valuable alternative to the breeding constraints of this highly heterozygous crop for improved trait development of cassava germplasm. Cassava transformation remains difficult and time-consuming because of limitations in selecting transgenic tissues and regeneration of transgenic plantlets. We have recently reported an efficient and robust cassava transformation protocol using the hygromycin phosphotransferase II (hptII) gene as selection marker and the aminoglycoside hygromycin at optimal concentrations to maximize the regeneration of transgenic plantlets. In the present work, we expanded the transformation protocol to the use of the neomycin phosphotransferase II (nptII) gene as selection marker. Several aminoglycosides compatible with the use of nptII were tested and optimal concentrations for cassava transformation were determined. Given its efficiency equivalent to hptII as selection marker with the described protocol, the use of nptII opens new possibilities to engineer transgenic cassava lines with multiple T-DNA insertions and to produce transgenic cassava with a resistance marker gene that is already deregulated in several commercial transgenic crops. PMID:22179195

  10. Coupling Physiology and Gene Regulation in Bacteria: The Phosphotransferase Sugar Uptake System Delivers the Signals

    NASA Astrophysics Data System (ADS)

    Stülke, Jörg; Hillen, Wolfgang

    In many bacteria a crucial link between metabolism and regulation of catabolic genes is based on the phosphotransferase sugar uptake system (PTS). We summarize the mechanisms of the signaling pathways originating from PTS and leading to regulation of transcription. A protein domain, called PTS regulation domain (PRD), is linked to many antiterminators and transcriptional activators and regulates their activity depending on its state of phosphorylation. Two sites can be phosphorylated in most PRDs: HPr-dependent modification at one site leads to activation while enzyme II dependent phosphorylation of the other site renders it inactive. In addition, PTS components are used to generate cofactors for regulators of transcription. The paradigm is the enzyme II dependent activity of adenylate cyclase determining the cyclic AMP level in Escherichia coli and thereby the activity of the catabolite activator protein. In many gram-positive bacteria catabolite repression is mediated by the catabolite control protein CcpA, which requires HPr Ser-46 phosphate as a cofactor to regulate transcription of catabolic genes. HPr Ser-46 phosphate is produced by HPr kinase, the activity of which is under metabolic control via the concentrations of glycolytic intermediates. These recent results establish a multifaceted regulatory role for PTS in addition to its well-established function in active sugar uptake.

  11. Distribution and Functions of Phosphotransferase System Genes in the Genome of the Lactic Acid Bacterium Oenococcus oeni

    PubMed Central

    Jamal, Zohra; Miot-Sertier, Cécile; Thibau, François; Dutilh, Lucie; Lonvaud-Funel, Aline; Ballestra, Patricia; Le Marrec, Claire

    2013-01-01

    Oenococcus oeni, the lactic acid bacterium primarily responsible for malolactic fermentation in wine, is able to grow on a large variety of carbohydrates, but the pathways by which substrates are transported and phosphorylated in this species have been poorly studied. We show that the genes encoding the general phosphotransferase proteins, enzyme I (EI) and histidine protein (HPr), as well as 21 permease genes (3 isolated ones and 18 clustered into 6 distinct loci), are highly conserved among the strains studied and may form part of the O. oeni core genome. Additional permease genes differentiate the strains and may have been acquired or lost by horizontal gene transfer events. The core pts genes are expressed, and permease gene expression is modulated by the nature of the bacterial growth substrate. Decryptified O. oeni cells are able to phosphorylate glucose, cellobiose, trehalose, and mannose at the expense of phosphoenolpyruvate. These substrates are present at low concentrations in wine at the end of alcoholic fermentation. The phosphotransferase system (PTS) may contribute to the perfect adaptation of O. oeni to its singular ecological niche. PMID:23524676

  12. Nucleotide and deduced amino acid sequences of the lacR, lacABCD, and lacFE genes encoding the repressor, tagatose 6-phosphate gene cluster, and sugar-specific phosphotransferase system components of the lactose operon of Streptococcus mutans.

    PubMed Central

    Rosey, E L; Stewart, G C

    1992-01-01

    The complete nucleotide sequences of lacRABCDF and partial nucleotide sequence of lacE from the lactose operon of Streptococcus mutans are presented. Comparison of the streptococcal lac determinants with those of Staphylococcus aureus and Lactococcus lactis indicate exceptional protein and nucleotide identity. The deduced polypeptides also demonstrate significant, but lower, sequence similarity with the corresponding lactose proteins of Lactobacillus casei. Additionally, LacR has sequence homology with the repressor (DeoR) of the Escherichia coli deoxyribonucleotide operon, while LacC is similar to phosphokinases (FruK and PfkB) from E. coli. The primary translation products of the lacRABCDFE genes are polypeptides of 251 (M(r) 28,713), 142 (M(r) 15,610), 171 (M(r) 18,950), 310 (M(r) 33,368), 325 (M(r) 36,495), 104 (M(r) 11,401), and 123 (NH2-terminal) amino acids, respectively. As inferred from their direct homology to the staphylococcal lac genes, these determinants would encode the repressor of the streptococcal lactose operon (LacR), galactose-6-phosphate isomerase (LacA and LacB), tagatose-6-phosphate kinase (LacC), tagatose-1,6-bisphosphate aldolase (LacD), and the sugar-specific components enzyme III-lactose (LacF) and enzyme II-lactose (LacE) of the S. mutans phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence encompassing the S. mutans lac promoter appears to contain repeat elements analogous to those of S. aureus, suggesting that repression and catabolite repression of the lactose operons may be similar in these organisms. Images PMID:1400164

  13. Comparative Genomic Analyses of the Bacterial Phosphotransferase System

    PubMed Central

    Barabote, Ravi D.; Saier, Milton H.

    2005-01-01

    We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer. PMID:16339738

  14. Deletion Mapping of the Genes Coding for HPr and Enzyme I of the Phosphoenolpyruvate: Sugar Phosphotransferase System in Salmonella typhimurium

    PubMed Central

    Cordaro, J. Christopher; Roseman, Saul

    1972-01-01

    Sugars transported by a bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) require two soluble proteins: HPr, a low-molecular-weight phosphate-carrier protein, and enzyme I. The structural genes coding for HPr (ptsH) and Enzyme I (ptsI) are shown to be cotransducible in Salmonella typhimurium. The gene order of this region of the Salmonella chromosome is cysA-trzA-ptsH-ptsI...(crr). A method for the isolation of trzA-pts deletion is described. One class of pts deletions extends through ptsH and into ptsI; a second class includes both ptsH and ptsI and extends into or through the crr gene. The crr gene either codes for or regulates the synthesis of a third PTS protein (factor III) which is sugar-specific. A hypothesis is presented for a mechanism of deletion formation. PMID:4562394

  15. Comparison of individual component deletions in a glucose-specific phosphotransferase system revealed their different applications

    PubMed Central

    Liang, Quanfeng; Zhang, Fengyu; Li, Yikui; Zhang, Xu; Li, Jiaojiao; Yang, Peng; Qi, Qingsheng

    2015-01-01

    The phosphoenolpyruvate-dependent glucose-specific phosphotransferase system (PTSGlc) is the main glucose uptake pathway in Escherichia coli that affects both substrate assimilation and metabolism leading to the product formation. In this study, the effect of single PTSGlc mutation on cell growth and substrate consumption was investigated by knocking out the genes involved in the phosphotransfer cascade of the PTSGlc. In addition, the distribution of the metabolites of mutants was analyzed. Each mutant was confirmed to have different adaptability in the presence of both glucose and xylose with different ratios, and a substrate mixture with high xylose content can be completely consumed in short time when the ptsI mutant is employed. Finally, ptsH deletion was for the first time applied for succinate production due to its well performance under anaerobic condition. Strain YL104H, in which ptsH was deleted, exhibited considerably increased succinate yield under both aerobic and anaerobic conditions. The succinate titer and overall productivity reached 511.11 mM and 1.01 g/L/h after 60 h during the whole-phase fermentation in a mineral salt medium. The present results demonstrated the glucose and xylose co-utilization efficiency and the product yield and productivity can be significantly improved if a suitable PTSGlc deletion mutant was selected. PMID:26285685

  16. A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression

    PubMed Central

    Millet, Yves A.; Chao, Michael C.; Sasabe, Jumpei; Davis, Brigid M.

    2015-01-01

    Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression of Vibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression of tcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulate tcpA expression, the screen yielded ptsI and ptsH, which encode the enzyme I (EI) and Hpr components of the V. cholerae phosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlc protein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression of tcpPH and aphAB, which themselves control expression of toxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that the V. cholerae PTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks. PMID:26056384

  17. Multiple sugar: phosphotransferase system permeases participate in catabolite modification of gene expression in Streptococcus mutans.

    PubMed

    Zeng, Lin; Burne, Robert A

    2008-10-01

    Streptococcus mutans is particularly well adapted for high-affinity, high-capacity catabolism of multiple carbohydrate sources. S. mutansenzyme II (EII(Lev)), a fructose/mannose permease encoded by the levDEFG genes, and fruA, which encodes a hydrolase that releases fructose from fructan polymers, are transcriptionally regulated by the LevQRST four-component signal transduction system. Here, we demonstrate that: (i) levDEFGX are co-transcribed and the levE/F intergenic region is required for optimal expression of levFGX; (ii) D-mannose is a potent inducer of the levD and fruA operons; (iii) CcpA regulates levD expression in a carbohydrate-specific manner; (iv) deletion of the genes for the fructose/mannose-EII enzymes of S. mutans (manL, fruI and levD) enhances levD expression; (v) repression of the LevQRST regulon by EII enzymes depends on the presence of their substrates and requires LevR, but not LevQST; and (vi) CcpA inhibits expression of the manL and fruI genes to indirectly control the LevQRST regulon. Further, the manL, ccpA, fruI/fruCD and levD gene products differentially exert control over the cellobiose and lactose operons. Collectively, the results reveal the existence of a global regulatory network in S. mutans that governs the utilization of non-preferred carbohydrates in response to the availability and source of multiple preferred carbohydrates. PMID:18699864

  18. Cloning, sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system, glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998.

    PubMed

    Qazi, P H; Johri, S; Verma, V; Khan, L; Qazi, G N

    2004-09-01

    A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation. PMID:15560368

  19. In70 of plasmid pAX22, a bla(VIM-1)-containing integron carrying a new aminoglycoside phosphotransferase gene cassette.

    PubMed

    Riccio, M L; Pallecchi, L; Fontana, R; Rossolini, G M

    2001-04-01

    An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides. PMID:11257042

  20. In70 of Plasmid pAX22, a blaVIM-1-Containing Integron Carrying a New Aminoglycoside Phosphotransferase Gene Cassette

    PubMed Central

    Riccio, Maria Letizia; Pallecchi, Lucia; Fontana, Roberta; Rossolini, Gian Maria

    2001-01-01

    An Achromobacter xylosoxydans strain showing broad-spectrum resistance to β-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-β-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a blaVIM-1 determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the blaVIM-1 gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3′)-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3′)-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5′ and 3′ conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3′ conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most β-lactams and aminoglycosides. PMID:11257042

  1. The glycolytic genes pfk and pyk from Lactobacillus casei are induced by sugars transported by the phosphoenolpyruvate:sugar phosphotransferase system and repressed by CcpA.

    PubMed

    Viana, Rosa; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

    2005-09-01

    In Lactobacillus casei BL23, phosphofructokinase activity was higher in cells utilizing sugars transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphofructokinase gene (pfk) was cloned from L. casei and shown to be clustered with the gene encoding pyruvate kinase (pyk). pfk and pyk genes are cotranscribed and induced upon growth on sugars transported by the PTS. Contrarily to the model proposed for Lactococcus lactis, where the global catabolite regulator protein (CcpA) is involved in PTS-induced transcription of pfk and pyk, a ccpA mutation resulted in a slight increase in pfk-pyk expression in L. casei. This weak regulation was evidenced by CcpA binding to a region of the pfk-pyk promoter which contained two cre sequences significantly deviated from the consensus. The PTS induction of pfk-pyk seems to be counteracted by the CcpA-mediated repression. Our results suggest that the need to accommodate the levels of pfk-pyk mRNA to the availability of sugars is fulfilled in L. casei by a PTS/CcpA-mediated signal transduction different from L. lactis. PMID:16075200

  2. Functional genomics to discover antibiotic resistance genes: The paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14.

    PubMed

    Telke, Amar A; Rolain, Jean-Marc

    2015-12-01

    Shewanella algae MARS 14 is a colistin-resistant clinical isolate retrieved from bronchoalveolar lavage of a hospitalised patient. A functional genomics strategy was employed to discover the molecular support for colistin resistance in S. algae MARS 14. A pZE21 MCS-1 plasmid-based genomic expression library was constructed in Escherichia coli TOP10. The estimated library size was 1.30×10(8) bp. Functional screening of colistin-resistant clones was carried out on Luria-Bertani agar containing 8 mg/L colistin. Five colistin-resistant clones were obtained after complete screening of the genomic expression library. Analysis of DNA sequencing results found a unique gene in all selected clones. Amino acid sequence analysis of this unique gene using the Integrated Microbial Genomes (IMG) and KEGG databases revealed that this gene encodes ethanolamine phosphotransferase (EptA, or so-called PmrC). Reverse transcription PCR analysis indicated that resistance to colistin in S. algae MARS 14 was associated with overexpression of EptA (27-fold increase), which plays a crucial role in the arrangement of outer membrane lipopolysaccharide. PMID:26498987

  3. Novel activator of mannose-specific phosphotransferase system permease expression in Listeria innocua, identified by screening for pediocin AcH resistance.

    PubMed

    Xue, Junfeng; Hunter, Ian; Steinmetz, Tori; Peters, Adam; Ray, Bibek; Miller, Kurt W

    2005-03-01

    To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 microg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EIItMan, which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by sigma54 factor and ManR (a sigma54-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN (sigma54) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators. PMID:15746330

  4. Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases.

    PubMed

    Lindner, Steffen N; Seibold, Gerd M; Henrich, Alexander; Krämer, Reinhard; Wendisch, Volker F

    2011-06-01

    Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield. PMID:21478323

  5. Pyrophosphate-Dependent Fructose-6-Phosphate 1-Phosphotransferase Induction and Attenuation of Hsp Gene Expression during Endosperm Modification in Quality Protein Maize1[C][W][OA

    PubMed Central

    Guo, Xiaomei; Ronhovde, Kyla; Yuan, Lingling; Yao, Bo; Soundararajan, Madhavan P.; Elthon, Thomas; Zhang, Chi; Holding, David R.

    2012-01-01

    Quality Protein Maize (QPM) is a hard-endosperm version of the high-lysine opaque2 (o2) maize (Zea mays) mutant, but the genes involved in modification of the soft o2 endosperm are largely unknown. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the ATP-independent conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. We found a large increase in transcript and protein levels of the α-regulatory subunit of PFP (PFPα) in QPM endosperm. In vitro enzyme assays showed a significant increase in forward PFP activity in developing endosperm extracts of QPM relative to the wild type and o2. An expressed retrogene version of PFPα of unknown function that was not up-regulated in QPM was also identified. The elevated expression levels of a number of ATP-requiring heat shock proteins (Hsps) in o2 endosperm are ameliorated in QPM. PFPα is also coinduced with Hsps in maize roots in response to heat, cold, and the unfolded protein response stresses. We propose that reduced ATP availability resulting from the generalized Hsp response in addition to the reduction of pyruvate, orthophosphate dikinase activity in o2 endosperm is compensated in part by increased PFP activity in QPM. PMID:22158678

  6. An esterase gene from Lactobacillus casei cotranscribed with genes encoding a phosphoenolpyruvate:sugar phosphotransferase system and regulated by a LevR-like activator and sigma54 factor.

    PubMed

    Yebra, María J; Viana, Rosa; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar

    2004-01-01

    A new esterase-encoding gene was found in the draft genome sequence of Lactobacillus casei BL23 (CECT5275). It is located in an operon together with genes encoding the EIIA, EIIB, EIIC, and EIID proteins of a mannose class phosphoenolpyruvate:sugar phosphotransferase system. After overproduction in Escherichia coli and purification, the esterase could hydrolyze acetyl sugars, hence the operon was named esu for esterase-sugar uptake genes. Upstream of the genes encoding the EII components (esuABCD) and the esterase (esuE), two genes transcribed in the opposite sense were found which encode a Bacillus subtilis LevR-like transcriptional activator (esuR) and a sigma54-like transcriptional factor (rpoN). As compared with the wild-type strain, elevated fructose phosphorylation was detected in L. casei mutants constitutively expressing the esu operon. However, none of the many sugars tested could induce the esu operon. The fact that EsuE exhibits esterase activity on acetyl sugars suggests that this operon could be involved in the uptake and metabolism of esterified sugars. Expression of the esu operon is similar to that of the B. subtilis lev operon: it contains a -12,-24 consensus promoter typical of sigma54-regulated genes, and EsuR and RpoN are essential for its transcription which is negatively regulated by EIIB(Esu). The esuABCDE transcription unit represents the first sigma54-regulated operon in lactobacilli. Furthermore, replacement of His852 in the phosphoenolpyruvate:sugar phosphotransferase system regulation domain II of EsuR with Ala indicated that the transcription activator function of EsuR is inhibited by EIIB(Esu)-mediated phosphorylation at His852. PMID:15925903

  7. Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli.

    PubMed Central

    Lai, X; Ingram, L O

    1993-01-01

    Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-beta-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for

  8. Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum.

    PubMed

    Ikeda, Masato; Mizuno, Yuta; Awane, Shin-ichi; Hayashi, Masahiro; Mitsuhashi, Satoshi; Takeno, Seiki

    2011-05-01

    Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS sugars: glucose, fructose, and sucrose. However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WTΔptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using Escherichia coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WTΔptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2 abolished growth on glucose, whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WTΔptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157

  9. The Phosphotransferase System of Streptomyces coelicolor Is Biased for N-Acetylglucosamine Metabolism

    PubMed Central

    Nothaft, Harald; Dresel, Dagmar; Willimek, Andreas; Mahr, Kerstin; Niederweis, Michael; Titgemeyer, Fritz

    2003-01-01

    Mutation of the crr-ptsI gene locus revealed that Streptomyces coelicolor uses the phosphotransferase system (PTS) for N-acetylglucosamine uptake. crr, ptsI, and ptsH, which encode the three general PTS phosphotransferases, are induced by N-acetylglucosamine but not by other PTS substrates. Thus, the S. coelicolor PTS is biased for N-acetylglucosamine utilization, a novel feature that distinguishes this PTS from others. PMID:14617669

  10. Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris.

    PubMed Central

    Inamine, J M; Lee, L N; LeBlanc, D J

    1986-01-01

    Lac+ plasmid DNA from Streptococcus cremoris H2 was subcloned with an Escherichia coli vector on a 3.5-kilobase-pair PstI-AvaI fragment. Genetic analysis of the cloned DNA was possible because linear Lac+ DNA fragments were productive in the S. sanguis transformation system. Complementation of S. sanguis Lac-mutants showed that the 3.5-kilobase-pair fragment included the structural gene for 6-phospho-beta-D-galactosidase and either enzyme II-lac or factor III-lac of the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system. Expression of the S. cremoris-like 40,000-dalton 6-phospho-beta-D-galactosidase in S. sanguis Lac+ transformants, rather than the 52,000-dalton wild-type S. sanguis enzyme, demonstrated the occurrence of gene replacement and not gene repair. The evidence supports chromosomal integration as the mechanism by which S. sanguis Lac- recipients are converted to a Lac+ phenotype after transformation with Lac+ DNA. Southern blot data suggest that the Lac+ DNA does not reside on a transposon, but that integration always occurs within a specific HincII fragment of the recipient chromosome. Hybridization experiments demonstrate homology between the S. cremoris Lac+ DNA and cellular DNA from Lac+ strains of Streptococcus lactis, S. mutans, S. faecalis, and S. sanguis. Images PMID:3091581

  11. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    SciTech Connect

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-04-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

  12. Purification, Crystallization And Preliminary X-Ray Analysis of Aminoglycoside-2 ''-Phosphotransferase-Ic [APH(2 '')-Ic] From Enterococcus Gallinarum

    SciTech Connect

    Byrnes, L.J.; Badarau, A.; Vakulenko, S.B.; Smith, C.A.; /SLAC, SSRL

    2009-04-30

    Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2{double_prime}-phosphotransferase-Ic [APH(2{double_prime})-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2{double_prime})-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris-HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 {angstrom}, {beta} = 108.8{sup o}. X-ray diffraction data were collected to approximately 2.15 {angstrom} resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  13. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

    PubMed Central

    Postma, P W; Lengeler, J W; Jacobson, G R

    1993-01-01

    Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the

  14. Phosphotransferase-mediated transport of the osmolyte 2-O-alpha-mannosyl-D-glycerate in Escherichia coli occurs by the product of the mngA (hrsA) gene and is regulated by the mngR (farR) gene product acting as repressor.

    PubMed

    Sampaio, Maria-Manuel; Chevance, Fabienne; Dippel, Renate; Eppler, Tanja; Schlegel, Anja; Boos, Winfried; Lu, Ying-Jie; Rock, Charles O

    2004-02-13

    2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake is mediated by the P-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 microm, and the Vmax was 0.65 nmol/min/10(9) cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with alpha-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system. PMID:14645248

  15. Molecular characteristics of phosphoenolpyruvate: mannose phosphotransferase system in Streptococcus bovis.

    PubMed

    Asanuma, Narito; Yoshii, Takahiro; Hino, Tsuneo

    2004-07-01

    To elucidate the regulatory mechanism of catabolite control in Streptococcus bovis, we investigated the molecular properties and gene expression of the mannose-specific phosphoenolpyruvate (PEP)-dependent sugar: phosphotransferase system (PTS). The mannose PTS gene cluster (man) was found to comprise a gene encoding enzyme (E) II AB (manL) and genes encoding EIIC (manM), EIID (manN), and a putative regulator (manO). The gene cluster (man operon) was transcribed from one transcriptional start site, which was located 40 bp upstream of the manL start codon. However, two transcriptional start sites were found between manN and manO in primer extension analysis, and the manO may be transcribed independently from the man operon. The man operon and manO were constitutively transcribed without being affected by culture conditions, such as the sugar supplied (glucose, galactose, fructose, maltose, lactose, sucrose, or mannose), growth rate, or pH. PMID:15297922

  16. Regulation of Lactobacillus casei sorbitol utilization genes requires DNA-binding transcriptional activator GutR and the conserved protein GutM.

    PubMed

    Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J

    2008-09-01

    Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTS(Gut)). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIB(Gat) domain) and a mannitol/fructose-specific EIIA-like domain (EIIA(Mtl) domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBC(Gut) negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

  17. Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM▿

    PubMed Central

    Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J.

    2008-01-01

    Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

  18. Transferable amikacin resistance in Acinetobacter spp. due to a new type of 3'-aminoglycoside phosphotransferase.

    PubMed Central

    Lambert, T; Gerbaud, G; Courvalin, P

    1988-01-01

    Acinetobacter baumannii BM2580 resistant to kanamycin and structurally related antibiotics, including amikacin, was isolated from a clinical specimen. A phosphocellulose paper-binding assay and DNA annealing studies indicated that resistance to aminoglycosides in BM2580 was due to synthesis of a new type of 3'-aminoglycoside phosphotransferase. The gene conferring resistance to kanamycin-amikacin in this strain was carried by a 63-kilobase plasmid, pIP1841, self-transferable to A. baumannii, A. haemolyticus, and A. lwoffii but not to Escherichia coli. The aminoglycoside resistance gene of pIP1841 was cloned in E. coli, where it was expressed. Images PMID:2831812

  19. A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria

    PubMed Central

    Spanogiannopoulos, Peter; Waglechner, Nicholas; Koteva, Kalinka; Wright, Gerard D.

    2014-01-01

    Many environmental bacteria are multidrug-resistant and represent a reservoir of ancient antibiotic resistance determinants, which have been linked to genes found in pathogens. Exploring the environmental antibiotic resistome, therefore, reveals the diversity and evolution of antibiotic resistance and also provides insight into the vulnerability of clinically used antibiotics. In this study, we describe the identification of a highly conserved regulatory motif, the rifampin (RIF) -associated element (RAE), which is found upstream of genes encoding RIF-inactivating enzymes from a diverse collection of actinomycetes. Using gene expression assays, we confirmed that the RAE is involved in RIF-responsive regulation. By using the RAE as a probe for new RIF-associated genes in several actinomycete genomes, we identified a heretofore unknown RIF resistance gene, RIF phosphotransferase (rph). The RPH enzyme is a RIF-inactivating phosphotransferase and represents a new protein family in antibiotic resistance. RPH orthologs are widespread and found in RIF-sensitive bacteria, including Bacillus cereus and the pathogen Listeria monocytogenes. Heterologous expression and in vitro enzyme assays with purified RPHs from diverse bacterial genera show that these enzymes are capable of conferring high-level resistance to a variety of clinically used rifamycin antibiotics. This work identifies a new antibiotic resistance protein family and reinforces the fact that the study of resistance in environmental organisms can serve to identify resistance elements with relevance to pathogens. PMID:24778229

  20. Identification of a glucose-mannose phosphotransferase system in Clostridium beijerinckii.

    PubMed

    Essalem, Mohemed E E; Mitchell, Wilfrid J

    2016-04-01

    Effective uptake of fermentable substrates is a fundamentally important aspect of any fermentation process. The solventogenic bacterium Clostridium beijerinckii is noted for its ability to ferment a wide range of carbohydrates, yet few of its sugar transport systems have been characterized. In common with other anaerobes, C. beijerinckii shows a marked dependence on the PEP-dependent phosphotransferase system (PTS) for sugar accumulation. In this study, the gene cbe0751 encoding the sugar-specific domains of a phosphotransferase belonging to the glucose family was cloned into an Escherichia coli strain lacking the ability to take up and phosphorylate glucose. Transformants gained ability to ferment glucose, and also mannose, and further analysis of a selected transformant demonstrated that it could take up and phosphorylate glucose, confirming that cbe0751 encodes a glucose PTS which also recognizes mannose as a substrate. RT-PCR analysis showed that cbe0751 was expressed in cultures grown on both substrates, but also to varying extents during growth on some other carbon sources. Although analogue inhibition studies suggested that Cbe0751 is not the only glucose PTS in C. beijerinckii, this system should nevertheless be regarded as a potential target for metabolic engineering to generate a strain showing improved sugar fermentation properties. PMID:26940293

  1. Dual Substrate Specificity of an N-Acetylglucosamine Phosphotransferase System in Clostridium beijerinckii

    PubMed Central

    Al Makishah, Naief H.

    2013-01-01

    The solventogenic clostridia have a considerable capacity to ferment carbohydrate substrates with the production of acetone and butanol, making them attractive organisms for the conversion of waste materials to valuable products. In common with other anaerobes, the clostridia show a marked dependence on the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) to accumulate sugars and sugar derivatives. In this study, we demonstrate that extracts of Clostridium beijerinckii grown on N-acetylglucosamine (GlcNAc) exhibit PTS activity for the amino sugar. The PTS encoded by the divergent genes cbe4532 (encoding the IIC and IIB domains) and cbe4533 (encoding a IIA domain) was shown to transport and phosphorylate GlcNAc and also glucose. When the genes were recombined in series under the control of the lac promoter in pUC18 and transformed into a phosphotransferase mutant (nagE) of Escherichia coli lacking GlcNAc PTS activity, the ability to take up and ferment GlcNAc was restored, and extracts of the transformant showed PEP-dependent phosphorylation of GlcNAc. The gene products also complemented an E. coli mutant lacking glucose PTS activity but were unable to complement the same strain for PTS-dependent mannose utilization. Both GlcNAc and glucose induced the expression of cbe4532 and cbe4533 in C. beijerinckii, and consistent with this observation, extracts of cells grown on glucose exhibited PTS activity for GlcNAc, and glucose did not strongly repress utilization of GlcNAc by growing cells. On the basis of the phylogeny and function of the encoded PTS, we propose that the genes cbe4532 and cbe4533 should be designated nagE and nagF, respectively. PMID:23995920

  2. Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase

    PubMed Central

    Beassoni, Paola R.; Gallarato, Lucas A.; Boetsch, Cristhian; Garrido, Mónica N.; Lisa, Angela T.

    2015-01-01

    Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4+ is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or NH4+ ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity. PMID:26576296

  3. Identification of the Operon for the Sorbitol (Glucitol) Phosphoenolpyruvate:Sugar Phosphotransferase System in Streptococcus mutans

    PubMed Central

    Boyd, David A.; Thevenot, Tracy; Gumbmann, Markus; Honeyman, Allen L.; Hamilton, Ian R.

    2000-01-01

    Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol P-enolpyruvate phosphotransferase transport system (PTS) of Streptococcus mutans LT11. The first gene, srlD, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by srlR, coding for a transcriptional regulator; srlM, coding for a putative activator; and the srlA, srlE, and srlB genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the srlD gene is found after the genes coding for the EII components; thus, the location of the gene in S. mutans is unique. The SrlR protein is similar to several transcriptional regulators found in Bacillus spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865–874, 1998), and its gene overlaps the srlM gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria. PMID:10639465

  4. Variability of a glucose phosphotransferase system permease in Mycoplasma mycoides subsp. mycoides Small Colony.

    PubMed

    Gaurivaud, Patrice; Persson, Anja; Grand, Dominique Le; Westberg, Joakim; Solsona, Michel; Johansson, Karl-Erik; Poumarat, François

    2004-12-01

    Intraclonal antigenic variation in pathogenic mycoplasma species is considered an important feature of host-pathogen interaction. Such intraclonal protein variation was observed for the interaction of Mycoplasma mycoides subsp. mycoides Small Colony, the agent of contagious bovine pleuropneumonia, with mAb 3F3. Colony immunostaining allows the definition of 3F3 ON- and 3F3 OFF-type variants, which revert at low frequency. Targets of mAb 3F3 were shown to be surface located, and resided on multiple polypeptides in the 58-68 kDa size range. Phage display and a genomic database were combined to determine the gene encoding the proteins recognized by mAb 3F3. A gene encoding the putative permease of the glucose phosphotransferase system was identified. Genome sequence analysis of strain PG1 revealed two highly similar copies of this gene, resulting from duplication of the chromosomal region carrying the gene. Southern blot analysis demonstrated the presence of this duplication in almost every African strain tested, but not in European strains. DNA analysis revealed that ON/OFF switching is governed by a base substitution occurring upstream of the coding region for the 3F3 epitope. This event generates a stop codon that results in the premature termination of the PtsG protein. PMID:15583154

  5. Rifampin phosphotransferase is an unusual antibiotic resistance kinase.

    PubMed

    Stogios, Peter J; Cox, Georgina; Spanogiannopoulos, Peter; Pillon, Monica C; Waglechner, Nicholas; Skarina, Tatiana; Koteva, Kalinka; Guarné, Alba; Savchenko, Alexei; Wright, Gerard D

    2016-01-01

    Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds. PMID:27103605

  6. Rifampin phosphotransferase is an unusual antibiotic resistance kinase

    PubMed Central

    Stogios, Peter J.; Cox, Georgina; Spanogiannopoulos, Peter; Pillon, Monica C.; Waglechner, Nicholas; Skarina, Tatiana; Koteva, Kalinka; Guarné, Alba; Savchenko, Alexei; Wright, Gerard D.

    2016-01-01

    Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds. PMID:27103605

  7. Some properties of rat-liver glucose--adenosine triphosphate phosphotransferases.

    PubMed

    McLean, P; Brown, J

    1966-09-01

    In normal rat liver hexokinase (EC 2.7.1.1) activity usually accounts for not more than 30% of the total glucose-ATP phosphotransferase activity, the remainder being due to glucokinase (EC 2.7.1.2). In the present work it was found that in normal rat liver the relative activities of these two enzymes were occasionally very different from those usually found even though the total glucose-ATP phosphotransferase activity was within the normal range. In some cases almost the entire glucose-ATP phosphotransferase was accounted for by the low-K(m) enzyme hexokinase. Some properties of this enzyme system are reported. It is suggested that this shift in favour of the low-K(m) enzyme without change in the total glucose-ATP phosphotransferase activity may represent a regulatory mechanism. PMID:5969293

  8. Role of Secondary Transporters and Phosphotransferase Systems in Glucose Transport by Oenococcus oeni ▿

    PubMed Central

    Kim, Ok Bin; Richter, Hanno; Zaunmüller, Tanja; Graf, Sabrina; Unden, Gottfried

    2011-01-01

    Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose- or fructose-grown bacteria catalyzed uptake of [14C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a high- and low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, Δp. Expression of the genes OEOE_0819 and OEOE_1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably Δp dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE_0464 [0464] for the permease) with similarity to the fructose- and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH. PMID:22020640

  9. Lcp1 Is a Phosphotransferase Responsible for Ligating Arabinogalactan to Peptidoglycan in Mycobacterium tuberculosis

    PubMed Central

    Harrison, James; Lloyd, Georgina; Joe, Maju; Lowary, Todd L.; Reynolds, Edward; Walters-Morgan, Hannah; Bhatt, Apoorva; Lovering, Andrew; Besra, Gurdyal S.

    2016-01-01

    ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The main structural elements of the cell wall consist of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are covalently attached to a complex polysaccharide, arabinogalactan (AG), via a unique α-l-rhamnopyranose–(1→3)-α-d-GlcNAc-(1→P) linker unit. While the molecular genetics associated with PG and AG biosynthetic pathways have been largely delineated, the mechanism by which these two major pathways converge has remained elusive. In Gram-positive organisms, the LytR-CpsA-Psr (LCP) family of proteins are responsible for ligating cell wall teichoic acids to peptidoglycan, through a linker unit that bears a striking resemblance to that found in mycobacterial arabinogalactan. In this study, we have identified Rv3267 as a mycobacterial LCP homolog gene that encodes a phosphotransferase which we have named Lcp1. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is capable of ligating AG to PG in a cell-free radiolabeling assay. PMID:27486192

  10. Glucose Transporter Mutants of Escherichia coli K-12 with Changes in Substrate Recognition of IICBGlc and Induction Behavior of the ptsG Gene

    PubMed Central

    Zeppenfeld, Tim; Larisch, Christina; Lengeler, Joseph W.; Jahreis, Knut

    2000-01-01

    In Escherichia coli K-12, the major glucose transporter with a central role in carbon catabolite repression and in inducer exclusion is the phosphoenolpyruvate-dependent glucose:phosphotransferase system (PTS). Its membrane-bound subunit, IICBGlc, is encoded by the gene ptsG; its soluble domain, IIAGlc, is encoded by crr, which is a member of the pts operon. The system is inducible by d-glucose and, to a lesser degree, by l-sorbose. The regulation of ptsG transcription was analyzed by testing the induction of IICBGlc transporter activity and of a single-copy Φ(ptsGop-lacZ) fusion. Among mutations found to affect directly ptsG expression were those altering the activity of adenylate cyclase (cyaA), the repressor DgsA (dgsA; also called Mlc), the general PTS proteins enzyme I (ptsI) and histidine carrier protein HPr (ptsH), and the IIAGlc and IIBGlc domains, as well as several authentic and newly isolated UmgC mutations. The latter, originally thought to map in the repressor gene umgC outside the ptsG locus, were found to represent ptsG alleles. These affected invariably the substrate specificity of the IICBGlc domain, thus allowing efficient transport and phosphorylation of substrates normally transported very poorly or not at all by this PTS. Simultaneously, all of these substrates became inducers for ptsG. From the analysis of the mutants, from cis-trans dominance tests, and from the identification of the amino acid residues mutated in the UmgC mutants, a new regulatory mechanism involved in ptsG induction is postulated. According to this model, the phosphorylation state of IIBGlc modulates IICGlc which, directly or indirectly, controls the repressor DgsA and hence ptsG expression. By the same mechanism, glucose uptake and phosphorylation also control the expression of the pts operon and probably of all operons controlled by the repressor DgsA. PMID:10913077

  11. Single-cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli.

    PubMed

    Westermayer, Sonja A; Fritz, Georg; Gutiérrez, Joaquín; Megerle, Judith A; Weißl, Mira P S; Schnetz, Karin; Gerland, Ulrich; Rädler, Joachim O

    2016-05-01

    The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution. Using time-lapse microscopy in perfused microchannels, we analyzed the competition between N-acetyl-glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co-expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co-expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner-take-all behaviour and stochastic co-expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions. PMID:26784570

  12. The global regulatory system Csr senses glucose through the phosphoenolpyruvate: carbohydrate phosphotransferase system.

    PubMed

    Pérez-Morales, Deyanira; Bustamante, Víctor H

    2016-02-01

    A novel connection between two regulatory systems controlling crucial biological processes in bacteria, the carbon storage regulator (Csr) system and the glucose-specific phosphotransferase system (PTS), is reported by Leng et al. in this issue. This involves the interaction of unphosphorylated EIIA(Glc), a component of the glucose-specific PTS, with the CsrD protein, which accelerates the decay of the CsrB and CsrC small RNAs via RNase E in Escherichia coli. As unphosphorylated EIIA(G) (lc) is generated in the presence of glucose, the PTS thus acts as a sensor of glucose for the Csr system. Interestingly, another pathway can operate for communication between the Csr system and the glucose-specific PTS. The absence of glucose generates phosphorylated EIIA(Glc) , which activates the enzyme adenylate cyclase to produce cyclic adenosine monophosphate (cAMP) that, in turn, binds to the regulator cAMP receptor protein (CRP). Leng et al. show that the complex cAMP-CRP modestly reduces CsrB decay independently of CsrD. On the other hand, a previous study indicates that the complex cAMP-CRP positively regulates the transcription of CsrB and CsrC in Salmonella enterica. Therefore, EIIA(G) (lc) could work as a molecular switch that regulates the activity of the Csr system, in response to its phosphorylation state determined by the presence or absence of glucose, in order to control gene expression. PMID:26593223

  13. Control of Transposon-mediated Directed Mutation by the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System

    PubMed Central

    Saier, Milton H.; Zhang, Zhongge

    2015-01-01

    The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances, it also regulates mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutation have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of Insertion Sequence-5 (IS5) hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing growth of wild type cells with glycerol as a carbon source in the presence of non-metabolizable glucose analogues which normally block glycerol utilization. The sugar transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cyclic AMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated. PMID:26159081

  14. Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli

    PubMed Central

    Somavanshi, Rahul; Ghosh, Bhaswar; Sourjik, Victor

    2016-01-01

    The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in Escherichia coli and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in E. coli, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy. PMID:27557415

  15. Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli.

    PubMed

    Somavanshi, Rahul; Ghosh, Bhaswar; Sourjik, Victor

    2016-08-01

    The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in Escherichia coli and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in E. coli, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy. PMID:27557415

  16. Sugar transport by the bacterial phosphotransferase system. In vivo regulation of lactose transport in Escherichia coli by IIIGlc, a protein of the phosphoenolpyruvate:glycose phosphotransferase system.

    PubMed

    Mitchell, W J; Saffen, D W; Roseman, S

    1987-11-25

    Escherichia coli and Salmonella typhimurium preferentially utilize sugar substrates of the phosphoenol-pyruvate:glycose phosphotransferase system (PTS) when the growth medium also contains other sugars. This phenomenon, diauxic growth, is regulated by the crr gene, which encodes the PTS protein IIIGlc (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 16241-16253). We have proposed that non-PTS permeases are regulated by their interaction with IIIGlc, and in vitro studies from other laboratories have provided support for this model, but the in vivo effects of excess IIIGlc are not known. In the present studies, transformed cells that overproduced IIIGlc 2- and 10-fold, respectively, were constructed from a pts+ strain of E. coli and plasmids containing the crr gene. In the 2-fold overproducer, fermentation of, and growth on the non-PTS carbohydrates glycerol, lactose, maltose, and melibiose was generally more sensitive to the glucose analogue methyl-alpha-D-glucopyranoside than in a control strain containing normal levels of IIIGlc. In addition, inhibition of lactose permease activity by methyl-alpha-glucoside (inducer exclusion) was more effective in the 2-fold overproducer than in the control strain, particularly when the permease activity was high. The 10-fold IIIGlc overproducing strain had a requirement for the amino acids methionine, isoleucine, leucine, and valine that may or may not be related to the increased concentration of IIIGlc. Fermentation of non-PTS carbohydrates was also poor in the latter strain. Finally, lactose permease activity was 50% of that in control cells containing the same levels of beta-galactosidase, and the lactose permease activity in the IIIGlc overproducer was reduced to an extremely low level in the presence of methyl alpha-glucoside. Thus there is an inverse relationship between the cellular concentration of IIIGlc and the ability to metabolize non-PTS substrates. The results are consistent with

  17. Phosphotransferase system-mediated glucose uptake is repressed in phosphoglucoisomerase-deficient Corynebacterium glutamicum strains.

    PubMed

    Lindner, Steffen N; Petrov, Dimitar P; Hagmann, Christian T; Henrich, Alexander; Krämer, Reinhard; Eikmanns, Bernhard J; Wendisch, Volker F; Seibold, Gerd M

    2013-04-01

    Corynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of C. glutamicum amino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted the pgi gene in the type strain C. glutamicum ATCC 13032. The resulting strain, C. glutamicum Δpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake in C. glutamicum Δpgi. Furthermore, Northern blot analyses revealed that expression of ptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimized l-lysine production in the model strain C. glutamicum DM1729 by deletion of pgi and overexpression of plasmid-encoded ptsG. l-Lysine yields and productivity with C. glutamicum Δpgi(pBB1-ptsG) were significantly higher than those with C. glutamicum Δpgi(pBB1). These results show that ptsG overexpression is required to overcome the repressed activity of PTS-mediated glucose uptake in pgi-deficient C. glutamicum strains, thus enabling efficient as well as fast l-lysine production. PMID:23396334

  18. The phosphotransferase system of Lactobacillus casei: regulation of carbon metabolism and connection to cold shock response.

    PubMed

    Monedero, Vicente; Mazé, Alain; Boël, Grégory; Zúñiga, Manuel; Beaufils, Sophie; Hartke, Axel; Deutscher, Josef

    2007-01-01

    Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium possesses numerous carbohydrate transport systems probably reflecting its capacity to proliferate under varying environmental conditions. Many carbohydrate transporters belong to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but all different kinds of non-PTS transporters are present as well and their substrates are known in a few cases. In L. casei regulation of carbohydrate transport and carbon metabolism is mainly achieved by PTS proteins. Carbon catabolite repression (CCR) is mediated via several mechanisms, including the major P-Ser-HPr/catabolite control protein A (CcpA)-dependent mechanism. Catabolite response elements, the target sites for the P-Ser-HPr/CcpA complex, precede numerous genes and operons. PTS regulation domain-containing antiterminators and transcription activators are also present in both L. casei strains. Their activity is usually controlled by two PTS-mediated phosphorylation reactions exerting antagonistic effects on the transcription regulators: P~EIIB-dependent phosphorylation regulates induction of the corresponding genes and P~His-HPr-mediated phosphorylation plays a role in CCR. Carbohydrate transport of L. casei is also regulated via inducer exclusion and inducer expulsion. The presence of glucose, fructose, etc. leads to inhibition of the transport or metabolism of less favorable carbon sources (inducer exclusion) or to the export of accumulated non-metabolizable carbon sources (inducer expulsion). While P-Ser-HPr is essential for inducer exclusion of maltose, it is not necessary for the expulsion of accumulated thio-methyl-beta-D-galactopyranoside. Surprisingly, recent evidence suggests that the PTS of L. casei also plays a role in cold shock response. PMID:17183208

  19. Cellobiose-Specific Phosphotransferase System of Klebsiella pneumoniae and Its Importance in Biofilm Formation and Virulence

    PubMed Central

    Wu, Meng-Chuan; Chen, Ying-Chun; Lin, Tzu-Lung; Hsieh, Pei-Fang

    2012-01-01

    Klebsiella pneumoniae is a Gram-negative bacillus belonging to the family Enterobacteriaceae. In the past 20 years, K. pneumoniae has become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA). The formation of biofilm facilitates bacterial colonization and has been implicated in reduced susceptibility to the host immune response. To investigate genes related to biofilm formation in a PLA-associated K. pneumoniae strain, a transposon mutant library was screened by microtiter plate assay to identify isolates impaired for biofilm formation. One of the mutants was disrupted in celB, encoding the putative cellobiose-specific subunit IIC of enzyme II (EIIC) of a carbohydrate phosphotransferase system (PTS). This transmembrane protein is responsible for recognizing and binding specific sugars and transporting them across the cell membrane into the cytoplasm. Deletion and chromosomal complementation of celB confirmed, by microtiter plate and slide culture assays, that celB was indeed responsible for biofilm formation. Cellobiose-specific PTS activities of deletion mutants grown in LB broth and 0.005% cellobiose minimal medium were markedly lower than that of the wild-type strain grown under the same conditions, thereby confirming the involvement of celB in cellobiose transport. In 0.005% cellobiose minimal medium, the celB mutant showed a delay in growth compared to the wild-type strain. In a mouse model of intragastric infection, deletion of the celB gene increased the survival rate from 12.5% to 87.5%, which suggests that the celB deletion mutant also exhibited reduced virulence. Thus, the celB locus of K. pneumoniae may contribute to biofilm formation and virulence through the metabolism of cellobiose. PMID:22566508

  20. Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli

    PubMed Central

    Kornberg, Hans L.; Lambourne, Linda T. M.; Sproul, Andrew A.

    2000-01-01

    From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth ≈8 mM but with Vmax for generation time ≈1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme IIGlc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG+ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax ≈7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme IIMan (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl α-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence. PMID:10677538

  1. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  2. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  3. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  4. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  5. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  6. Phosphoenolpyruvate-dependent protein kinase from skeletal muscle

    SciTech Connect

    Khandelwal, R.L.; Bhanot, P.; Waygood, E.B.

    1986-05-01

    Soluble extracts of skeletal muscle from rat, rabbit and hamster when incubated with 0.1 mM (/sup 32/P)phosphoenolpyruvate give rise to a similar set of phosphoproteins as resolved by SDS-PAGE with Mr 25,000, 35,000, 37,000, 43,000 and 59,000. The phosphorylation of these proteins is neither inhibited by excess ATP nor achieved by incubation with (..gamma..-/sup 32/P)ATP. Except for the Mr 43,000 phosphoprotein, the phosphorylation of the other proteins dramatically increased in the presence of 0.1 mM CTP. Although phosphatase inhibits such as NaF and PPi were not effective, CTP may act to inhibit phosphatase activity rather than activating a protein kinase. The phosphoamino acids produced in these phosphoproteins were acid stable and only phosphoserine has been routinely identified. Using DEAE-cellulose, CM-Sephadex and Ultrogel AcA44 chromatography, the Mr 37,000 phosphoprotein has been purified from rabbit skeletal muscle to near homogeneity. No physiological role for either the protein kinase or its substrates has yet been found.

  7. Purification, crystallization and preliminary X-ray analysis of Enterococcus casseliflavus aminoglycoside-2′′-phosphotransferase-IVa

    PubMed Central

    Toth, Marta; Vakulenko, Sergei; Smith, Clyde A.

    2010-01-01

    The deactivation of aminoglycoside antibiotics by chemical modification is one of the major sources of bacterial resistance to this family of therapeutic compounds, which includes the clinically relevant drugs streptomycin, kanamycin and gentamicin. The aminoglycoside phosphotransferases (APHs) form one such family of enzymes responsible for this resistance. The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] from Enterococcus casseliflavus, has been cloned and the protein (comprising 306 amino-acid residues) has been expressed in Escherichia coli and purified. The enzyme was crystallized in three substrate-free forms. Two of the crystal forms belonged to the orthorhombic space group P212121 with similar unit-cell parameters, although one of the crystal forms had a unit-cell volume that was approximately 13% smaller than the other and a very low solvent content of around 38%. The third crystal form belonged to the monoclinic space group P21 and preliminary X-ray diffraction analysis was consistent with the presence of two molecules in the asymmetric unit. The orthorhombic crystal forms of apo APH(2′′)-IVa both diffracted to 2.2 Å resolution and the monoclinic crystal form diffracted to 2.4 Å resolution; synchrotron diffraction data were collected from these crystals at SSRL (Stanford, California, USA). Structure determination by molecular replacement using the structure of the related enzyme APH(2′′)-IIa is proceeding. PMID:20057078

  8. Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon.

    PubMed

    Yamada, M; Saier, M H

    1987-04-25

    The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene. PMID:3553176

  9. Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

    PubMed Central

    Cochu, Armelle; Vadeboncoeur, Christian; Moineau, Sylvain; Frenette, Michel

    2003-01-01

    In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABMan as well as membrane fragments containing IICMan and IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the

  10. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a...

  11. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a...

  12. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a...

  13. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a...

  14. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a...

  15. Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes.

    PubMed

    van Meel, Eline; Lee, Wang-Sik; Liu, Lin; Qian, Yi; Doray, Balraj; Kornfeld, Stuart

    2016-04-01

    The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif. PMID:26833567

  16. Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes.

    PubMed

    Liao, Cheng-Heng; Yao, Lili; Xu, Ya; Liu, Wei-Bing; Zhou, Ying; Ye, Bang-Ce

    2015-12-22

    The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals. PMID:26644570

  17. Purification and Characterization of Aminoglycoside Phosphotransferase APH(6)-Id, a Streptomycin Inactivating Enzyme

    PubMed Central

    Ashenafi, Meseret; Ammosova, Tatiana; Nekhai, Sergei; Byrnes, W. Malcolm

    2014-01-01

    As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified and characterized the enzyme. When expressed in E. coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration (MIC) needed to inhibit cell growth. Size exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of 32P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady state kinetic analysis gave the following results: Km(streptomycin) of 0.38 ± 0.13 mM, Km(ATP) of 1.03 ± 0.1 mM, Vmax of 3.2 ± 1.1 μmol/min/mg and kcat of 1.7 ± 0.6 s−1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin. PMID:24248535

  18. The Histidine-Phosphocarrier Protein of the Phosphoenolpyruvate: Sugar Phosphotransferase System of Bacillus sphaericus Self-Associates

    PubMed Central

    Doménech, Rosa; Hernández-Cifre, José G.; Bacarizo, Julio; Díez-Peña, Ana I.; Martínez-Rodríguez, Sergio; Cavasotto, Claudio N.; de la Torre, José García; Cámara-Artigás, Ana; Velázquez-Campoy, Adrián; Neira, José L.

    2013-01-01

    The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. Bacillus sphaericus, a bacterium with the ability to produce insecticidal proteins, is unable to use hexoses and pentoses as the sole carbon source, but it has ptsHI genes encoding the two general proteins of the PTS: enzyme I (EI) and the histidine phosphocarrier (HPr). In this work, we describe the biophysical and structural properties of HPr from B. sphaericus, HPrbs, and its affinity towards EI of other species to find out whether there is inter-species binding. Conversely to what happens to other members of the HPr family, HPrbs forms several self-associated species. The conformational stability of the protein is low, and it unfolds irreversibly during heating. The protein binds to the N-terminal domain of EI from Streptomyces coelicolor, EINsc, with a higher affinity than that of the natural partner of EINsc, HPrsc. Modelling of the complex between EINsc and HPrbs suggests that binding occurs similarly to that observed in other HPr species. We discuss the functional implications of the oligomeric states of HPrbs for the glycolytic activity of B. sphaericus, as well as a strategy to inhibit binding between HPrsc and EINsc. PMID:23922699

  19. Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the γ Subunit of GlcNAc-1-Phosphotransferase.

    PubMed

    van Meel, Eline; Kornfeld, Stuart

    2016-07-01

    The lysosomal storage disorder ML III γ is caused by defects in the γ subunit of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the enzyme that tags lysosomal enzymes with the mannose 6-phosphate lysosomal targeting signal. In patients with this disorder, most of the newly synthesized lysosomal enzymes are secreted rather than being sorted to lysosomes, resulting in increased levels of these enzymes in the plasma. Several missense mutations in GNPTG, the gene encoding the γ subunit, have been reported in mucolipidosis III γ patients. However, in most cases, the impact of these mutations on γ subunit function has remained unclear. Here, we report that the variants c.316G>A (p.G106S), c.376G>A (p.G126S), and c.425G>A (p.C142Y) cause misfolding of the γ subunit, whereas another variant, c.857C>T (p.T286M), does not appear to alter γ subunit function. The misfolded γ subunits were retained in the ER and failed to rescue the lysosomal targeting of lysosomal acid glycosidases. PMID:27038293

  20. YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12.

    PubMed

    Becker, Ann-Katrin; Zeppenfeld, Tim; Staab, Ariane; Seitz, Sabine; Boos, Winfried; Morita, Teppei; Aiba, Hiroji; Mahr, Kerstin; Titgemeyer, Fritz; Jahreis, Knut

    2006-08-01

    The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity. PMID:16855233

  1. Recombinant organisms capable of fermenting cellobiose

    DOEpatents

    Ingram, Lonnie O.; Lai, Xiaokuang; Moniruzzaman, Mohammed; York, Sean W.

    2000-01-01

    This invention relates to a recombinant microorganism which expresses pyruvate decarboxylase, alcohol dehydrogenase, Klebsiella phospho-.beta.-glucosidase and Klebsiella (phosphoenolpyruvate-dependent phosphotransferase system) cellobiose-utilizing Enzyme II, wherein said phospho-.beta.-glucosidase and said (phosphoenolpyruvate-dependent phosphotransferase) cellobiose-utilizing Enzyme II are heterologous to said microorganism and wherein said microorganism is capable of utilizing both hemicellulose and cellulose, including cellobiose, in the production of ethanol.

  2. Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease

    PubMed Central

    Zeng, Lin

    2013-01-01

    Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA. PMID:23222725

  3. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation.

    PubMed

    Boyko, Konstantin M; Gorbacheva, Marina A; Korzhenevskiy, Dmitry A; Alekseeva, Maria G; Mavletova, Dilara A; Zakharevich, Natalia V; Elizarov, Sergey M; Rudakova, Natalia N; Danilenko, Valery N; Popov, Vladimir O

    2016-09-01

    Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. PMID:27338640

  4. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    SciTech Connect

    Byrnes, Laura J.; Badarau, Adriana; Vakulenko, Sergei B.; Smith, Clyde A.

    2008-02-01

    APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal. Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)-Ic variants were crystallized in the presence of 14–20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris–HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X-ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  5. In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis.

    PubMed Central

    Thompson, J

    1978-01-01

    Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates.Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3. Images PMID:101523

  6. Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli

    SciTech Connect

    Iino, Daisuke; Takakura, Yasuaki; Kuroiwa, Mika; Kawakami, Ryouta; Sasaki, Yasuyuki; Hoshino, Takayuki; Ohsawa, Kanju; Nakamura, Akira; Yajima, Shunsuke

    2007-08-01

    The crystallization and preliminary X-ray studies of the aminoglycoside antibiotic-modifying enzyme hygromycin B phosphotransferase from E. coli are reported. Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3{sub 2}21, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein.

  7. Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc

    PubMed Central

    Hariharan, Parameswaran; Balasubramaniam, Dhandayuthapani; Peterkofsky, Alan; Kaback, H. Ronald

    2015-01-01

    In a variety of bacteria, the phosphotransferase protein IIAGlc plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H+, using a mechanism in which sugar- and H+-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIAGlc (inducer exclusion). Here we report the thermodynamic features of the IIAGlc–LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIAGlc binds to LacY with a Kd of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIAGlc binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIAGlc blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIAGlc binding. The precise mechanism of the inhibition of LacY by IIAGlc elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIAGlc. PMID:25675534

  8. Synbiotic impact of tagatose on viability of Lactobacillus rhamnosus strain GG mediated by the phosphotransferase system (PTS).

    PubMed

    Koh, Ji Hoon; Choi, Seung Hye; Park, Seung Won; Choi, Nag-Jin; Kim, Younghoon; Kim, Sae Hun

    2013-10-01

    Synbiotics, the combination of prebiotics and probiotics, has been shown to produce synergistic effects that promote gastrointestinal well-being of host. Tagatose is a low calorie food ingredient with putative health-promoting benefits. Herein, we investigated its synbiotic impact on the viability of Lactobacillus casei 01 and Lactobacillus rhamnosus strain GG and the potential mechanism involved. Tagatose, as a synbiotic substrate, enhanced the growth of L. casei 01 and L. rhamnosus strain GG compared to other prebiotics. Other gut-indigenous such as Clostridium spp. readily utilized fructooligosaccharide (FOS), the most widely used functional prebiotics, but not tagatose. Additionally, tagatose enhanced probiotic functions of L. casei 01 and L. rhamnosus strain GG by reinforcing their attachment on HT-29 intestine epithelial cells and enhancing their cholesterol-lowering activities. Whole transcriptome study and quantitative real-time polymerase chain reaction (qRT-PCR) test showed that the presence of tagatose in L. rhamnosus strain GG caused induction of a large number of genes associated with carbohydrate metabolism including the phosphotransferase system (PTS). Collectively, these results indicate the tagatose enhanced the growth of L. casei 01 and L. rhamnosus strain GG and their probiotic activities by activating tagatose-associated PTS networks. Importantly, this study highlights the potential application of tagatose and L. casei 01 and/or L. rhamnosus strain GG as a synbiotic partner in functional dairy foods (i.e. yogurt and cheese) and therapeutic dietary supplements. PMID:23764214

  9. Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima, a Member of the ASKHA Superfamily of Phosphotransferases

    SciTech Connect

    Diao, Jiasheng; Hasson, Miriam S.

    2009-04-01

    The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-{angstrom} crystal structure of Buk2 complexed with ({beta},{gamma}-methylene) adenosine 5'-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

  10. Rapid and Liquid-Based Selection of Genetic Switches Using Nucleoside Kinase Fused with Aminoglycoside Phosphotransferase

    PubMed Central

    Kawai-Noma, Shigeko; Saito, Kyoichi; Umeno, Daisuke

    2015-01-01

    The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3’)-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling. PMID:25790096

  11. The phosphoenolpyruvate:sugar phosphotransferase system is involved in sensitivity to the glucosylated bacteriocin sublancin.

    PubMed

    Garcia De Gonzalo, C V; Denham, E L; Mars, R A T; Stülke, J; van der Donk, W A; van Dijl, J M

    2015-11-01

    The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin. PMID:26282429

  12. The Phosphoenolpyruvate:Sugar Phosphotransferase System Is Involved in Sensitivity to the Glucosylated Bacteriocin Sublancin

    PubMed Central

    Garcia De Gonzalo, C. V.; Denham, E. L.; Mars, R. A. T.; Stülke, J.

    2015-01-01

    The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin. PMID:26282429

  13. Glycogen contains phosphodiester groups that can be introduced by UDPglucose: glycogen glucose 1-phosphotransferase.

    PubMed

    Lomako, J; Lomako, W M; Whelan, W J; Marchase, R B

    1993-08-30

    Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose: glycogen glucose 1-phosphotransferase that positions glucose 1-phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate. PMID:8396041

  14. Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation.

    PubMed

    Paganelli, Fernanda L; Huebner, Johannes; Singh, Kavindra V; Zhang, Xinglin; van Schaik, Willem; Wobser, Dominique; Braat, Johanna C; Murray, Barbara E; Bonten, Marc J M; Willems, Rob J L; Leavis, Helen L

    2016-07-15

    Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis. PMID:26984142

  15. Impact of the core components of the phosphoenolpyruvate-carbohydrate phosphotransferase system, HPr and EI, on differential protein expression in Ralstonia eutropha H16.

    PubMed

    Kaddor, Chlud; Voigt, Birgit; Hecker, Michael; Steinbüchel, Alexander

    2012-07-01

    In Ralstonia eutropha H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/EI deletion mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner-Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16ΔptsHI. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16ΔptsHI, and to gain insight into the molecular response of R. eutropha to the deletion of HPr/EI. PMID:22630130

  16. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    SciTech Connect

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter; Chow, Joseph W.; Lerner, Stephen; Vakulenko, Sergei; Smith, Clyde A.

    2005-04-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

  17. Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase αβ and γ subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology.

    PubMed

    Flanagan-Steet, Heather; Matheny, Courtney; Petrey, Aaron; Parker, Joshua; Steet, Richard

    2016-09-01

    Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and ß subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg(-/-) embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg(-/-) embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg(-/-) embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal. PMID:27241848

  18. Product inhibition of potato tuber pyrophosphate:fructose-6-phosphate phosphotransferase by phosphate and pyrophosphate.

    PubMed

    Stitt, M

    1989-02-01

    The product inhibition of potato (Solanum tuberosum) tuber pyrophosphate:fructose-6-phosphate phosphotransferase by inorganic pyrophosphate and inorganic phosphate has been studied. The binding of substrates for the forward (glycolytic) and the reverse (gluconeogenic) reaction is random order, and occurs with only weak competition between the substrate pair fructose-6-phosphate and pyrophosphate, and between the substrate pair fructose-1,6-bisphosphate and phosphate. Pyrophosphate is a powerful inhibitor of the reverse reaction, acting competitively to fructose-1,6-biphosphate and noncompetitively to phosphate. At the concentrations needed for catalysis of the reverse reaction, phosphate inhibits the forward reaction in a largely noncompetitive mode with respect to both fructose-6-phosphate and pyrophosphate. At higher concentrations, phosphate inhibits both the forward and the reverse reaction by decreasing the affinity for fructose-2,6-bisphosphate and thus, for the other three substrates. These results allow a model to be proposed, which describes the interactions between the substrates at the catalytic site. They also suggest the enzyme may be regulated in vivo by changes of the relation between metabolites and phosphate and could act as a means of controlling the cytosolic pyrophosphate concentration. PMID:16666593

  19. The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

    PubMed Central

    Govindarajan, Sutharsan; Elisha, Yair; Nevo-Dinur, Keren; Amster-Choder, Orna

    2013-01-01

    ABSTRACT The bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near the Escherichia coli cell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shaped E. coli cells are comparable, implying that negatively curved “pole-like” sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein from Bacillus subtilis at strongly curved sites in both B. subtilis and E. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness. PMID:24129255

  20. Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

    SciTech Connect

    Fong, D.; Lemke, C; Huang, J; Xiong, B; Berghuis, A

    2010-01-01

    Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3{prime}) isozymes and an APH(2{double_prime}) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3{prime}) and APH(2{double_prime}) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.

  1. Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate: Mannitol phosphotransferase system

    SciTech Connect

    Mueller, E.G.; Knowles, J.R. ); Khandekar, S.S.; Jacobson, G.R. )

    1990-07-24

    The authors have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EII{sup Mtl}) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EII{sup Mtl}, the phospho group from ((R)-{sup 16}O, {sup 17}O, {sup 18}O)PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a single site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EII{sup Mtl} must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EII{sup Mtl} intermediates have been demonstrated biochemically, the results imply that EII{sup Mtl} is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EII{sup Mtl} intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein.

  2. Structure and function of the mannitol permease of the Escherichia coli phosphotransferase sugar transport system

    SciTech Connect

    Stephan, M.M.

    1988-01-01

    The mannitol permease, or mannitol enzyme II, is responsible for the phosphorylation and transmembrane transport of the hexitol mannitol via the phosphotransferase sugar transport system (PTS) in Escherichia coli. Neither the detailed molecular mechanisms by which this protein carries out these functions nor its three dimensional structure in the membrane are known. An in vivo selective radiolabeling system was used to study the enzyme's subunits interactions as they related to function, as well as its membrane topography, by polyacrylamide gel electrophoresis. The intramembrane topography of the mannitol enzyme II was investigated using proteases as probes of enzyme structure in the membrane. The enzyme was found to have two distinct domains, a very hydrophobic, membrane-bound, N-terminal domain, and a relatively hyprophilic C-terminal domain which protrudes into the cytoplasm. The membrane-bound domain was further dissected, and an extra-membrane loop region was identified using peptide-specific antibodies. The cytoplasmic domain was found to contain a site of covalent phosphorylation using (/sup 32/p)-labeled PEP, as well as the binding site for the phosphodonor HPr.

  3. In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake in Streptomyces coelicolor

    PubMed Central

    Nothaft, Harald; Parche, Stephan; Kamionka, Annette; Titgemeyer, Fritz

    2003-01-01

    HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent Km of 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor. PMID:12533468

  4. QUANTIFICATION OF TRANSGENIC PLANT MARKER GENE PERSISTENCE IN THE FIELD

    EPA Science Inventory

    Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT-II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, com...

  5. Efficient production of xylitol from hemicellulosic hydrolysate using engineered Escherichia coli.

    PubMed

    Su, Buli; Wu, Mianbin; Zhang, Zhe; Lin, Jianping; Yang, Lirong

    2015-09-01

    A metabolically engineered Escherichia coli has been constructed for the production of xylitol, one of the top 12 platform chemicals from agricultural sources identified by the US Department of Energy. An optimal plasmid was constructed to express xylose reductase from Neurospora crassa with almost no inclusion bodies at relatively high temperature. The phosphoenolpyruvate-dependent glucose phosphotransferase system (ptsG) was disrupted to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by deleting the xylose isomerase (xylA) and xylulose kinase (xylB) genes. The putative pathway for xylitol phosphorylation was also blocked by disrupting the phosphoenolpyruvate-dependent fructose phosphotransferase system (ptsF). The xylitol producing recombinant E. coli allowed production of 172.4 g L(-1) xylitol after 110 h of fed-batch cultivation with an average productivity of 1.57 g L(-1) h(-1). The molar yield of xylitol to glucose reached approximately 2.2 (mol xylitol mol(-1) glucose). Furthermore, the recombinant strain also produced about 150 g L(-1) xylitol from hemicellulosic sugars in modified M9 minimal medium and the overall productivity was 1.40 g L(-1) h(-1), representing the highest xylitol concentration and productivity reported to date from hemicellulosic sugars using bacteria. Thus, this engineered E. coli is a candidate for the development of efficient industrial-scale production of xylitol from hemicellulosic hydrolysate. PMID:26197036

  6. How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†

    PubMed Central

    Deutscher, Josef; Francke, Christof; Postma, Pieter W.

    2006-01-01

    The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. PMID:17158705

  7. The mechanism of the reaction catalysed by adenosine triphosphate–creatine phosphotransferase

    PubMed Central

    Morrison, J. F.; James, Elizabeth

    1965-01-01

    1. The forward and reverse reactions catalysed by ATP–creatine phosphotransferase have been studied kinetically at pH8·0 in the presence and absence of products, under conditions in which the free Mg2+ concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP2−+creatine0⇌MgADP−+phosphocreatine2−2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme–MgADP–creatine and enzyme–MgATP–phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme–MgADP–creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme–MgATP–phosphocreatine complex, where each reactant hinders the combination of the other. PMID:16749122

  8. Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

    PubMed Central

    Reizer, J.; Hoischen, C.; Reizer, A.; Pham, T. N.; Saier, M. H.

    1993-01-01

    We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7686067

  9. Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12

    PubMed Central

    Göhler, Anna-Katharina; Staab, Ariane; Gabor, Elisabeth; Homann, Karina; Klang, Elisabeth; Kosfeld, Anne; Muus, Janna-Eleni; Wulftange, Jana Selina

    2012-01-01

    The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICBGlc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICBGlc, Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICBGlc, by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions. PMID:22178967

  10. Construction of Listeria monocytogenes mutants with in-frame deletions in the phosphotransferase transport system (PTS) and analysis of their growth under stress conditions.

    PubMed

    Liu, Yanhong; Ceruso, Marina; Jiang, Yuji; Datta, Atin R; Carter, Laurenda; Strain, Errol; Pepe, Tiziana; Anastasi, Aniello; Fratamico, Pina

    2013-09-01

    Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ∆LMOf2365_0445 was increased under nisin (125 μg/mL) treatments compared to the wild-type (P < 0.01). The growth of ∆LMOf2365_0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ∆LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ∆LMOf2365_0442 and ∆LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food. PMID:23909479

  11. Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani.

    PubMed

    Schmidt-Arras, Dirk; Leclercq, Olivier; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Faigle, Wolfgang; Loew, Damarys; Späth, Gerald F

    2011-08-24

    The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase-substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development. PMID:21443974

  12. Characterization of a novel GDP-mannose:Serine-protein mannose-1-phosphotransferase from Leishmania mexicana.

    PubMed

    Moss, J M; Reid, G E; Mullin, K A; Zawadzki, J L; Simpson, R J; McConville, M J

    1999-03-01

    Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Manalpha1-PO4-Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Manalpha1-P to serine residues in a range of defined peptides. The presence and location of the Manalpha1-PO4-Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn2+, utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO4 residues in the in vitro assays. Similarly, Man-PO4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and

  13. Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

    PubMed Central

    Bourand, A.; Yebra, M. J.; Boël, G.; Mazé, A.

    2013-01-01

    Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD+ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation. PMID:23564164

  14. Utilization of D-ribitol by Lactobacillus casei BL23 requires a mannose-type phosphotransferase system and three catabolic enzymes.

    PubMed

    Bourand, A; Yebra, M J; Boël, G; Mazé, A; Deutscher, J

    2013-06-01

    Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation. PMID:23564164

  15. Characterization of factor IIIGLc in catabolite repression-resistant (crr) mutants of Salmonella typhimurium.

    PubMed Central

    Scholte, B J; Schuitema, A R; Postma, P W

    1982-01-01

    crr mutants of Salmonella typhimurium are thought to be defective in the regulation of adenylate cyclase and a number of transport systems by the phosphoenolpyruvate-dependent sugar phosphotransferase system, crr mutants are also defective in the enzymatic activity of factor IIIGlc (IIIGlc), a protein component of the phosphotransferase system involved in glucose transport. Therefore, it has been proposed that IIIGlc is the primary effector of phosphotransferase system-mediated regulation of cell metabolism. We characterized crr mutants with respect to the presence and function of IIIGlc by using an immunochemical approach. All of the crr mutants tested had low (0 to 30%) levels of IIIGlc compared with wild-type cells, as determined by rocket immunoelectrophoresis. The IIIGlc isolated from one crr mutant was investigated in more detail and showed abnormal aggregation behavior, which indicated a structural change in the protein. These results supported the hypothesis that a crr mutation directly affects IIIGlc, probably by altering the structural gene of IIIGlc. Several crr strains which appeared to be devoid of IIIGlc in immunoprecipitation assays were still capable of in vitro phosphorylation and transport of methyl alpha-glucoside. This phosphorylation activity was sensitive to specific anti-IIIGlc serum. Moreover, the membranes of crr mutants, as well as those of wild-type cells, contained a protein that reacted strongly with our anti-IIIGlc serum. We propose that S. typhimurium contains a membrane-bound form of IIIGlc which may be involved in phosphotransferase system activity. Images PMID:7035434

  16. Control of Pyrophosphated-Fructose-6-Phosphate 1-Phosphotransferase Activity in the Cotyledons of Citrullus lanatus1

    PubMed Central

    Botha, Anna-Maria; Botha, Frederik C.

    1990-01-01

    After initiation of radicle elongation, the pyrophosphate:d-fructose-6-phosphate 1-phosphotransferase (PFP) activity sharply increases in the cotyledons of Citrullus lanatus. Removal of the radicle early during incubation prevents the increase in PFP activity in the cotyledons evident in the control. Removal of the radicle at any stage after germination results in a decrease in PFP activity in the cotyledons. Application of kinetin (0.5 micromolar) or 2-chlorophosphonic acid (0.1 micromolar) to isolated cotyledons replaces the effect of the radicle. Gibberellic acid (0.09 micromolar GA3) also partially mimics the presence of the radicle. Anaerobic conditions, as well as cycloheximide application (0.18 micromolar) to intact embryos or to kinetin and ethrel treated isolated cotyledons prevent the increase in PFP activity evident in the control. PMID:16667523

  17. High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

    PubMed

    Torres, C; Perlin, M H; Baquero, F; Lerner, D L; Lerner, S A

    2000-08-01

    This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed. PMID:10929874

  18. Structure, dynamics and biophysics of the cytoplasmic protein–protein complexes of the bacterial phosphoenolpyruvate: Sugar phosphotransferase system

    SciTech Connect

    Clore, G. Marius; Venditti, Vincenzo

    2013-10-01

    The bacterial phosphotransferase system (PTS) couples phosphoryl transfer, via a series of bimolecular protein–protein interactions, to sugar transport across the membrane. The multitude of complexes in the PTS provides a paradigm for studying protein interactions, and for understanding how the same binding surface can specifically recognize a diverse array of targets. Fifteen years of work aimed at solving the solution structures of all soluble protein–protein complexes of the PTS has served as a test bed for developing NMR and integrated hybrid approaches to study larger complexes in solution and to probe transient, spectroscopically invisible states, including encounter complexes. We review these approaches, highlighting the problems that can be tackled with these methods, and summarize the current findings on protein interactions.

  19. Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study

    PubMed Central

    Kaplan, Elise; Guichou, Jean-François; Chaloin, Laurent; Kunzelmann, Simone; Leban, Nadia; Serpersu, Engin H.; Lionne, Corinne

    2016-01-01

    Background Aminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step. Methods We used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements. Results For the first time, Kd values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio Kd/Km, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction. Conclusions APH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2′ and 6′, but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3′)-IIIa, an intermediate containing product is preponderant during the steady state. General significance This intermediate may constitute a good target for future drug design. PMID:26802312

  20. Solution NMR Structure of the 48-kDa IIAMannose-HPr Complex of the Escherichia coli Mannose Phosphotransferase System*

    PubMed Central

    Williams, David C.; Cai, Mengli; Suh, Jeong-Yong; Peterkofsky, Alan; Clore, G. Marius

    2005-01-01

    The solution structure of the 48-kDa IIAMan-HPr complex of the mannose branch of the Escherichia coli phosphotransferase system has been solved by NMR using conjoined rigid body/torsion angle-simulated annealing on the basis of intermolecular nuclear Overhauser enhancement data and residual dipolar couplings. IIAMan is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr, formed primarily by helices 1 and 2, interacts with a deep groove at the interface of the two subunits of IIAMan. The interaction surface on IIAMan is predominantly helical, comprising helix 3 from the subunit that bears the active site His-10 and helices 1, 4, and 5 from the other subunit. The total buried accessible surface area at the protein-protein interface is 1450 Å2. The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic, and charged residues. The active site histidines, His-10 of IIAMan and His-15 (italics indicate HPr residues) of HPr, are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-ε2 atom of His-10 and the N-δ1 atom of His-15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparing the structures of complexes of HPr with three other structurally unrelated phosphotransferase system proteins, enzymes I, IIAglucose, and IIAmannitol, reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins. PMID:15788390

  1. Phosphotransferase system-dependent extracellular growth of listeria monocytogenes is regulated by alternative sigma factors σL and σH.

    PubMed

    Wang, Siyun; Orsi, Renato H; Tang, Silin; Zhang, Wei; Wiedmann, Martin; Boor, Kathryn J

    2014-12-01

    Alternative sigma (σ) factors and phosphotransferase systems (PTSs) play pivotal roles in the environmental adaptation and virulence of Listeria monocytogenes. The growth of the L. monocytogenes parent strain 10403S and 15 isogenic alternative σ factor mutants was assessed in defined minimal medium (DM) with PTS-dependent or non-PTS-dependent carbon sources at 25°C or 37°C. Overall, our results suggested that the regulatory effect of alternative σ factors on the growth of L. monocytogenes is dependent on the temperature and the carbon source. One-way analysis of variance (one-way ANOVA) showed that the factor "strain" had a significant effect on the maximum growth rate (μmax), lag phase duration (λ), and maximum optical density (ODmax) in PTS-dependent carbon sources (P < 0.05) but not in a non-PTS-dependent carbon source. Also, the ODmax was not affected by strain for any of the three PTS-dependent carbon sources at 25°C but was affected by strain at 37°C. Monitoring by quantitative real-time PCR (qRT-PCR) showed that transcript levels for lmo0027, a glucose-glucoside PTS permease (PTS(Glc)-1)-encoding gene, were higher in the absence of σ(L), and lower in the absence of σ(H), than in the parent strain. Our data thus indicate that σ(L) negatively regulates lmo0027 and that the increased μmax observed for the ΔsigL strain in DM with glucose may be associated with increased expression of PTS(Glc)-1 encoded by lmo0027. Our findings suggest that σ(H) and σ(L) mediate the PTS-dependent growth of L. monocytogenes through complex transcriptional regulations and fine-tuning of the expression of specific pts genes, including lmo0027. Our findings also reveal a more important and complex role of alternative σ factors in the regulation of growth in different sugar sources than previously assumed. PMID:25281379

  2. Mobilization properties of small ColE1-like plasmids carrying kanamycin resistance gene isolated from Salmonella enterica serotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Previously we isolated and characterized various groups of small kanamycin resistance (KanR) ColE1-like plasmids from different serotypes of Salmonella enterica isolates. These plasmids all carried the aph(3)-I gene encoding the aminoglycoside phosphotransferase responsible for the kanam...

  3. Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus.

    PubMed

    Ronneau, Séverin; Petit, Kenny; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTS(Ntr)) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EI(Ntr)) of PTS(Ntr). Decreasing intracellular glutamine concentration triggers phosphorylation of EI(Ntr) and its downstream components HPr and EIIA(Ntr). Once phosphorylated, both HPr∼P and EIIA(Ntr)∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth. PMID:27109061

  4. Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus

    PubMed Central

    Ronneau, Séverin; Petit, Kenny; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTSNtr) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EINtr) of PTSNtr. Decreasing intracellular glutamine concentration triggers phosphorylation of EINtr and its downstream components HPr and EIIANtr. Once phosphorylated, both HPr∼P and EIIANtr∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth. PMID:27109061

  5. Crystallographic Studies of Two Bacterial AntibioticResistance Enzymes: Aminoglycoside Phosphotransferase (2')-Ic and GES-1\\beta-lactamase

    SciTech Connect

    Brynes, Laura; /Rensselaer Poly.

    2007-10-31

    Guiana Extended-Spectrum-1 (GES-1) and Aminoglycoside phosphotransferase (2')-Ic (APH(2')-Ic) are two bacteria-produced enzymes that essentially perform the same task: they provide resistance to an array of antibiotics. Both enzymes are part of a growing resistance problem in the medical world. In order to overcome the ever-growing arsenal of antibiotic-resistance enzymes, it is necessary to understand the molecular basis of their action. Accurate structures of these proteins have become an invaluable tool to do this. Using protein crystallography techniques and X-ray diffraction, the protein structure of GES-1 bound to imipenem (an inhibitor) has been solved. Also, APH(2')-Ic has been successfully crystallized, but its structure was unable to be solved using molecular replacement using APH(2')-Ib as a search model. The structure of GES-1, with bound imipenem was solved to a resolution of 1.89A, and though the inhibitor is bound with only moderate occupancy, the structure shows crucial interactions inside the active site that render the enzyme unable to complete the hydrolysis of the {beta}-lactam ring. The APH(2')-Ic dataset could not be matched to the model, APH(2')-Ib, with which it shares 25% sequence identity. The structural information gained from GES-1, and future studies using isomorphous replacement to solve the APH(2')-Ic structure can aid directly to the creation of novel drugs to combat both of these classes of resistance enzymes.

  6. Fructose-1,6-Bisphosphate Is an Allosteric Activator of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase.

    PubMed

    Nielsen, T. H.

    1995-05-01

    The activity of highly purified pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) from barley (Hordeum vulgare) leaves was studied under conditions where the catalyzed reaction was allowed to approach equilibrium. The activity of PFP was monitored by determining the changes in the levels of fructose-6-phosphate, orthophosphate, and fructose-1,6-bisphosphate (Fru-1,6-bisP). Under these conditions PFP activity was not dependent on activation by fructose-2,6-bisphosphate (Fru-2,6-bisP). Inclusion of aldolase in the reaction mixture temporarily restored the dependence of PFP on Fru-2,6-bisP. Alternatively, PFP was activated by Fru-1,6-bisP in the presence of aldolase. It is concluded that Fru-1,6-bisP is an allosteric activator of barley PFP, which can substitute for Fru-2,6-bisP as an activator. A significant activation was observed at a concentration of 5 to 25 [mu]M Fru-1,6-bisP, which demonstrates that the allosteric site of barley PFP has a very high affinity for Fru-1,6-bisP. The high affinity for Fru-1,6-bisP at the allosteric site suggests that the observed activation of PFP by Fru-1,6-bisP constitutes a previously unrecognized in vivo regulation mechanism. PMID:12228454

  7. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  8. Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum.

    PubMed

    Lindner, Steffen N; Seibold, Gerd M; Krämer, Reinhard; Wendisch, Volker F

    2011-01-01

    Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes. PMID:22008639

  9. The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

    PubMed

    Deutscher, Josef; Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-06-01

    The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  10. Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

    PubMed

    Karstens, Katja; Zschiedrich, Christopher P; Bowien, Botho; Stülke, Jörg; Görke, Boris

    2014-04-01

    EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha. PMID:24515609

  11. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  12. Streptococcus salivarius mutants defective in mannose phosphotransferase systems show reduced sensitivity to mutacins I-T9 and R-3B.

    PubMed

    Nicolas, Guillaume G; Frenette, Michel; Lavoie, Marc C

    2010-08-01

    Twenty-four mutacin-producing Streptococcus mutans strains were screened for their propensity to produce class II one-peptide bacteriocin using a deferred antagonism assay. Streptococcus salivarius and 3 mutants defective in their mannose phosphotransferase systems (mannose-PTS) were used as sensitive strains to identify which mannose-PTS could act as the docking site for class II one-peptide bacteriocin activity. We observed that only 2 strains of S. mutans, T9 and 3B, potentially produce class II one-peptide bacteriocin, namely mutacins I-T9 and R-3B, but with no preference for any mannose-PTS complex as a target. PMID:20725132

  13. Histidine to aspartate phosphotransferase activity of nm23 proteins: phosphorylation of aldolase C on Asp-319.

    PubMed Central

    Wagner, P D; Vu, N D

    2000-01-01

    nm23 genes have been implicated in the suppression of tumour metastasis and cell motility; however, the biochemical mechanisms for these suppressions are not known. We have previously described the transfer of phosphate from the catalytic histidine residues of nm23 proteins to an aspartic or a glutamic residue on one or more 43 kDa proteins in detergent extracts of bovine brain membranes. To gain a better understanding of this transferase activity, we partly purified this 43 kDa protein and identified aldolases A and C as the major 43 kDa proteins present in the preparation. Aldolase was purified from brain cytosol; its phosphorylation by rat liver nm23 proteins and by recombinant human nm23-H1 was examined. The site of phosphorylation was identified as Asp-319 on aldolase C. The equivalent residue on aldolase A, a glutamic residue, was not phosphorylated. Aldolase C was rapidly phosphorylated by wild-type nm23-H1 but was not phosphorylated, or was phosphorylated very slowly, by either nm23-H1(P96S) or nm23-H1(S120G), mutants of nm23-H1 that do not suppress cell motility. This is the first identification of a protein that is phosphorylated on an aspartic residue by nm23 proteins. The sequence around Asp-319 of aldolase C has some similarities to those around the histidine residues on ATP-citrate lyase and succinic thiokinase that are phosphorylated by nm23 proteins. PMID:10698688

  14. Synthesis of HPr(Ser-P)(His-P) by enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius.

    PubMed

    Casabon, Israël; Couture, Manon; Vaillancourt, Katy; Vadeboncoeur, Christian

    2006-05-30

    HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosphorylation. However, streptococci contain significant amounts of HPr(Ser-P)(His approximately P) during exponential growth, and recent studies suggest that phosphorylation of HPr(Ser-P) by EI is involved in the recycling of HPr(Ser-P)(His approximately P). We report in this paper a study on the phosphorylation of Streptococcus salivarius HPr, HPr(Ser-P), and HPr(S46D) by EI. Our results indicate that (i) the specificity constant (k(cat)/K(m)) of EI for HPr(Ser-P) at pH 7.9 was approximately 5000-fold smaller than that observed for HPr, (ii) no metabolic intermediates were able to stimulate HPr(Ser-P) phosphorylation, (iii) the rate of HPr phosphorylation decreased at pHs below 6.5, while that of HPr(Ser-P) increased and was almost 10-fold higher at pH 6.1 than at pH 7.9, (iv) HPr(S46D), a mutated HPr alleged to mimic HPr(Ser-P), was also phosphorylated more efficiently under acidic conditions, and, lastly, (v) phosphorylation of Bacillus subtilis HPr(Ser-P) by B. subtilis EI was also stimulated at acidic pH. Our results suggest that the high levels of HPr(Ser-P)(His approximately P) in streptococci result from the combination of two factors, a high physiological concentration of HPr(Ser-P) and stimulation of HPr(Ser-P) phosphorylation by EI at acidic pH, an intracellular condition that occurs in response to the acidification of the external medium during growth of the culture. PMID:16716080

  15. Helical shifts generate two distinct conformers in the atomic resolution structure of the CheA phosphotransferase domain from Thermotoga maritima.

    PubMed

    Quezada, Cindy M; Gradinaru, Cristian; Simon, Melvin I; Bilwes, Alexandrine M; Crane, Brian R

    2004-08-27

    Helical histidine phosphotransferase (HPt) domains play a central role in many aspects of bacterial signal transduction. The 0.98 A resolution crystallographic structure of the amino-terminal HPt domain (P1) from the chemotaxis kinase CheA of Thermotoga maritima reveals a remarkable degree of structural heterogeneity within a four-helix bundle. Two of the four helices have alternate main-chain conformations that differ by a 1.3-1.7A shift along the bundle axis. These dual conformers were only resolved with atomic resolution diffraction data and their inclusion significantly improved refinement statistics. Neither conformer optimizes packing within the helical core, consistent with their nearly equal refined occupancies. Altered hydrogen bonding within an inter-helical loop may facilitate transition between conformers. Two discrete structural states rather than a continuum of closely related conformations indicates an energetic barrier to conversion between conformers in the crystal at 100K, although many more states are expected in solution at physiological temperatures. Anisotropic atomic thermal B factors within the two conformers indicate modest overall atomic displacement that is largest perpendicular to the helical bundle and not along the direction of apparent motion. Despite the conformational heterogeneity of P1 in the crystal at low temperature, the protein displays high thermal stability in solution (T(m)=100 degrees C). Addition of a variable C-terminal region that corresponds to a mobile helix in other CheA structures significantly narrows the temperature width of the unfolding transition and may affect domain dynamics. Helices that compose the kinase recognition site and contain the phospho-accepting His45 do not have alternate conformations. In this region, atomic resolution provides detailed structural parameters for a conserved hydrogen-bonding network that tunes the reactivity of His45. A neighboring glutamate (E67), essential for

  16. Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination.

    PubMed

    Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Toki, Seiichi

    2016-01-01

    Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice. PMID:27557691

  17. Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

    PubMed Central

    Peterson, J L; McBride, O W

    1980-01-01

    The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity. Images PMID:6929511

  18. New Construct Approaches for Efficient Gene Silencing in Plants

    PubMed Central

    Yan, Hua; Chretien, Robert; Ye, Jingsong; Rommens, Caius M.

    2006-01-01

    An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts. PMID:16766670

  19. Kinetic studies of the reverse reaction catalysed by adenosine triphosphate–creatine phosphotransferase. The inhibition by magnesium ions and adenosine diphosphate

    PubMed Central

    Morrison, J. F.; O'Sullivan, W. J.

    1965-01-01

    1. Kinetic investigations of the reaction catalysed by ATP–creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg2+ at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion–nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg2+ is a non-competitive inhibitor of the enzyme with respect to both MgADP− and phosphocreatine. 4. ADP3− is a competitive inhibitor of the enzyme with respect to MgADP− and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants. PMID:14342234

  20. Marker-free plasmids for gene therapeutic applications--lack of antibiotic resistance gene substantially improves the manufacturing process.

    PubMed

    Mairhofer, Jürgen; Cserjan-Puschmann, Monika; Striedner, Gerald; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Grabherr, Reingard

    2010-04-01

    Plasmid DNA is being considered as a promising alternative to traditional protein vaccines or viral delivery methods for gene therapeutic applications. DNA-based products are highly flexible, stable, are easily stored and can be manufactured on a large scale. Although, much safer than viral approaches, issues have been raised with regard to safety due to possible integration of plasmid DNA into cellular DNA or spread of antibiotic resistance genes to intestinal bacteria by horizontal gene transfer. Accordingly, there is interest in methods for the production of plasmid DNA that lacks the antibiotic resistance gene to further improve their safety profile. Here, we report for the first time the gram-scale manufacturing of a minimized plasmid that is devoid of any additional sequence elements on the plasmid backbone, and merely consists of the target expression cassette and the bacterial origin of replication. Three different host/vector combinations were cultivated in a fed-batch fermentation process, comparing the progenitor strain JM108 to modified strains JM108murselect, hosting a plasmid either containing the aminoglycoside phosphotransferase which provides kanamycin resistance, or a marker-free variant of the same plasmid. The metabolic load exerted by expression of the aminoglycoside phosphotransferase was monitored by measuring ppGpp- and cAMP-levels. Moreover, we revealed that JM108 is deficient of the Lon protease and thereby refined the genotype of JM108. The main consequences of Lon-deficiency with regard to plasmid DNA production are discussed herein. Additionally, we found that the expression of the aminoglycoside phosphotransferase, conferring resistance to kanamycin, was very high in plasmid DNA producing processes that actually inclusion bodies were formed. Thereby, a severe metabolic load on the host cell was imposed, detrimental for overall plasmid yield. Hence, deleting the antibiotic resistance gene from the vector backbone is not only beneficial

  1. Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: A sup 31 P NMR study

    SciTech Connect

    Shashidhar, M.S.; Kuppe, A. ); Volwerk, J.J.; Griffith, O.H.

    1990-09-04

    The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

  2. Crystal structures of phosphotransferase system enzymes PtxB (IIB(Asc)) and PtxA (IIA(Asc)) from Streptococcus mutans.

    PubMed

    Lei, Jian; Li, Lan-Fen; Su, Xiao-Dong

    2009-02-20

    Streptococcus mutans is the primary etiological agent of dental caries in man and other mammalian organisms. This bacterium metabolizes carbohydrates actively and thrives under anaerobic conditions by fermenting l-ascorbate (Asc) via the sga operon, which includes SgaT, PtxB, and PtxA. These three proteins are members of the Asc family of enzyme II (EII) complexes of the bacterial phosphotransferase system. Here, we report the crystal structure of PtxB, solved by single-wavelength anomalous dispersion phasing, and that of PtxA, solved by molecular replacement, from S. mutans. PtxB provides the first crystal structure of an EIIB from the Asc family, composed of a central beta sheet of parallel strands flanked by alpha helices on both sides. The structure of PtxB is similar to the structures of IIB(Mtl) (IIB subunit of mannitol PTS) and IIB(Cel) (IIB subunit of cellobiose) in Escherichia coli despite the low sequence identity. PtxA adopts a globular alpha/beta sandwich structure. The phosphorylation-site His68 is situated between beta2 and beta3, within a hydrophobic pocket. We found that the hydrogen bond on N(delta1) of the active-site histidine is a common means of ensuring that phosphate is on the correct N(varepsilon2) site in many EIIA families. Finally, a model of the PtxB-PtxA complex was constructed, and a PtxA-phospho-PtxB state is proposed. Analyses of the two structures shed light on the catalytic mechanism of the phosphotransferase system. PMID:19135450

  3. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

    PubMed Central

    Mizrachi Nebenzahl, Yaffa; Blau, Karin; Kushnir, Tatyana; Shagan, Marilou; Portnoi, Maxim; Cohen, Aviad; Azriel, Shalhevet; Malka, Itai; Adawi, Asad; Kafka, Daniel; Dotan, Shahar; Guterman, Gali; Troib, Shany; Fishilevich, Tali; Gershoni, Jonathan M; Braiman, Alex; Mitchell, Andrea M; Mitchell, Timothy J; Porat, Nurith; Goliand, Inna; Chalifa Caspi, Vered; Swiatlo, Edwin; Tal, Michael; Ellis, Ronald; Elia, Natalie; Dagan, Ron

    2016-01-01

    In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development. PMID:26990554

  4. Analyses of disease-related GNPTAB mutations define a novel GlcNAc-1-phosphotransferase interaction domain and an alternative site-1 protease cleavage site

    PubMed Central

    Velho, Renata Voltolini; De Pace, Raffaella; Klünder, Sarah; Sperb-Ludwig, Fernanda; Lourenço, Charles Marques; Schwartz, Ida V. D.; Braulke, Thomas; Pohl, Sandra

    2015-01-01

    Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/β-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/β-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/β-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive β-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype–phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity. PMID:25788519

  5. Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

    PubMed Central

    Thomas, Suzanne; Brochu, Denis; Vadeboncoeur, Christian

    2001-01-01

    In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for α-galactosidase, β-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of α-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and

  6. Co-induction of beta-galactosidase and the lactose-P-enolpyruvate phosphotransferase system in Streptococcus salivarius and Streptococcus mutans.

    PubMed Central

    Hamilton, I R; Lo, G C

    1978-01-01

    The addition of lactose, galactose, or isopropyl-beta-D-thiogalactoside (IPTG) to glucose-grown cells of Streptococcus salivarius 25975 resulted in the co-induction of both the lactose-P-enolpyruvate phosphotransferase system (lactose-PTS) and beta-galactosidase, with the latter the predominant metabolic system. With various strains of Streptococcus mutans and Streptococcus sanguis 10556, on the other hand, the lactose-PTS was the major metabolic pathway with beta-galactosidase induced either to low or negligible levels. In all cases, induction of the lactose-PTS resulted in the concomitant induction of 6-P-beta-galactosidase. The induction by lactose of both the lactose-PTS and beta-galactosidase in all strains was repressed by glucose and other catabolites, notably, fructose. Induction of beta-galactosidase in S. salivarius 25975 by IPTG was, however, relatively resistant to glucose repression. Induction experiments with IPTG and lactose suggested that a cellular metabolite of lactose metabolism was a repressor of enzyme activity. Exogenous cAMP was shown to reverse the transient repression by glucose of beta-galactosidase induction in cells of S. salivarius 25975 receiving lactose, provided the cells were grown with small amounts of toluene to overcome the permeability barrier to this nucleotide, cAMP, was however, unable to overcome the permanent repression of beta-galactosidase activity to a significant extent under these conditions. PMID:214423

  7. Effect of Growth Rate and Glucose Concentration on the Activity of the Phosphoenolpyruvate Phosphotransferase System in Streptococcus mutans Ingbritt Grown in Continuous Culture

    PubMed Central

    Ellwood, D. C.; Phipps, P. J.; Hamilton, I. R.

    1979-01-01

    Streptococcus mutans Ingbritt was grown anaerobically in a chemostat with a glucose limitation, as well as with an excess of glucose (amino acid limitation) at dilution rates (D) between 0.05 and 0.4 h−1 (mean generation time = 12 to 1.5 h). The glucose-limited culture produced cells having 1.5- to 6.0-fold greater glycolytic activity than the cells from the glucose-excess culture. The preferred substrate for these cells was glucose, with the glycolytic rate for sucrose being only slightly lower; the rate for fructose was half that of glucose. The glycolytic rate of the glucose-limited cells was maximum at D = 0.1 h−1, with a decline in rate as the growth rate approached D = 0.4 h−1. A comparison of the activity of phosphoenolpyruvate phosphotransferase system (PTS) in the two types of cells showed that the glucose-limited cells had 1.7- to 5.6-fold greater PTS activity for the three sugars than the glucose-excess-grown cells. Whereas little difference was seen between the three sugars with the latter cells, the glucose-PTS had the greatest activity with glucose-limited cells, with the maximum in cells grown at D = 0.1 h−1. Comparison of the rate of sugar uptake in the chemostat with the rate of PTS transport activity in the cells at each growth rate demonstrated that only under conditions of slow growth with a glucose limitation was the PTS system capable of supporting growth on glucose. Furthermore, PTS activity in cells grown with an excess of glucose was insignificant when compared with glucose uptake during growth in the chemostat. This evidence supports the observation that S. mutans possesses at least one other system, in addition to the PTS, for the transport of glucose into the cell. The organism was, however, devoid of glucose-proton symport transport activity. PMID:33901

  8. Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

    PubMed Central

    Li, Yue Zhen; Wang, Dan; Feng, Xue Ying; Jiao, Jian; Chen, Wen Xin

    2015-01-01

    The nitrogen phosphotransferase system (PTSNtr) consists of EINtr, NPr, and EIIANtr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EINtr and NPr to EIIANtr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EINtr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIANtr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIANtr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EINtr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTSNtr in symbiosis was also observed when related PTSNtr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTSNtr in addition to the negative role of unphosphorylated EIIANtr. PMID:26682851

  9. The Phosphoenolpyruvate Phosphotransferase System in Group A Streptococcus Acts To Reduce Streptolysin S Activity and Lesion Severity during Soft Tissue Infection

    PubMed Central

    Gera, Kanika; Le, Tuquynh; Jamin, Rebecca; Eichenbaum, Zehava

    2014-01-01

    Obtaining essential nutrients, such as carbohydrates, is an important process for bacterial pathogens to successfully colonize host tissues. The phosphoenolpyruvate phosphotransferase system (PTS) is the primary mechanism by which bacteria transport sugars and sense the carbon state of the cell. The group A streptococcus (GAS) is a fastidious microorganism that has adapted to a variety of niches in the human body to elicit a wide array of diseases. A ΔptsI mutant (enzyme I [EI] deficient) generated in three different strains of M1T1 GAS was unable to grow on multiple carbon sources (PTS and non-PTS). Complementation with ptsI expressed under its native promoter in single copy was able to rescue the growth defect of the mutant. In a mouse model of GAS soft tissue infection, all ΔptsI mutants exhibited a significantly larger and more severe ulcerative lesion than mice infected with the wild type. Increased transcript levels of sagA and streptolysin S (SLS) activity during exponential-phase growth was observed. We hypothesized that early onset of SLS activity would correlate with the severity of the lesions induced by the ΔptsI mutant. In fact, infection of mice with a ΔptsI sagB double mutant resulted in a lesion comparable to that of either the wild type or a sagB mutant alone. Therefore, a functional PTS is not required for subcutaneous skin infection in mice; however, it does play a role in coordinating virulence factor expression and disease progression. PMID:24379283

  10. Transgenic Arabidopsis tester lines with dominant marker genes.

    PubMed

    Van Lijsebettens, M; Wang, X; Cnops, G; Boerjan, W; Desnos, T; Höfte, H; Van Montagu, M

    1996-06-12

    The map positions of a set of eight T-DNA insertions in the Arabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar), beta-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). The neo, hpt and bar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing the hpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites. PMID:8676880

  11. Regulation of CsrB/C sRNA decay by EIIA(Glc) of the phosphoenolpyruvate: carbohydrate phosphotransferase system.

    PubMed

    Leng, Yuanyuan; Vakulskas, Christopher A; Zere, Tesfalem R; Pickering, Bradley S; Watnick, Paula I; Babitzke, Paul; Romeo, Tony

    2016-02-01

    Csr is a conserved global regulatory system, which uses the sequence-specific RNA-binding protein CsrA to activate or repress gene expression by binding to mRNA and altering translation, stability and/or transcript elongation. In Escherichia coli, CsrA activity is regulated by two sRNAs, CsrB and CsrC, which bind to multiple CsrA dimers, thereby sequestering this protein away from its mRNA targets. Turnover of CsrB/C sRNAs is tightly regulated by a GGDEF-EAL domain protein, CsrD, which targets them for cleavage by RNase E. Here, we show that EIIA(Glc) of the glucose-specific PTS system is also required for the normal decay of these sRNAs and that it acts by binding to the EAL domain of CsrD. Only the unphosphorylated form of EIIA(Glc) bound to CsrD in vitro and was capable of activating CsrB/C turnover in vivo. Genetic studies confirmed that this mechanism couples CsrB/C sRNA decay to the availability of a preferred carbon source. These findings reveal a new physiological influence on the workings of the Csr system, a novel function for the EAL domain, and an important new way in which EIIA(Glc) shapes global regulatory circuitry in response to nutritional status. PMID:26507976

  12. Mannitol transport in Streptococcus mutans.

    PubMed Central

    Maryanski, J H; Wittenberger, C L

    1975-01-01

    A hexitol-inducible, phosphoenolpyruvate-dependent phosphotransferase system was demonstrated in Streptococcus mutans. Cell-free extracts obtained from mannitol-grown cells from a representative strain of each of the five S. mutans serotypes (AHT, BHT, C-67-1, 6715, and LM7) were capable of converting mannitol to mannitol-1-phosphate by a reaction which required phosphoenolpyruvate and Mg2+. Mannitol and sorbitol phosphotransferase activities were found in cell-free extracts prepared from cells grown on the respective substrate, but neither hexitol phosphotransferase activity was present in extracts obtained from cells grown on other substrates examined. A heat-stable, low-molecular-weight component was partially purified from glucose-grown cells and found to stimulate the mannitol phosphotransferase system. Divalent cations Mn2+ and Ca2+ partially replaced Mg2+, while Zn2+ was found to be highly inhibitory. PMID:1194241

  13. Characterization and Nucleotide Sequence of the Cryptic Cel Operon of Escherichia Coli K12

    PubMed Central

    Parker, L. L.; Hall, B. G.

    1990-01-01

    Wild-type Escherichia coli are not able to utilize β-glucoside sugars because the genes for utilization of these sugars are cryptic. Spontaneous mutations in the cel operon allow its expression and enable the organism to ferment cellobiose, arbutin and salicin. In this report we describe the structure and nucleotide sequence of the cel operon. The cel operon consists of five genes: celA, whose function is unknown; celB and celC which encode phosphoenolpyruvate-dependent phosphotransferase system enzyme II(cel) and enzyme III(cel), respectively, for the transport and phosphorylation of β-glucoside sugars; celD, which encodes a negative regulatory protein; and celF, which encodes a phospho-β-glucosidase that acts on phosphorylated cellobiose, arbutin and salicin. The mutationally activated cel operon is induced in the presence of its substrates, and is repressed in their absence. A comparison of proteins encoded by the cel operon with functionally equivalent proteins of the bgl operon, another cryptic E. coli gene system responsible for the catabolism of β-glucoside sugars, revealed no significant homology between these two systems despite common functional characteristics. The celD and celF encoded repressor and phospho-β-glucosidase proteins are homologous to the melibiose regulatory protein and to the melA encoded α-galactosidase of E. coli, respectively. Furthermore, the celC encoded PEP-dependent phosphotransferase system enzyme III(cel) is strikingly homologous to an enzyme III(lac) of the Gram-positive organism Staphylococcus aureus. We conclude that the genes for these two enzyme IIIs diverged much more recently than did their hosts, indicating that E. coli and S. aureus have undergone relatively recent exchange of chromosomal genes. PMID:2179047

  14. Distribution of proteins similar to IIIManH and IIIManL of the Streptococcus salivarius phosphoenolpyruvate:mannose-glucose phosphotransferase system among oral and nonoral bacteria.

    PubMed Central

    Pelletier, M; Frenette, M; Vadeboncoeur, C

    1995-01-01

    In Streptococcus salivarius, the phosphoenolpyruvate (PEP):mannose-glucose phosphotransferase system, which concomitantly transports and phosphorylates mannose, glucose, fructose, and 2-deoxyglucose, is composed of the general energy-coupling proteins EI and HPr, the specific membrane-bound IIIMan, and two forms of a protein called IIIMan, with molecular weights of 38,900 (IIIManH) and 35,200 (IIIManL), that are found in the cytoplasm as well as associated with the membrane. Several lines of evidence suggest that IIIManH and/or IIIManL are involved in the control of sugar metabolism. To determine whether other bacteria possess these proteins, we tested for their presence in 28 oral streptococcus strains, 3 nonoral streptococcus strains, 2 lactococcus strains, 2 enterococcus strains, 2 bacillus strains, 1 lactobacillus strain, Staphylococcus aureus, and Escherichia coli. Three approaches were used to determine whether the IIIMan proteins were present in these bacteria: (i) Western blot (immunoblot) analysis of cytoplasmic and membrane proteins, using anti-IIIManH and anti-IIIManH rabbit polyclonal antibodies; (ii) analysis of PEP-dependent phosphoproteins by polyacrylamide gel electrophoresis; and (iii) inhibition by anti-IIIMan antibodies of the PEP-dependent phosphorylation of 2-deoxyglucose (a mannose analog) by crude cellular extracts. Only the species S. salivarius and Streptococcus vestibularis possessed the two forms of IIIMan. Fifteen other streptococcal species possessed one protein with a molecular weight between 35,200 and 38,900 that cross-reacted with both antibodies. In the case of 9 species, a protein possessing the same electrophoretic mobility was phosphorylated at the expense of PEP. No such phosphoprotein, however, could be detected in the other six species. A III(Man)-like protein with a molecular weight of 35,500 was also detected in Lactobacillus casei by Western blot experiments as well as by PEP-dependent phosphoprotein analysis, and a

  15. Uptake and transient expression of chimeric genes in seed-derived embryos.

    PubMed Central

    Töpfer, R; Gronenborn, B; Schell, J; Steinbiss, H H

    1989-01-01

    Uptake of DNA in dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. Gene expression vectors used in this study contained a neomycin phosphotransferase (NPT) II reporter gene fused to various promoters. Some of the chimeric "neo" genes were shown to yield reproducibly NPT II activity in germinating embryos. This NPT II activity was increased markedly when the neo genes were carried by a vector capable of autonomous replication. Dimers of wheat dwarf virus, a monopartite gemini virus, were thus shown to be effective in amplifying the transient expressed NPT II activity in embryos of several cereals. These and other observations indicate that the observed transient expression really results from DNA uptake and expression in plant embryo cells and is not due to contaminating microorganisms. PMID:2562504

  16. Genomic characterization of symbiotic mycoplasmas from the stomach of deep-sea isopod bathynomus sp.

    PubMed

    Wang, Yong; Huang, Jiao-Mei; Wang, Shao-Lu; Gao, Zhao-Ming; Zhang, Ai-Qun; Danchin, Antoine; He, Li-Sheng

    2016-09-01

    Deep-sea isopod scavengers such as Bathynomus sp. are able to live in nutrient-poor environments, which is likely attributable to the presence of symbiotic microbes in their stomach. In this study we recovered two draft genomes of mycoplasmas, Bg1 and Bg2, from the metagenomes of the stomach contents and stomach sac of a Bathynomus sp. sample from the South China Sea (depth of 898 m). Phylogenetic trees revealed a considerable genetic distance to other mycoplasma species for Bg1 and Bg2. Compared with terrestrial symbiotic mycoplasmas, the Bg1 and Bg2 genomes were enriched with genes encoding phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) and sodium-driven symporters responsible for the uptake of sugars, amino acids and other carbohydrates. The genome of mycoplasma Bg1 contained sialic acid lyase and transporter genes, potentially enabling the bacteria to attach to the stomach sac and obtain organic carbons from various cell walls. Both of the mycoplasma genomes contained multiple copies of genes related to proteolysis and oligosaccharide degradation, which may help the host survive in low-nutrient conditions. The discovery of the different types of mycoplasma bacteria in the stomach of this deep-sea isopod affords insights into symbiotic model of deep-sea animals and genomic plasticity of mycoplasma bacteria. PMID:27312602

  17. The sim operon facilitates the transport and metabolism of sucrose isomers in Lactobacillus casei ATCC 334.

    PubMed

    Thompson, John; Jakubovics, Nicholas; Abraham, Bindu; Hess, Sonja; Pikis, Andreas

    2008-05-01

    Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with M(r)s of approximately 50,000 and approximately 17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the approximately 50-kDa protein as an NAD(+)- and metal ion-dependent phospho-alpha-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-alpha-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to approximately 1.5- and approximately 1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon. PMID:18310337

  18. Genetic transformation and gene expression in white pine (pinus strobus)

    SciTech Connect

    Minocha, R.

    1987-10-01

    The objectives of the study were: (1) to develop protocols for transformation of white pine (Pinus strobus) embryonic tissue; and (2) to analyze the regulation of foreign gene expression in Pinus strobus. A number of Agrobacterium tumefaciens strains containing chimeric genes for neomycin phosphotransferase (NPTII for kanamycin resistance) and chloramphenicol acetyl transferase (CAT) under the control of either a constitutive promoter (NOS-nopaline synthase) or light-inducible promoters (RuBisCO small subunit and chlorophyll a/b binding protein) were used. A variety of tissues from white pine seedlings and mature trees was used. The techniques for transformation were modified from those used for tobacco transformation. The results show that white pine tissue from young seedlings is high suitable for transformation by A. tumefaciens. Whereas the normal tissues are very sensitive to kanamycin, transformed callus was quite resistant to this antibiotic.

  19. Co-transformation of grapevine somatic embryos to produce transgenic plants free of marker genes.

    PubMed

    Dutt, Manjul; Li, Zhijian T; Dhekney, Sadanand A; Gray, Dennis J

    2012-01-01

    A cotransformation system using somatic embryos was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. "Thompson Seedless" somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bidirectional dual promoter complex. Our technique included a short positive selection phase of cotransformed somatic embryos on liquid medium containing 100 mg/L kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg/L 5-fluorocytosine. PMID:22351010

  20. The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

    SciTech Connect

    Tan, K.; Borovilos, M.; Zhou, M; Horer, S; Clancy, S; Moy, S; Volkart, LL; Sassoon, J; Baumann, U; Joachimiak, A

    2009-12-25

    Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representing a novel all {alpha}-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.

  1. Transcriptional analysis of prebiotic uptake and catabolism by Lactobacillus acidophilus NCFM.

    PubMed

    Andersen, Joakim Mark; Barrangou, Rodolphe; Hachem, Maher Abou; Lahtinen, Sampo J; Goh, Yong-Jun; Svensson, Birte; Klaenhammer, Todd R

    2012-01-01

    The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota. PMID:23028535

  2. Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824.

    PubMed

    Yu, Yang; Tangney, Martin; Aass, Hans C; Mitchell, Wilfrid J

    2007-03-01

    Although the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolize a wide range of carbohydrates offers the potential for revival based on the use of cheap, low-grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolyzed by a phospho-beta-galactosidase. These activities are induced during growth on lactose but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho-beta-galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed. PMID:17209069

  3. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  4. [Cloning of HAL1 gene and characterization for salt tolerance tomato].

    PubMed

    Zhang, Q; Wang, S F; Zhao, Y X; Zhao, K F; Zhang, H

    2001-11-01

    The HAL1 gene was cloned by PCR strategy and confirmed by sequencing. Its open read frame is 879 bp, encoding a peptide of 294 amino acids (32 kD Protein). A chimaeric construct of HAL1 and Npt II (neomycin phosphotransferase II) was constructed and introduced into commercial cultivars of tomato (Zhong SU No. 5: Lycopersicon escullentum) by Agrobacterium tumefacien-mediated gene transformation. Transformants were selected for their ability to grow and root on media containing kanamycin. Transformation was confirmed by analysis of PCR, Southern blot and RT-PCR. The salt tolerance of transgenic tomato is evaluated by comparing the fresh weight, dry weight, Na+, K+ content of transgenic tomato and control tomato. It is concluded that the over-expressing of HAL1 in tomato could enhance the salt tolerance of the transgenic tomato. PMID:11910760

  5. Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.).

    PubMed

    Vermeulen, A; Vaucheret, H; Pautot, V; Chupeau, Y

    1992-06-01

    Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. PMID:24203132

  6. Different 3' end regions strongly influence the level of gene expression in plant cells.

    PubMed Central

    Ingelbrecht, I L; Herman, L M; Dekeyser, R A; Van Montagu, M C; Depicker, A G

    1989-01-01

    We have investigated the functional role of a 3' end region on the expression of a reporter gene in plant cells. In stably transformed plants, expression of the reporter gene without a plant gene 3' end is variable and depends on the fortuitous presence of polyadenylation signals in the downstream sequences. When the reporter gene is flanked by pBR322 DNA, 3'-processing and polyadenylation occurs at (a) cryptic site(s) within these vector sequences. Using a transient gene expression system, we present a deletion analysis of the 3' end of the octopine synthase gene showing that the most proximal polyadenylation signal per se is not sufficient to ensure expression but that a downstream (G)T-rich sequence is also required. Optimal expression of the fusion gene requires more than 98 base pairs and at most 142 base pairs downstream from the most distal polyadenylation site. We analyzed the expression of chimeric genes with 3' end sequences originating from different plant genes. In the transient expression assay, all constructs direct similar neomycin phosphotransferase II activities. However, in stably transformed tissue, the gene constructs displayed characteristic expression levels which varied as much as 60-fold. This result suggests a role for 3' end sequences in post-transcriptional processes such as efficiency of 3'-processing and/or mRNA stability. PMID:2562510

  7. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA.

    PubMed

    Teixeira, Bertinellys; Rodulfo, Hectorina; Carreño, Numirin; Guzmán, Militza; Salazar, Elsa; De Donato, Marcos

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America. PMID:27007556

  8. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA

    PubMed Central

    TEIXEIRA, Bertinellys; RODULFO, Hectorina; CARREÑO, Numirin; GUZMÁN, Militza; SALAZAR, Elsa; DONATO, Marcos DE

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America. PMID:27007556

  9. Mutations affecting transport of the hexitols D-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping.

    PubMed Central

    Lengeler, J

    1975-01-01

    Mutants of Escherichia coli K-12 unable to grow on any of the three naturally occurring hexitols D-manitol, D-glucitol, and galactitol and, among these specifically, mutants with altered transport and phosphorylating activity have been isolated. Different isolation procedures have been utilized, including suicide by D-[3H]mannitol, chemotaxis, and resistance to the toxic hexitol analogue 2-deoxy-arabino-hexitol. Mutations thus obtained have been mapped in four distinct operons. (i) Mutations affecting an enzyme II-complexmt1 activity of the phosphoenolpyruvate-dependent phosphotransferase system all map in gene mtlA. This gene has previously been shown (Solomon and Lin, 1972) to be part of an operon, mtl, located at 71 min on the E. coli linkage map containing, in addition to mtlA, the cis-dominant regulatory gene mtlC and mtlD, the structural gene for the enzyme D-mannitol-1-phosphate dehydrogenase. The gene order in this operon, induced by D-mannitol, is mtlC A D. (ii) Mutations in gene gutA affecting a second enzyme II-complexgut of the phosphotransferase system map at 51 min, clustered in operon gutC A D together with the cis-dominant regulatory gene gutC and the structural gene gutD for the enzyme D-glucitol-6-phosphate dehydrogenase. The gut operon, previously called sbl or srl, is induced by D-glucitol. (iii) Mutations affecting the transport and catabolism of galactitol are clustered in a third operon, gatC A D, located at 40.5 min. This operon again contains a cis-dominant regulatory gene, gatC, the structural gene gatD for galactitol-1-phosphate dehydrogenase, and gene gatA coding for a thrid hexitol-specific enzyme II-complexgat. Other genes coding for two additional enzymes involved in galactitol catabolism apparently are not linked to gatC A D. (iv) A fourth class of mutants pleiotropically negative for hexitol growth and transport maps in the pts operon. Triple-negative mutants (mtlA gutA gatA) do not have further transport or phosphorylating activity

  10. Genes and Gene Therapy

    MedlinePlus

    ... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  11. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  12. A study of lactose metabolism in Lactococcus garvieae reveals a genetic marker for distinguishing between dairy and fish biotypes.

    PubMed

    Fortina, Maria Grazia; Ricci, Giovanni; Borgo, Francesca

    2009-06-01

    Dairy and fish isolates of Lactococcus garvieae were tested for their ability to utilize lactose and to grow in milk. Fish isolates were unable to assimilate lactose, but unexpectedly, they possessed the ability to grow in milk. Genetic studies, carried out constructing different vectorette libraries, provided evidence that in fish isolates, no genes involved in lactose utilization were present. For L. garvieae dairy isolates, a single system for the catabolism of lactose was found. It consists of a lactose transport and hydrolysis depending on a phosphoenolpyruvate-dependent phosphotransferase system combined with a phospho-beta-galactosidase. The genes involved were highly similar at the nucleotide sequence level to their counterparts in Lactococcus lactis; however, while in many L. lactis strains these genes are plasmid encoded, in L. garvieae they are chromosomally located. Thus, in the species L. garvieae, the phospho-beta-galactosidase gene, detectable in all strains of dairy origin but lacking in fish isolates, can be considered a reliable genetic marker for distinguishing biotypes in the two diverse ecological niches. Moreover, we obtained information regarding the complete nucleotide sequence of the gal operon in L. garvieae, consisting of a galactose permease and the Leloir pathway enzymes. This is one of the first reports concerning the determination of the nucleotide sequences of genes (other than the 16S rDNA gene) in L. garvieae and should be considered a step in a continuous effort to explore the genome of this species, with the aim of determining the real relationship between the presence of L. garvieae in dairy products and food safety. PMID:19610335

  13. Genotypic and phenotypic evaluation of the evolution of high-level daptomycin nonsusceptibility in vancomycin-resistant Enterococcus faecium.

    PubMed

    Humphries, Romney M; Kelesidis, Theodoros; Tewhey, Ryan; Rose, Warren E; Schork, Nicholas; Nizet, Victor; Sakoulas, George

    2012-11-01

    Whole-genome sequencing and cell membrane studies of three clonal Enterococcus faecium strains with daptomycin MICs of 4, 32, and 192 μg/ml were performed, revealing nonsynonymous single nucleotide variants in eight open reading frames, including those predicted to encode a phosphoenolpyruvate-dependent, mannose-specific phosphotransferase system, cardiolipin synthetase, and EzrA. Membrane studies revealed a higher net surface charge among the daptomycin-nonsusceptible isolates and increased septum formation in the isolate with a daptomycin MIC of 192 μg/ml. PMID:22948885

  14. Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

    PubMed Central

    Song, Yajian; Xue, Yanfen; Ma, Yanhe

    2013-01-01

    The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

  15. Bifidobacterium longum Requires a Fructokinase (Frk; ATP:d-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

    PubMed Central

    Caescu, Cristina I.; Vidal, Olivier; Krzewinski, Frédéric; Artenie, Vlad; Bouquelet, Stéphane

    2004-01-01

    Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co2+-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Kmfructose = 0.739 ± 0.18 mM and KmATP = 0.756 ± 0.08 mM), is highly specific for d-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for d-fructose. PMID:15375133

  16. A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.

    PubMed

    Nishizawa-Yokoi, Ayako; Nonaka, Satoko; Osakabe, Keishi; Saika, Hiroaki; Toki, Seiichi

    2015-09-01

    Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species. PMID:26143254

  17. The 5 A projection structure of the transmembrane domain of the mannitol transporter enzyme II.

    PubMed

    Koning, R I; Keegstra, W; Oostergetel, G T; Schuurman-Wolters, G; Robillard, G T; Brisson, A

    1999-04-16

    The uptake of mannitol in Escherichia coli is controlled by the phosphoenolpyruvate dependent phosphotransferase system. Enzyme II mannitol (EIIMtl) is part of the phosphotransferase system and consists of three covalently bound domains. IICMtl, the integral membrane domain of EIIMtl, is responsible for mannitol transport across the cytoplasmic membrane. In order to understand this molecular process, two-dimensional crystals of IICMtl were grown by reconstitution into lipid bilayers and their structure was investigated by cryo-electron crystallography. The IICMtl crystals obey p22121 symmetry and have a unit cell of 125 Ax65 A, gamma=90 degrees. A projection structure was determined at 5 A resolution using both electron images and electron diffractograms. The unit cell contains two IICMtl dimers with a size of about 40 Ax90 A, which are oriented up and down in the crystal. Each monomer exhibits six domains of high density which most likely correspond to transmembrane alpha-helices and cytoplasmic loops. PMID:10222194

  18. Metagenomic analysis reveals that bacteriophages are reservoirs of antibiotic resistance genes.

    PubMed

    Subirats, Jéssica; Sànchez-Melsió, Alexandre; Borrego, Carles M; Balcázar, José Luis; Simonet, Pascal

    2016-08-01

    A metagenomics approach was applied to explore the presence of antibiotic resistance genes (ARGs) in bacteriophages from hospital wastewater. Metagenomic analysis showed that most phage sequences affiliated to the order Caudovirales, comprising the tailed phage families Podoviridae, Siphoviridae and Myoviridae. Moreover, the relative abundance of ARGs in the phage DNA fraction (0.26%) was higher than in the bacterial DNA fraction (0.18%). These differences were particularly evident for genes encoding ATP-binding cassette (ABC) and resistance-nodulation-cell division (RND) proteins, phosphotransferases, β-lactamases and plasmid-mediated quinolone resistance. Analysis of assembled contigs also revealed that blaOXA-10, blaOXA-58 and blaOXA-24 genes belonging to class D β-lactamases as well as a novel blaTEM (98.9% sequence similarity to the blaTEM-1 gene) belonging to class A β-lactamases were detected in a higher proportion in phage DNA. Although preliminary, these findings corroborate the role of bacteriophages as reservoirs of resistance genes and thus highlight the necessity to include them in future studies on the emergence and spread of antibiotic resistance in the environment. PMID:27312355

  19. Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.

    PubMed

    Sanson, Misu; Makthal, Nishanth; Gavagan, Maire; Cantu, Concepcion; Olsen, Randall J; Musser, James M; Kumaraswami, Muthiah

    2015-06-01

    Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis. PMID:25824840

  20. Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

    PubMed Central

    Sanson, Misu; Makthal, Nishanth; Gavagan, Maire; Cantu, Concepcion; Olsen, Randall J.; Musser, James M.

    2015-01-01

    Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis. PMID:25824840

  1. Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism, and products.

    PubMed Central

    Thompson, J; Nguyen, N Y; Robrish, S A

    1992-01-01

    Studies of sucrose utilization by Fusobacterium mortiferum ATCC 25557 have provided the first definitive evidence for phosphoenolpyruvate-dependent sugar:phosphotransferase activity in the family Bacteroidaceae. The phosphoenolpyruvate-dependent sucrose:phosphotransferase system and the two enzymes required for the dissimilation of sucrose 6-phosphate are induced specifically by growth of F. mortiferum on the disaccharide. Monomeric sucrose 6-phosphate hydrolase (M(r), 52,000) and a dimeric ATP-dependent fructokinase (subunit M(r), 32,000) have been purified to electrophoretic homogeneity. The physicochemical and catalytic properties of these enzymes have been examined, and the N-terminal amino acid sequences for both proteins are reported. The characteristics of sucrose 6-phosphate hydrolase and fructokinase from F. mortiferum are compared with the same enzymes from both gram-positive and gram-negative species. Butyric, acetic, and D-lactic acids are the end products of sucrose fermentation by F. mortiferum. A pathway is proposed for the translocation, phosphorylation, and metabolism of sucrose by this anaerobic pathogen. Images PMID:1533618

  2. Emergence of macrolide resistance gene mph(B) in Streptococcus uberis and cooperative effects with rdmC-like gene.

    PubMed

    Achard, Adeline; Guérin-Faublée, Véronique; Pichereau, Vianney; Villers, Corinne; Leclercq, Roland

    2008-08-01

    Streptococcus uberis UCN60 was resistant to spiramycin (MIC = 8 microg/ml) but susceptible to erythromycin (MIC = 0.06 microg/ml), azithromycin (MIC = 0.12 microg/ml), josamycin (MIC = 0.25 microg/ml), and tylosin (MIC = 0.5 microg/ml). A 2.5-kb HindIII fragment was cloned from S. uberis UCN60 DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to spiramycin by inactivation. The sequence analysis of the fragment showed the presence of an rdmC-like gene that putatively encoded a protein belonging to the alpha/beta hydrolase family and of the first 196 nucleotides of the mph(B) gene putatively encoding a phosphotransferase known to inactivate 14-, 15-, and 16-membered macrolides in E. coli. The entire mph(B) gene was then identified in S. uberis UCN60. The two genes were expressed alone or in combination in E. coli, Staphylococcus aureus, and Enterococcus faecalis. Analysis of MICs revealed that rdmC-like alone did not confer resistance to erythromycin, tylosin, and josamycin in those three hosts. It conferred resistance to spiramycin in E. coli and E. faecalis but not in S. aureus. mph(B) conferred resistance in E. coli to erythromycin, tylosin, josamycin, and spiramycin but only low levels of resistance in E. faecalis and S. aureus to spiramycin (MIC = 8 microg/ml). The combination of mph(B) and rdmC-like genes resulted in a resistance to spiramycin and tylosin in the three hosts that significantly exceeded the mere addition of the resistance levels conferred by each resistance mechanism alone. PMID:18519724

  3. Gene expression profiling of a nisin-sensitive Listeria monocytogenes Scott A ctsR deletion mutant.

    PubMed

    Liu, Yanhong; Morgan, Shannon; Ream, Amy; Huang, Lihan

    2013-05-01

    Listeria monocytogenes is a food-borne pathogen of significant threat to public health. Nisin is the only bacteriocin that can be used as a food preservative. Due to its antimicrobial activity, it can be used to control L. monocytogenes in food; however, the antimicrobial mechanism of nisin activity against L. monocytogenes is not fully understood. The CtsR (class III stress gene repressor) protein negatively regulates the expression of class III heat shock genes. A spontaneous pressure-tolerant ctsR deletion mutant that showed increased sensitivity to nisin has been identified. Microarray technology was used to monitor the gene expression profiles of the ctsR mutant under treatments with nisin. Compared to the nisin-treated wild type, 113 genes were up-regulated (>2-fold increase) in the ctsR deletion mutant whereas four genes were down-regulated (<-2-fold decrease). The up-regulated genes included genes that encode for ribosomal proteins, membrane proteins, cold-shock domain proteins, translation initiation and elongation factors, cell division, an ATP-dependent ClpC protease, a putative accessory gene regulator protein D, transport and binding proteins, a beta-glucoside-specific phosphotransferase system IIABC component, as well as hypothetical proteins. The down-regulated genes consisted of genes that encode for virulence, a transcriptional regulator, a stress protein, and a hypothetical protein. The gene expression changes determined by microarray assays were confirmed by quantitative real-time PCR analyses. Moreover, an in-frame deletion mutant for one of the induced genes (LMOf2365_1877) was constructed in the wild-type L. monocytogenes F2365 background. ΔLMOf2365_1877 had increased nisin sensitivity compared to the wild-type strain. This study enhances our understanding of how nisin interacts with the ctsR gene product in L. monocytogenes and may contribute to the understanding of the antibacterial mechanisms of nisin. PMID:23494707

  4. Genome reconstruction and gene expression of "Candidatus Accumulibacter phosphatis" Clade IB performing biological phosphorus removal.

    PubMed

    Mao, Yanping; Yu, Ke; Xia, Yu; Chao, Yuanqing; Zhang, Tong

    2014-09-01

    We report the first integrated metatranscriptomic and metagenomic analysis of enhanced biological phosphorus removal (EBPR) sludge. A draft genome of Candidatus Accumulibacter spp. strain HKU-1, a member of Clade IB, was retrieved. It was estimated to be ∼90% complete and shared average nucleotide identities of 83% and 88% with the finished genome CAP IIA UW-1 and the draft genome CAP IA UW-2, respectively. Different from CAP IIA UW-1, the phosphotransferase (pap) in polyphosphate metabolism and V-ATPase in orthophosphate transport were absent from CAP IB HKU-1. Additionally, unlike CAP IA UW-2, CAP IB HKU-1 carried the genes for carbon fixation and nitrogen fixation. Despite these differences, the key genes required for acetate uptake, glycolysis and polyhydroxyalkanoate (PHA) synthesis were conserved in all these Accumulibacter genomes. The preliminary metatranscriptomic results revealed that the most significantly up-regulated genes of CAP IB HKU-1 from the anaerobic to the aerobic phase were responsible for assimilatory sulfate reduction, genetic information processing and phosphorus absorption, while the down-regulated genes were related to N2O reduction, PHA synthesis and acetyl-CoA formation. This study yielded another important Accumulibacter genome, revealed the functional difference within the Accumulibacter Type I, and uncovered the genetic responses to EBPR stimuli at a higher resolution. PMID:25089581

  5. Degenerate PCR primers for detecting putative priming glycosyltransferase genes in Bifidobacterium strains.

    PubMed

    Hidalgo-Cantabrana, C; Ordoñez, I; Ruas-Madiedo, P; Margolles, A

    2015-01-01

    A new PCR-based method to detect putative exopolysaccharide (EPS) producers from the genus Bifidobacterium was developed based on the detection of two priming glycosyltransferase genes: rfbP (undecaprenyl-phosphate sugar phospho-transferase) and cpsD (galactosyl-transferase). An in silico analysis of the genomes of 28 bifidobacterial strains, belonging to 8 different species, allowed us to detect rfbP, cpsD, or both, in the large majority of the genomes. Based on DNA sequence homology studies, 24 degenerated primers were synthesised in order to select the primer pairs with the broadest capacity to detect the presence of these genes. Four primer pairs targeting internal regions of rfbP and cpsD were selected, allowing the detection of at least one of the two genes in 63 out of 99 bifidobacterial strains analysed, whereas control strains from other genera yielded negative results, suggesting that these genes are widely spread in this genus. The use of these primers is recommended to screen for the potential of Bifidobacterium strains to produce EPS. PMID:25653152

  6. Expression of Foreign Genes in Transgenic Yellow-Poplar Plants 1

    PubMed Central

    Wilde, H. Dayton; Meagher, Richard B.; Merkle, Scott A.

    1992-01-01

    Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves. ImagesFigure 1Figure 2 PMID:16668600

  7. Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes

    SciTech Connect

    Scorza, R.; Zimmerman, T.W.; Cordts, J.M.; Footen, K.J. ); Ravelonandro, M. . Station de Pathologie Vegetale)

    1994-09-01

    Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), [beta]-glucuronidase (GUS).

  8. Genes and gene regulation

    SciTech Connect

    MacLean, N.

    1988-01-01

    Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

  9. Seryl-phosphorylated HPr regulates CcpA-independent carbon catabolite repression in conjunction with PTS permeases in Streptococcus mutans.

    PubMed

    Zeng, Lin; Burne, Robert A

    2010-03-01

    Carbohydrate catabolite repression (CCR) in Streptococcus mutans can be independent of catabolite control protein A (CcpA) and requires specific components of phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) permeases. Here, the effects of various ptsH (HPr) and hprK (HPr kinase/phosphatase) mutations on growth and CCR were evaluated. An hprKV265F mutation, which enhanced Ser46 phosphorylation of HPr, inhibited growth on multiple PTS sugars. A ptsHS46A mutation reversed the effects of hprKV265F in most cases. A strain carrying a ptsHS46D mutation, which mimics HPr(Ser-P), presented with more severe growth defects than the hprKV265F mutant. The hprKV265F mutant enhanced CCR of the fruA and levD operons, a phenotype reversible by the ptsHS46A mutation. The effects of the hprKV265F mutation on fruA and levD expression were independent of CcpA, but dependent on ManL (IIAB(Man)) and, to a lesser extent, on FruI (IIABC(Fru)), in a carbohydrate-specific fashion. Expression of the Bacillus subtilis ptsG gene in the manL mutant did not restore CCR of the lev or fru operons. The hprKV265F mutation inhibited growth on cellobiose and lactose, but only the transcription of the cel operon was decreased. Thus, in S. mutans, serine-phosphorylated HPr functions in concert with particular PTS permeases to prioritize carbohydrate utilization by modulating sugar transport and transcription of catabolic operons. PMID:20487301

  10. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  11. Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity.

    PubMed

    Chen, Chen; Zhao, Guozhong; Chen, Wei; Guo, Benheng

    2015-11-01

    Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. PMID:26319882

  12. Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

    PubMed Central

    Chen, Chen; Zhao, Guozhong

    2015-01-01

    Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. PMID:26319882

  13. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. PMID:26672454

  14. Characterization of a Multiresistant Mosaic Plasmid from a Fish Farm Sediment Exiguobacterium sp. Isolate Reveals Aggregation of Functional Clinic-Associated Antibiotic Resistance Genes

    PubMed Central

    Yang, Jing; Wang, Chao; Wu, Jinyu; Liu, Li; Zhang, Gang

    2014-01-01

    The genus Exiguobacterium can adapt readily to, and survive in, diverse environments. Our study demonstrated that Exiguobacterium sp. strain S3-2, isolated from marine sediment, is resistant to five antibiotics. The plasmid pMC1 in this strain carries seven putative resistance genes. We functionally characterized these resistance genes in Escherichia coli, and genes encoding dihydrofolate reductase and macrolide phosphotransferase were considered novel resistance genes based on their low similarities to known resistance genes. The plasmid G+C content distribution was highly heterogeneous. Only the G+C content of one block, which shared significant similarity with a plasmid from Exiguobacterium arabatum, fit well with the mean G+C content of the host. The remainder of the plasmid was composed of mobile elements with a markedly lower G+C ratio than the host. Interestingly, five mobile elements located on pMC1 showed significant similarities to sequences found in pathogens. Our data provided an example of the link between resistance genes in strains from the environment and the clinic and revealed the aggregation of antibiotic resistance genes in bacteria isolated from fish farms. PMID:24362420

  15. Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy.

    PubMed

    Themis, M; May, D; Coutelle, C; Newbold, R F

    2003-09-01

    Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy. PMID:12923569

  16. Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis.

    PubMed

    Pichard, Scott L; Paul, John H

    1991-06-01

    A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences. PMID:16348507

  17. Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton

    PubMed Central

    Bajwa, Kamran S.; Shahid, Ahmad A.; Rao, Abdul Q.; Bashir, Aftab; Aftab, Asia; Husnain, Tayyab

    2015-01-01

    Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification. PMID:26583018

  18. RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells.

    PubMed

    Grishchenko, O V; Kiselev, K V; Tchernoded, G K; Fedoreyev, S A; Veselova, M V; Bulgakov, V P; Zhuravlev, Y N

    2016-09-01

    Maackia amurensis Rupr. et Maxim is a valuable leguminous tree grown in the Russian Far East, in China, and in Korea. Polyphenols from the heartwood of this species (primarily stilbenes and isoflavonoids) possess strong hepatoprotective activity. Callus culture of M. amurensis produced isoflavonoids and their derivatives. In pharmacological experiments, the callus complex was at least as effective, as the plant complex. To increase the yield of isoflavonoids, calli were transformed with the rolB gene of Agrobacterium rhizogenes. Neomycin phosphotransferase (nptII) gene was used for transgenic cell selection. Three rolB transgenic callus lines with different levels of the rolB gene expression were established. Insertion of the rolB gene caused alterations in callus structure, growth, and isoflavonoid production, and stronger alterations were observed with higher expression levels. MB1, MB2, and MB4 cultures accumulated 1.4, 1.5, and 2.1 % of dry weight (DW) isoflavonoids, respectively. In contrast, the empty vector-transformed MV culture accumulated 1.22 % DW. Isoflavonoid productivity of the obtained MB1, MB2, and MB4 cultures was equal to 117, 112, and 199 mg/L of medium, respectively, comparing to 106 mg/L for the MV culture. High level of expression of the rolB gene in MB4 culture led to a 2-fold increase in the isoflavonoid content and productivity and reliably increased dry biomass accumulation. Lower expression levels of the rolB gene in MB1 and MB2 calli did not significantly enhance biomass accumulation and isoflavonoid content, although the rolB gene activated isoflavonoid biosynthesis during the early growth stages and caused the increased content of several distinct compounds. PMID:27063013

  19. Long-term transplantation of canine keratinocytes made resistant to G418 through retrovirus-mediated gene transfer.

    PubMed Central

    Flowers, M E; Stockschlaeder, M A; Schuening, F G; Niederwieser, D; Hackman, R; Miller, A D; Storb, R

    1990-01-01

    We studied cultured canine keratinocytes to determine whether they could serve as targets for retrovirus-mediated gene transfer and whether infected cells could persist after transplantation into dogs, a large random-bred model for gene transfer studies. Canine keratinocytes obtained from skin biopsy samples were cultured in vitro with lethally irradiated NIH 3T3 cells used as a feeder layer. The keratinocyte colonies consisted of squamous epithelium with numerous desmosomes, tonofilaments, and keratohyalin granules. In addition, the cells were strongly reactive with monoclonal antibodies to cytokeratin intermediate filament proteins. For the infection studies, we grew the keratinocytes on a feeder layer of lethally irradiated PA317 retrovirus packaging cells, which produced a helper-free amphotropic retroviral vector containing the neomycin phosphotransferase (neo) gene. After cocultivation, 34% (range, 10-76%) of the keratinocytes were found to be resistant to the neomycin analogue G418. Infected keratinocytes were then transplanted into the dog of origin; 1% (range, less than 0.1-3%) of the keratinocytes obtained 27-130 days after transplantation from skin biopsy samples gave rise to G418-resistant colonies. We conclude that canine keratinocytes cultured in vitro can be infected efficiently with a neo gene-containing retroviral vector, and they show persistent G418 resistance for at least 130 days after transplantation into the skin donor. Images PMID:2315325

  20. Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

    PubMed

    Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

    2013-12-01

    Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops. PMID:23974493

  1. Molecular breeding of lignin-degrading brown-rot fungus Gloeophyllum trabeum by homologous expression of laccase gene.

    PubMed

    Arimoto, Misa; Yamagishi, Kenji; Wang, Jianqiao; Tanaka, Kanade; Miyoshi, Takanori; Kamei, Ichiro; Kondo, Ryuichiro; Mori, Toshio; Kawagishi, Hirokazu; Hirai, Hirofumi

    2015-12-01

    The basidiomycete Gloeophyllum trabeum KU-41 can degrade Japanese cedar wood efficiently. To construct a strain better suited for biofuel production from Japanese cedar wood, we developed a gene transformation system for G. trabeum KU-41 using the hygromycin phosphotransferase-encoding gene (hpt) as a marker. The endogenous laccase candidate gene (Gtlcc3) was fused with the promoter of the G. trabeum glyceraldehyde-3-phosphate dehydrogenase-encoding gene and co-transformed with the hpt-bearing pAH marker plasmid. We obtained 44 co-transformants, and identified co-transformant L#61, which showed the highest laccase activity among all the transformants. Moreover, strain L#61 was able to degrade lignin in Japanese cedar wood-containing medium, in contrast to wild-type G. trabeum KU-41 and to a typical white-rot fungus Phanerochaete chrysosporium. By using strain L#61, direct ethanol production from Japanese cedar wood was improved compared to wild type. To our knowledge, this study is the first report of the molecular breeding of lignin-degrading brown-rot fungus and direct ethanol production from softwoods by co-transformation with laccase overproduction constructs. PMID:26695948

  2. Analysis of an Ordered, Comprehensive STM Mutant Library in Infectious Borrelia burgdorferi: Insights into the Genes Required for Mouse Infectivity

    PubMed Central

    Lin, Tao; Gao, Lihui; Zhang, Chuhua; Odeh, Evelyn; Jacobs, Mary B.; Coutte, Loïc; Chaconas, George; Philipp, Mario T.; Norris, Steven J.

    2012-01-01

    The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM) analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease. PMID:23133514

  3. Genomic structure and chromosomal localization of the human deoxycytidine kinase gene

    SciTech Connect

    Song, J.J.; Walker, S.; Gribbin, T. ); Chen, E. Univ. of North Carolina, Chapel Hill ); Johnson, E.E.; Spychala, J.; Mitchell, B.S. )

    1993-01-15

    Deoxycytidine kinase (NTP:deoxycytidine 5[prime]-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5[prime] flanking regions and delinated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5[prime] flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5[prime] upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have >6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5[prime] sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression. 32 refs., 4 figs., 2 tabs.

  4. Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections

    SciTech Connect

    Gonsalves, C.; Xue, B.; Yepes, M.; Fuchs, M.; Ling, K.; Namba, S. . Dept. of Plant Pathology)

    1994-03-01

    A single regeneration procedure using cotyledon examples effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens or microprojectile bombardment methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), [beta]-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 [mu]m 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg[center dot]liter[sup [minus]1] and carbenicillin (Cb) at 500 mg[center dot]liter[sup [minus]1]. The authors' comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R[sub 0] plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes.

  5. Transcriptional Gene Silencing Mediated by a Plastid Inner Envelope Phosphoenolpyruvate/Phosphate Translocator CUE1 in Arabidopsis1[OA

    PubMed Central

    Shen, Jie; Ren, Xiaozhi; Cao, Rui; Liu, Jun; Gong, Zhizhong

    2009-01-01

    Mutations in REPRESSOR OF SILENCING1 (ROS1) lead to the transcriptional gene silencing (TGS) of ProRD29A:LUC (LUCIFERASE) and Pro35S:NPTII (Neomycin Phosphotransferase II) reporter genes. We performed a genetic screen to find suppressors of ros1 that identified two mutant alleles in the Arabidopsis (Arabidopsis thaliana) CHLOROPHYLL A/B BINDING PROTEIN UNDEREXPRESSED1 (CUE1) gene, which encodes a plastid inner envelope phosphoenolpyruvate/phosphate translocator. The cue1 mutations released the TGS of Pro35S:NPTII and the transcriptionally silent endogenous locus TRANSCRIPTIONAL SILENCING INFORMATION in a manner that was independent of DNA methylation but dependent on chromatin modification. The cue1 mutations did not affect the TGS of ProRD29A:LUC in ros1, which was dependent on RNA-directed DNA methylation. It is possible that signals from chloroplasts help to regulate the epigenetic status of a subset of genomic loci in the nucleus. PMID:19515789

  6. CcpA-mediated repression of Clostridium difficile toxin gene expression.

    PubMed

    Antunes, Ana; Martin-Verstraete, Isabelle; Dupuy, Bruno

    2011-02-01

    The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile. PMID:21299645

  7. Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Hamann, L; Nickel, R; Tannich, E

    1995-01-01

    To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568055

  8. A Gene System for Glucitol Transport and Metabolism in Clostridium beijerinckii NCIMB 8052

    PubMed Central

    Tangney, Martin; Brehm, John K.; Minton, Nigel P.; Mitchell, Wilfrid J.

    1998-01-01

    The gutD gene of Clostridium beijerinckii NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an Escherichia coli gutD mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order gutA1 gutA2 orfX gutB gutD were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from E. coli, while the remaining ORF (orfX) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which gutD was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The gutA1 and gutA2 genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by gutB. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the gutB gene. The gut genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the gut genes were not expressed until glucose was depleted. PMID:9572925

  9. Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement.

    PubMed Central

    Buckley, N D; Lee, L N; LeBlanc, D J

    1995-01-01

    The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in

  10. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

    PubMed

    Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

    2003-08-01

    An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167

  11. Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

    PubMed Central

    De Block, Marc; De Brouwer, Dirk; Tenning, Paul

    1989-01-01

    An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO3 was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed. Images Figure 1 Figure 2 PMID:16667089

  12. Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker.

    PubMed

    Chakraborty, M; Sairam Reddy, P; Laxmi Narasu, M; Krishna, Gaurav; Rana, Debashis

    2016-01-01

    Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l(-1)) and Paramomycin (70 mg l(-1)) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe. PMID:27186018

  13. Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum).

    PubMed

    Singh, H Ranjit; Deka, Manab; Das, Sudripta

    2015-07-01

    Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea. PMID:25772466

  14. Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Brenna, Andrea; Montanini, Barbara; Muggiano, Eleonora; Proietto, Marco; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275

  15. Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation

    PubMed Central

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi (“truffles”) with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275

  16. The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

    PubMed Central

    Brochu, Denis; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 μM and 1 mM, respectively. PMID:9922231

  17. A novel splice site mutation in the GNPTAB gene in an Iranian patient with mucolipidosis II α/β.

    PubMed

    Hashemi-Gorji, Feyzollah; Ghafouri-Fard, Soudeh; Salehpour, Shadab; Yassaee, Vahid Reza; Miryounesi, Mohammad

    2016-08-01

    Mucolipidosis type II α/β (ML II α/β) and mucolipidosis type III α/β (ML III α/β) have been shown to be caused by an absence or reduced level of uridine diphosphate (UDP)-N-acetylglucosamine-1-phosphotransferase enzyme (EC 2.7.8.17) activity, respectively. Both disorders are caused by mutations in the GNPTAB gene and are inherited in an autosomal recessive manner. Here we report a 2-year-old female patient being diagnosed as a case of ML II α/β due to coarse face, severe developmental delay, multiple dysostosis, noticeable increase of multiple lysosomal enzymes activity in plasma and normal acid mucopolysaccharides in urine. Mutational analysis of the GNPTAB gene has revealed a novel homozygous mutation in the patient (c.3250-2A>G) with both parents being heterozygote. Transcript analyses showed that this novel splice site mutation leads to exon 17 skipping and a frameshift afterwards (p.P1084_R1112del F1113Vfs*1). Consequently, we confirmed the association of this mutation with ML II α/β. Our finding expands the number of reported cases of this rare metabolic disorder and adds to the GNPTAB mutation database. PMID:27180337

  18. Silencing the HaAK Gene by Transgenic Plant-Mediated RNAi Impairs Larval Growth of Helicoverpa armigera

    PubMed Central

    Liu, Feng; Wang, Xiao-Dong; Zhao, Yi-Ying; Li, Yan-Jun; Liu, Yong-Chang; Sun, Jie

    2015-01-01

    Insect pests have caused noticeable economic losses in agriculture, and the heavy use of insecticide to control pests not only brings the threats of insecticide resistance but also causes the great pollution to foods and the environment. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been is currently developed for protection against insect pests. In this study, we used this technology to silence the arginine kinase (AK) gene of Helicoverpa armigera (HaAK), encoding a phosphotransferase that plays a critical role in cellular energy metabolism in invertebrate. Transgenic Arabidopsis plants producing HaAK dsRNA were generated by Agrobacterium-mediated transformation. The maximal mortality rate of 55% was reached when H. armigera first-instar larvae were fed with transgenic plant leaves for 3 days, which was dramatically higher than the 18% mortality recorded in the control group. Moreover, the ingestion of transgenic plants significantly retarded larval growth, and the transcript levels of HaAK were also knocked down by up to 52%. The feeding bioassays further indicated that the inhibition efficiency was correlated with the integrity and concentration of the produced HaAK dsRNA in transgenic plants. These results strongly show that the resistance to H. armigera was improved in transgenic Arabidopsis plants, suggesting that the RNAi targeting of AK has the potential for the control of insect pests. PMID:25552931

  19. Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

    SciTech Connect

    Martin, S.A.; Russell, J.B.

    1987-10-01

    Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments, indicated that separate phosphotransferases systems existed for glucose, maltose, and sucrose. (/sup 14/C)maltose transport was inhibited by excess glucose and to a lesser extent by sucrose. (/sup 14/C)glucose and (/sup 14/C)sucrose transports were not inhibited by an excess of maltose. Since (/sup 14/C)maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of P/sub i/ was increased from 0 to 100 mM, a maltose phosphorylase was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for ..cap alpha..-glucose 1-phosphate. Only sucrose-grown cells possessed sucrose hydrolase activity, and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.

  20. Tn5393d, a complex Tn5393 derivative carrying the PER-1 extended-spectrum beta-lactamase gene and other resistance determinants.

    PubMed

    Mantengoli, Elisabetta; Rossolini, Gian Maria

    2005-08-01

    In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum beta-lactamase, the bla(PER-1) gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the bla(PER-1) gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the bla(PER-1) gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the bla(PER-1) genes, respectively. The putative composite transposon carrying bla(PER-1), named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the bla(PER-1) gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons. PMID:16048938

  1. The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo.

    PubMed Central

    Gallie, D R; Sleat, D E; Watts, J W; Turner, P C; Wilson, T M

    1987-01-01

    A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA [defined as omega' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo. Images PMID:3575095

  2. The glucose transport system of the hyperthermophilic anaerobic bacterium Thermotoga neapolitana

    SciTech Connect

    Galperin, M.Y.; Noll, K.M.; Romano, A.H.

    1996-08-01

    The glucose transport system of the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DOG). T. neapolitana accumulated 2-DOG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external D-glucose. This active transport of 2-DOG was dependent upon the presence of sodium ion and an external source of energy, such as pyruvate, and was inhibited by arsenate and gramicidin D. There was no phosphoenolpyruvate-dependent phosphorylation of glucose, 2-DOG, or fructose by cell extracts or toluene-treated cells, indicating the absence of a phosphoenolpyruvate:sugar phosphotransferase system. These data indicate that D-glucose is taken up by T.neapolitana via an active transport system that is energized by an ion gradient generated by ATP, derived from substrate-level phosphorylation. 33 refs., 3 figs., 1 tab.

  3. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    SciTech Connect

    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.

    2011-12-31

    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  4. [Identification of the open reading frame coding transposase of Bordetella pertussis RS-element].

    PubMed

    Vorontsov, V V; Sivov, I G; Umiarov, A M; Siniashina, L N; Bol'shakova, T N; Dobrynina, O Iu; Molchanova, M L; Amelina, I P; Rudnev, I A; Karataev, G I

    2004-01-01

    A computer-aided analysis of the repeating sequence of Bordetella pertussis chromosome (RSBP3) revealed 3 open reading frames, one of whose (ORF1) can code a protein whose structure and properties are similar to those of transposasas, i.e. enzymes in charges for the traveling of migrating genetic elements of pro- and eukaryote. Mutants of the RSBP3 insertion sequence with the affected and unaffected ORF1 sequence were constructed in order to substantiate the above assumption. Two independent experimental models (formation of inter-plasmid co-integrates and of co-integrates between plasmid and E. coli chromosome) were used to show that the RSBP3-stimulated formation of co-integrates is only true for plasmids containing RSBP3 with the unaffected ORF1 sequence. An activity of the Hpr protein (a component of the phosphoenolpyruvate-dependent phosphotransferase) was proven to influence the formation process of inter-plasmid co-integrates. PMID:15164716

  5. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  6. Trichoderma genes

    DOEpatents

    Foreman, Pamela; Goedegebuur, Frits; Van Solingen, Pieter; Ward, Michael

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  7. Multiplex, construct-specific, and real-time PCR-based analytical methods for Bt rice with cry1Ac gene.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika

    2012-01-01

    Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptLL) marker gene, and an endogenous a-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise. PMID:22468358

  8. LacR is a repressor of lacABCD and LacT is an activator of lacTFEG, constituting the lac gene cluster in Streptococcus pneumoniae.

    PubMed

    Afzal, Muhammad; Shafeeq, Sulman; Kuipers, Oscar P

    2014-09-01

    Comparison of the transcriptome of Streptococcus pneumoniae strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose revealed the elevated expression of various genes and operons, including the lac gene cluster, which is organized into two operons, i.e., lac operon I (lacABCD) and lac operon II (lacTFEG). Deletion of the DeoR family transcriptional regulator lacR that is present downstream of the lac gene cluster revealed elevated expression of lac operon I even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of lac operon I, which encodes enzymes involved in the phosphorylated tagatose pathway in the absence of lactose or galactose. Deletion of lacR did not affect the expression of lac operon II, which encodes a lactose-specific phosphotransferase. This finding was further confirmed by β-galactosidase assays with PlacA-lacZ and PlacT-lacZ in the presence of either lactose or glucose as the sole carbon source in the medium. This suggests the involvement of another transcriptional regulator in the regulation of lac operon II, which is the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of lac operon II in the presence of lactose and CcpA-independent regulation of the lac gene cluster in S. pneumoniae. PMID:24951784

  9. [Language gene].

    PubMed

    Takahashi, Hiroshi

    2006-11-01

    The human capacity for acquiring speech and language must derive, at least in part, from the genome. Recent advance in the field of molecular genetics finally discovered 'Language Gene'. Disruption of FOXP2 gene, the firstly identified 'language gene' causes severe speech and language disorder. To elucidate the anatomical basis of language processing in the brain, we examined the expression pattern of FOXP2/Foxp2 genes in the monkey and rat brains through development. We found the preferential expression of FOXP2/Foxp2 in the striosomal compartment of the developing striatum. Thus, we suggest the striatum, particularly striosomal system may participate in neural information processing for language and speech. Our suggestion is consistent with the declarative/ procedural model of language proposed by Ullman (1997, 2001), which the procedural memory-dependent mental grammar is rooted in the basal ganglia and the frontal cortex, and the declarative memory-dependent mental lexicon is rooted in the temporal lobe. PMID:17432197

  10. Genes V.

    SciTech Connect

    Lewin, B.

    1994-12-31

    This fifth edition book encompasses a wide range of topics covering 1,272 pages. The book is arranged into nine parts with a total of 36 chapters. These nine parts include Introduction; DNA as a Store of Information; Translation; Constructing Cells; Control of Prokaryotypic Gene Expression; Perpetuation of DNA; Organization of the Eukaryotypic Genome; Eukaryotypic Transcription and RNA Processing; The Dynamic Genome; and Genes in Development.

  11. Nucleotide sequences of the arb genes, which control beta-glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new beta-glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans.

    PubMed Central

    el Hassouni, M; Henrissat, B; Chippaux, M; Barras, F

    1992-01-01

    The phytopathogenic bacterium Erwinia chrysanthemi, unlike other members of the family Enterobacteriaceae, is able to metabolize the beta-glucosides, arbutin, and salicin. A previous genetic analysis of the E. chrysanthemi arb genes, which mediate beta-glucoside metabolism, suggested that they were homologous to the Escherichia coli K-12 bgl genes. We have now determined the nucleotide sequence of a 5,065-bp DNA fragment containing three genes, arbG, arbF, and arbB. Deletion analysis, expression in minicell systems, and comparison with sequences of other proteins suggest that arbF and arbB encode a beta-glucoside-specific phosphotransferase system-dependent permease and a phospho-beta-glucosidase, respectively. The ArbF amino acid sequence shares 55% identity with that of the E. coli BglF permease and contains most residues thought to be important for a phosphotransferase. One change, however, was noted, since BglF Arg-625, presumably involved in phosphoryl transfer, was replaced by a Cys residue in ArbF. An analysis of the ArbB sequence led to the definition of a protein family which contained enzymes classified as phospho-beta-glucosidases, phospho-beta-galactosidases, beta-glucosidases, and beta-galactosidases and originating from gram-positive and gram-negative bacteria, archebacteria, and mammals, including humans. An analysis of this family allowed us (i) to speculate on the ways that these enzymes evolved, (ii) to identify a glutamate residue likely to be a key amino acid in the catalytic activity of each protein, and (iii) to predict that domain II of the human lactate-phlorizin hydrolase, which is involved in lactose intolerance, is catalytically nonactive. A comparison between the untranslated regions of the E. chrysanthemi arb cluster and the E. coli bgl operon revealed the conservation of two regions which, in the latter, are known to terminate transcription under noninducing conditions and be the target of the BglG transcriptional antiterminator under

  12. Genetic analysis around aminoalcohol dehydrogenase gene of Rhodococcus erythropolis MAK154: a putative GntR transcription factor in transcriptional regulation.

    PubMed

    Urano, Nobuyuki; Kataoka, Michihiko; Ishige, Takeru; Kita, Shinji; Sakamoto, Keiji; Shimizu, Sakayu

    2011-02-01

    NADP(+)-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 catalyzes the reduction of (S)-1-phenyl-1-keto-2-methylaminopropane ((S)-MAK) to d-pseudoephedrine, which is used as a pharmaceutical. AADH is suggested to participate in aminoalcohol or aminoketone metabolism in this organism because it is induced by the addition of several aminoalcohols, such as 1-amino-2-propanol. Genetic analysis of around the aadh gene showed that some open reading frames (ORFs) are involved in this metabolic pathway. Four of these ORFs might form a carboxysome-like polyhedral organelle, and others are predicted to encode aminotransferase, aldehyde dehydrogenase, phosphotransferase, and regulator protein. OrfE, a homologous ORF of the FadR subfamily of GntR transcriptional regulators, lies downstream from aadh. To investigate whether or not orfE plays a role in the regulation of aadh expression, the gene disruption mutant of R. erythropolis MAK154 was constructed. The ΔorfE strain showed higher AADH activity than wild-type strain. In addition, a transformed strain, which harbored multi-orfE, showed no AADH activity even in the induced condition with 1-amino-2-propanol. These results suggest that OrfE is a negative regulator that represses aadh expression in the absence of 1-amino-2-propanol. PMID:20953603

  13. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation.

    PubMed

    Nakagawa, Tsuyoshi; Kurose, Takayuki; Hino, Takeshi; Tanaka, Katsunori; Kawamukai, Makoto; Niwa, Yasuo; Toyooka, Kiminori; Matsuoka, Ken; Jinbo, Tetsuro; Kimura, Tetsuya

    2007-07-01

    We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research. PMID:17697981

  14. High-level resistance to class IIa bacteriocins is associated with one general mechanism in Listeria monocytogenes.

    PubMed

    Gravesen, Anne; Ramnath, Manilduth; Rechinger, K Björn; Andersen, Natalie; Jänsch, Lothar; Héchard, Yann; Hastings, John W; Knøchel, Susanne

    2002-08-01

    Class IIa bacteriocins may be used as natural food preservatives, yet resistance development in the target organisms is still poorly understood. In this study, the understanding of class IIa resistance development in Listeria monocytogenes is extended, linking the seemingly diverging results previously reported. Eight resistant mutants having a high resistance level (at least a 10(3)-fold increase in MIC), originating from five wild-type listerial strains, were independently isolated following exposure to four different class IIa bacteriocin-producing lactic acid bacteria (including pediocin PA-1 and leucocin A producers). Two of the mutants were isolated from food model systems (a saveloy-type sausage at 10 degrees C, and salmon juice at 5 degrees C). Northern blot analysis showed that the eight mutants all had increased expression of EII(Bgl) and a phospho-beta-glucosidase homologue, both originating from putative beta-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). However, disruption of these genes in a resistant mutant did not confer pediocin sensitivity. Comparative two-dimensional gel analysis of proteins isolated from mutant and wild-type strains showed that one spot was consistently missing in the gels from mutant strains. This spot corresponded to the MptA subunit of the mannose-specific PTS, found only in the gels of wild-type strains. The mptACD operon was recently shown to be regulated by the sigma(54) transcription factor in conjunction with the activator ManR. Class IIa bacteriocin-resistant mutants having defined mutations in mpt or manR also exhibited the two diverging PTS expression changes. It is suggested here that high-level class IIa resistance in L. monocytogenes and at least some other Gram-positive bacteria is developed by one prevalent mechanism, irrespective of wild-type strain, class IIa bacteriocin, or the tested environmental conditions. The changes in expression of the beta-glucoside-specific and

  15. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  16. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  17. Regulation of phosphotransferases in glucose- and xylose-fermenting yeasts

    SciTech Connect

    Yang, V.W.; Jeffries, T.W.

    1997-12-31

    This research examined the titers of hexokinase (HK), phosphofructokinase (PFK), and xylulokinase (XUK) in Saccharomyces cerevisiae and two xylose fermenting yeasts, Pachysolen tannophilus and Candida shehatae, following shifts in carbon source and aeration. Xylose-grown C. shehatae, glucose-grown P. tannophilus, and glucose-grown S. cerevisiae, had the highest specific activities of XUK, HK, and PFK, respectively. XUK was induced by xylose to moderate levels in both P. tannophilus and C. shehatae, but was present only in trace levels in S. cerevisiae. HK activities in P. tannophilus were two to three fold higher when cells were grown on glucose than when grown on xylose, but HK levels were less inducible in C. shehatae. The PFK activities in S. cerevisiae were 1.5 to 2 times higher than in the two xylose-fermenting yeasts. Transfer from glucose to xylose rapidly inactivated HK in P. tannophilus, and transfer from xylose to glucose inactivated XUK in C. shehatae. The patterns of induction and inactivation indicate that the basic regulatory mechanisms differ in the two xylose fermenting yeasts. 42 refs., 2 figs., 4 tabs.

  18. Elaboration of a reliable strategy based on real-time PCR to characterize genetically modified plantlets and to evaluate the efficiency of a marker gene removal in grape (Vitis spp.).

    PubMed

    Dalla Costa, Lorenza; Vaccari, Ilaria; Mandolini, Marco; Martinelli, Lucia

    2009-04-01

    We have developed an effective strategy based on real-time PCR assay for the molecular characterization of genetically modified grape and to quantify the efficiency of a marker gene removal. This research has been implemented in Vitis vinifera cv. Brachetto plantlets where exogenes were inserted during cocultures of embryogenic calli with Agrobacterium tumefaciens carrying the chemically inducible site-specific cre/loxP pX6 vector where the expression of the cre recombinase is regulated by 17-beta-estradiol. The neomycin phosphotransferase gene (nptII) for the kanamycin resistance trait was inserted as part of the gene transfer protocol, and this exogene was employed as a case study for carrying out our research. The 9-cis-epoxycarotenoid dioxygenase (nced2) and chalcone isomerase (chi) genes coding for two enzymes, involved respectively in abscisic acid and flavonoid biosynthesis, proved to be valuable reference endogenes for real-time PCR assays. Two types of duplo-target plasmids were exploited for building the standard curves: in one type (p-nptII/nced2) the nptII sequence is linked to the nced2 sequence; in the other (p-nptII/chi) it is linked to the chi. These calibrators were intended to simulate an ideal genetically modified plant carrying a homozygous single-copy exogene insertion. The repeatability test confirmed the suitability of both plasmid calibrators. Foreign gene stability can be monitored during long-term plant preservation, and the method proved to be suitable for quantifying the efficiency of nptII gene removal induced by 17-beta-estradiol. PMID:19265380

  19. Genes and Hearing Loss

    MedlinePlus

    ... Meeting Calendar Find an ENT Doctor Near You Genes and Hearing Loss Genes and Hearing Loss Patient ... mutation may only have dystopia canthorum. How Do Genes Work? Genes are a road map for the ...

  20. Compare Gene Profiles

    SciTech Connect

    2014-05-31

    Compare Gene Profiles (CGP) performs pairwise gene content comparisons among a relatively large set of related bacterial genomes. CGP performs pairwise BLAST among gene calls from a set of input genome and associated annotation files, and combines the results to generate lists of common genes, unique genes, homologs, and genes from each genome that differ substantially in length from corresponding genes in the other genomes. CGP is implemented in Python and runs in a Linux environment in serial or parallel mode.

  1. Temperature-Dependent Expression of phzM and Its Regulatory Genes lasI and ptsP in Rhizosphere Isolate Pseudomonas sp. Strain M18▿

    PubMed Central

    Huang, Jiaofang; Xu, Yuquan; Zhang, Hongyan; Li, Yaqian; Huang, Xianqing; Ren, Bin; Zhang, Xuehong

    2009-01-01

    Pseudomonas sp. strain M18, an effective biological control agent isolated from the melon rhizosphere, has a genetic background similar to that of the opportunistic human pathogen Pseudomonas aeruginosa PAO1. However, the predominant phenazine produced by strain M18 is phenazine-1-carboxylic acid (PCA) rather than pyocyanin (PYO); the quantitative ratio of PCA to PYO is 105 to 1 at 28°C in strain M18, while the ratio is 1 to 2 at 37°C in strain PAO1. We first provided evidence that the differential production of the two phenazines in strains M18 and PAO1 is related to the temperature-dependent and strain-specific expression patterns of phzM, a gene involved in the conversion of PCA to PYO. Transcriptional levels of phzM were measured by quantitative real-time PCR, and the activities of both transcriptional and translational phzM′-′lacZ fusions were determined in strains M18 and PAO1, respectively. Using lasI::Gm and ptsP::Gm inactivation M18 mutants, we further show that expression of the phzM gene is positively regulated by the quorum-sensing protein LasI and negatively regulated by the phosphoenolpyruvate phosphotransferase protein PtsP. Surprisingly, the lasI and ptsP regulatory genes were also expressed in a temperature-dependent and strain-specific manner. The differential production of the phenazines PCA and PYO by strains M18 and PAO1 may be a consequence of selective pressure imposed on P. aeruginosa PAO1 and its relative M18 in the two different niches over a long evolutionary process. PMID:19717631

  2. Gene gymnastics

    PubMed Central

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  3. Gene doping: gene delivery for olympic victory

    PubMed Central

    Gould, David

    2013-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  4. Autism and Genes

    ERIC Educational Resources Information Center

    National Institutes of Health, 2005

    2005-01-01

    This document defines and discusses autism and how genes play a role in the condition. Answers to the following questions are covered: (1) What are genes? (2) What is autism? (3) What causes autism? (4) Why study genes to learn about autism? (5) How do researchers look for the genes involved in autism? (screen the whole genome; conduct cytogenetic…

  5. Compare Gene Profiles

    Energy Science and Technology Software Center (ESTSC)

    2014-05-31

    Compare Gene Profiles (CGP) performs pairwise gene content comparisons among a relatively large set of related bacterial genomes. CGP performs pairwise BLAST among gene calls from a set of input genome and associated annotation files, and combines the results to generate lists of common genes, unique genes, homologs, and genes from each genome that differ substantially in length from corresponding genes in the other genomes. CGP is implemented in Python and runs in a Linuxmore » environment in serial or parallel mode.« less

  6. Evolution by gene loss.

    PubMed

    Albalat, Ricard; Cañestro, Cristian

    2016-07-01

    The recent increase in genomic data is revealing an unexpected perspective of gene loss as a pervasive source of genetic variation that can cause adaptive phenotypic diversity. This novel perspective of gene loss is raising new fundamental questions. How relevant has gene loss been in the divergence of phyla? How do genes change from being essential to dispensable and finally to being lost? Is gene loss mostly neutral, or can it be an effective way of adaptation? These questions are addressed, and insights are discussed from genomic studies of gene loss in populations and their relevance in evolutionary biology and biomedicine. PMID:27087500

  7. Human Gene Therapy: Genes without Frontiers?

    ERIC Educational Resources Information Center

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  8. Evolution of gene expression after gene amplification.

    PubMed

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-05-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat-maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  9. Evolution of Gene Expression after Gene Amplification

    PubMed Central

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-01-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat–maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  10. Reading and Generalist Genes

    ERIC Educational Resources Information Center

    Haworth, Claire M. A.; Meaburn, Emma L.; Harlaar, Nicole; Plomin, Robert

    2007-01-01

    Twin-study research suggests that many (but not all) of the same genes contribute to genetic influence on diverse learning abilities and disabilities, a hypothesis called "generalist genes". This generalist genes hypothesis was tested using a set of 10 DNA markers (single nucleotide polymorphisms [SNPs]) found to be associated with early reading…

  11. Accepting Foreign Genes.

    PubMed

    Boto, Luis

    2016-05-01

    Three recent papers underline the importance of the host genomic background in allowing the stable maintenance of horizontally acquired genes. These studies suggest that post-transfer changes in both host genome and acquired genes contribute to the stable integration of foreign genes. PMID:27075565

  12. A unique gene cluster for the utilization of the mucosal and human milk-associated glycans galacto-N-biose and lacto-N-biose in Lactobacillus casei.

    PubMed

    Bidart, Gonzalo N; Rodríguez-Díaz, Jesús; Monedero, Vicente; Yebra, María J

    2014-08-01

    The probiotic Lactobacillus casei catabolizes galacto-N-biose (GNB) and lacto-N-biose (LNB) by using a transport system and metabolic routes different from those of Bifidobacterium. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC (EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate : sugar phosphotransferase system (PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTS(Gnb) . Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-β-galactosidase) hydrolyses both disaccharides. However, GnbF (N-acetylgalactosamine-6P deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA (N-acetylglucosamine-6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose-6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose-6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes. PMID:24942885

  13. [Imprinted genes in plants].

    PubMed

    Zhang, Li-Geng; Yang, Ruo-Fei; Fu, Feng-Ling; Li, Wan-Chen

    2010-12-01

    The expression of imprinted genes is regulated by epigenetic mechanism. In plant endosperm, the allele of imprinted genes is expressed in a pattern of parent-of-origin-dependent. The expression of imprinted genes plays essential roles in the development of embryos and their annexe structures, as well as seed size, reproductive barriers and apomixis. Along with the progress of plant epigenetic research, the exploration of imprinted genes is becoming hotspot in epigenetic research. This review focused on the parental conflict theory about the origin of imprinted genes, and the latest research advances in expression regulation mechanism of plant imprinted genes, using the examples of the important imprinted genes MEA, FIS2, FWA, MPC, and PHE1 in Arabidopsis, and FIEI and FIE2 in maize. PMID:21513148

  14. Retrieval with gene queries

    PubMed Central

    Sehgal, Aditya K; Srinivasan, Padmini

    2006-01-01

    Background Accuracy of document retrieval from MEDLINE for gene queries is crucially important for many applications in bioinformatics. We explore five information retrieval-based methods to rank documents retrieved by PubMed gene queries for the human genome. The aim is to rank relevant documents higher in the retrieved list. We address the special challenges faced due to ambiguity in gene nomenclature: gene terms that refer to multiple genes, gene terms that are also English words, and gene terms that have other biological meanings. Results Our two baseline ranking strategies are quite similar in performance. Two of our three LocusLink-based strategies offer significant improvements. These methods work very well even when there is ambiguity in the gene terms. Our best ranking strategy offers significant improvements on three different kinds of ambiguities over our two baseline strategies (improvements range from 15.9% to 17.7% and 11.7% to 13.3% depending on the baseline). For most genes the best ranking query is one that is built from the LocusLink (now Entrez Gene) summary and product information along with the gene names and aliases. For others, the gene names and aliases suffice. We also present an approach that successfully predicts, for a given gene, which of these two ranking queries is more appropriate. Conclusion We explore the effect of different post-retrieval strategies on the ranking of documents returned by PubMed for human gene queries. We have successfully applied some of these strategies to improve the ranking of relevant documents in the retrieved sets. This holds true even when various kinds of ambiguity are encountered. We feel that it would be very useful to apply strategies like ours on PubMed search results as these are not ordered by relevance in any way. This is especially so for queries that retrieve a large number of documents. PMID:16630348

  15. Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages.

    PubMed Central

    Murphy, K M; Sweet, M J; Ross, I L; Hume, D A

    1993-01-01

    The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells

  16. Do Housekeeping Genes Exist?

    PubMed Central

    Sun, Bingyun

    2015-01-01

    The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body. PMID:25970694

  17. Do housekeeping genes exist?

    PubMed

    Zhang, Yijuan; Li, Ding; Sun, Bingyun

    2015-01-01

    The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body. PMID:25970694

  18. Towards Consensus Gene Ages.

    PubMed

    Liebeskind, Benjamin J; McWhite, Claire D; Marcotte, Edward M

    2016-01-01

    Correctly estimating the age of a gene or gene family is important for a variety of fields, including molecular evolution, comparative genomics, and phylogenetics, and increasingly for systems biology and disease genetics. However, most studies use only a point estimate of a gene's age, neglecting the substantial uncertainty involved in this estimation. Here, we characterize this uncertainty by investigating the effect of algorithm choice on gene-age inference and calculate consensus gene ages with attendant error distributions for a variety of model eukaryotes. We use 13 orthology inference algorithms to create gene-age datasets and then characterize the error around each age-call on a per-gene and per-algorithm basis. Systematic error was found to be a large factor in estimating gene age, suggesting that simple consensus algorithms are not enough to give a reliable point estimate. We also found that different sources of error can affect downstream analyses, such as gene ontology enrichment. Our consensus gene-age datasets, with associated error terms, are made fully available at so that researchers can propagate this uncertainty through their analyses (geneages.org). PMID:27259914

  19. Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

    PubMed

    Zheng, Si-Jun; Henken, Betty; de Maagd, Ruud A; Purwito, Agus; Krens, Frans A; Kik, Chris

    2005-06-01

    Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot. PMID:16145834

  20. Determination of aminoglycoside resistance in Staphylococcus aureus by DNA hybridization.

    PubMed Central

    Dickgiesser, N; Kreiswirth, B N

    1986-01-01

    A method is described for identification of the genes conferring aminoglycoside resistance in Staphylococcus aureus by dot-blot and Southern blot techniques. As radioactive probes, fragments of plasmids pAT48, pUBH2, and pH13, carrying the genes for an aminocyclitol-3'-phosphotransferase, an aminocyclitol-4'-adenylyltransferase, and an aminocyclitol-2''-phosphotransferase-aminocyclitol-6'-acetyltransferase, respectively, were used. Images PMID:3729351

  1. The gap gene network

    PubMed Central

    2010-01-01

    Gap genes are involved in segment determination during the early development of the fruit fly Drosophila melanogaster as well as in other insects. This review attempts to synthesize the current knowledge of the gap gene network through a comprehensive survey of the experimental literature. I focus on genetic and molecular evidence, which provides us with an almost-complete picture of the regulatory interactions responsible for trunk gap gene expression. I discuss the regulatory mechanisms involved, and highlight the remaining ambiguities and gaps in the evidence. This is followed by a brief discussion of molecular regulatory mechanisms for transcriptional regulation, as well as precision and size-regulation provided by the system. Finally, I discuss evidence on the evolution of gap gene expression from species other than Drosophila. My survey concludes that studies of the gap gene system continue to reveal interesting and important new insights into the role of gene regulatory networks in development and evolution. PMID:20927566

  2. Metastasis Suppressor Genes

    PubMed Central

    Yan, Jinchun; Yang, Qin; Huang, Qihong

    2014-01-01

    Metastasis is a major cause of cancer mortality. Metastasis is a complex process that requires the regulation of both metastasis-promoting and metastasis suppressor genes. The discovery of metastasis suppressor genes contributes significantly to our understanding of metastasis mechanisms and provides prognostic markers and therapeutic targets in clinical cancer management. In this review, we summarize the methods that have been used to identify metastasis suppressors and the potential clinical impact of these genes. PMID:23348381

  3. Towards Consensus Gene Ages

    PubMed Central

    Liebeskind, Benjamin J.; McWhite, Claire D.; Marcotte, Edward M.

    2016-01-01

    Correctly estimating the age of a gene or gene family is important for a variety of fields, including molecular evolution, comparative genomics, and phylogenetics, and increasingly for systems biology and disease genetics. However, most studies use only a point estimate of a gene’s age, neglecting the substantial uncertainty involved in this estimation. Here, we characterize this uncertainty by investigating the effect of algorithm choice on gene-age inference and calculate consensus gene ages with attendant error distributions for a variety of model eukaryotes. We use 13 orthology inference algorithms to create gene-age datasets and then characterize the error around each age-call on a per-gene and per-algorithm basis. Systematic error was found to be a large factor in estimating gene age, suggesting that simple consensus algorithms are not enough to give a reliable point estimate. We also found that different sources of error can affect downstream analyses, such as gene ontology enrichment. Our consensus gene-age datasets, with associated error terms, are made fully available at so that researchers can propagate this uncertainty through their analyses (geneages.org). PMID:27259914

  4. History of gene therapy.

    PubMed

    Wirth, Thomas; Parker, Nigel; Ylä-Herttuala, Seppo

    2013-08-10

    Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results. In this review, we will take a historical view and highlight some of the milestones that had an important impact on the development of gene therapy. We will also discuss briefly the safety and ethical aspects of gene therapy and address some concerns that have been connected with gene therapy as an important therapeutic modality. PMID:23618815

  5. [The gene or genes of allergic asthma?].

    PubMed

    Demoly, P; Bousquet, J; Godard, P; Michel, F B

    1993-05-15

    Asthma is a multifactorial disease in which the hereditary component has been demonstrated by familial and identical twin studies. Allergy is important in the aetiology of asthma and is characterized by a hyperreaction to allergens triggering predominantly the immunoglobulines E. The levels of these antibodies are found to be elevated even in non allergic asthmatics. The majority of genetic research in this area is focused on either the genes of the specific immune response or that of the non allergic response. These are the genes of the class II MHC, and the APY gene on chromosome 11q respectively. The modern techniques of molecular genetics and in particular those of inverse genetics have recently contributed to a more comprehensive understanding of this disease. PMID:8316547

  6. GENE EXPRESSION NETWORKS

    EPA Science Inventory

    "Gene expression network" is the term used to describe the interplay, simple or complex, between two or more gene products in performing a specific cellular function. Although the delineation of such networks is complicated by the existence of multiple and subtle types of intera...

  7. Your Genes, Your Choices

    MedlinePlus

    Table of Contents Your Genes, Your Choices describes the Human Genome Project, the science behind it, and the ethical, legal, and social issues that are ... Nothing could be further from the truth. Your Genes, Your Choices points out how the progress of ...

  8. What Is a Gene?

    MedlinePlus

    ... a new kind of medicine — so new that scientists are still doing experiments to see if it works. It uses the technology of genetic engineering to treat a disease caused by a gene that has changed in some way. One method being tested is replacing sick genes with healthy ...

  9. Gene expression networks.

    PubMed

    Thomas, Reuben; Portier, Christopher J

    2013-01-01

    With the advent of microarrays and next-generation biotechnologies, the use of gene expression data has become ubiquitous in biological research. One potential drawback of these data is that they are very rich in features or genes though cost considerations allow for the use of only relatively small sample sizes. A useful way of getting at biologically meaningful interpretations of the environmental or toxicological condition of interest would be to make inferences at the level of a priori defined biochemical pathways or networks of interacting genes or proteins that are known to perform certain biological functions. This chapter describes approaches taken in the literature to make such inferences at the biochemical pathway level. In addition this chapter describes approaches to create hypotheses on genes playing important roles in response to a treatment, using organism level gene coexpression or protein-protein interaction networks. Also, approaches to reverse engineer gene networks or methods that seek to identify novel interactions between genes are described. Given the relatively small sample numbers typically available, these reverse engineering approaches are generally useful in inferring interactions only among a relatively small or an order 10 number of genes. Finally, given the vast amounts of publicly available gene expression data from different sources, this chapter summarizes the important sources of these data and characteristics of these sources or databases. In line with the overall aims of this book of providing practical knowledge to a researcher interested in analyzing gene expression data from a network perspective, the chapter provides convenient publicly accessible tools for performing analyses described, and in addition describe three motivating examples taken from the published literature that illustrate some of the relevant analyses. PMID:23086841

  10. 4. AERIAL VIEW OF GENE WASH RESERVOIR AND GENE CAMP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. AERIAL VIEW OF GENE WASH RESERVOIR AND GENE CAMP LOOKING SOUTHWEST. DAM AND SPILLWAY VISIBLE IN BOTTOM OF PHOTO. - Gene Wash Reservoir & Dam, 2 miles west of Parker Dam, Parker Dam, San Bernardino County, CA

  11. Genes and Social Behavior

    PubMed Central

    Robinson, Gene E.; Fernald, Russell D.; Clayton, David F.

    2011-01-01

    What specific genes and regulatory sequences contribute to the organization and functioning of brain circuits that support social behavior? How does social experience interact with information in the genome to modulate these brain circuits? Here we address these questions by highlighting progress that has been made in identifying and understanding two key “vectors of influence” that link genes, brain, and social behavior: 1) social information alters gene readout in the brain to influence behavior; and 2) genetic variation influences brain function and social behavior. We also briefly discuss how evolutionary changes in genomic elements influence social behavior and outline prospects for a systems biology of social behavior. PMID:18988841

  12. GeneLab

    NASA Technical Reports Server (NTRS)

    Berrios, Daniel C.; Thompson, Terri G.

    2015-01-01

    NASA GeneLab is expected to capture and distribute omics data and experimental and process conditions most relevant to research community in their statistical and theoretical analysis of NASAs omics data.

  13. Terplex Gene Delivery System.

    PubMed

    Kim, Sung Wan

    2005-01-01

    Polymeric gene delivery systems have been developed to overcome problems caused by viral carriers. They are low cytotoxic, have no size limit, are convenient in handling, of low cost and reproducible. A Terplex gene delivery system consisting of plasmid DNA, low density lipoprotein and hydropholized poly-L-lysine was designed and characterized. The plasmid DNA, when formulated with stearyl PLL and LDL, forms a stable and hydrophobicity/charge-balanced Terplex system of optimal size for efficient cellular uptake. DNA is still intact after the Terplex formation. This information is expected to be utilized for the development of improved transfection vector for in vivo gene therapy. Terplex DNA complex showed significantly longer retention in the vascular space than naked DNA. This system was used in the augmentation of myocardial transfection at an infarction site with the VEGF gene. PMID:16243067

  14. Terplex gene delivery system.

    PubMed

    Kim, Sung Wan

    2005-01-01

    Polymeric gene delivery systems have been developed to overcome problems caused by viral carriers. They are low cytotoxic, have no size limit, are convenient in handling, of low cost and reproducible. A Terplex gene delivery system consisting of plasmid DNA, low density lipoprotein and hydropholized poly-L-lysine was designed and characterized. The plasmid DNA, when formulated with stearyl PLL and LDL, forms a stable and hydrophobicity/charge-balanced Terplex system of optimal size for efficient cellular uptake. DNA is still intact after the Terplex formation. This information is expected to be utilized for the development of improved transfection vector for in vivo gene therapy. Terplex DNA complex showed significantly longer retention in the vascular space than naked DNA. This system was used in the augmentation of myocardial transfection at an infarction site with the VEGF gene. PMID:16240997

  15. Vaginal gene therapy.

    PubMed

    Rodríguez-Gascón, Alicia; Del Pozo-Rodríguez, Ana; Isla, Arantxazu; Solinís, María Angeles

    2015-09-15

    In the last years, vaginal gene therapy has gained increasing attention mainly for the treatment and control of sexually transmitted infections. DNA delivery has been also suggested to improve reproductive outcomes for women with deficiencies in the female reproductive tract. Although no product has reached clinical phase, preclinical investigations reveal the potential of the vaginal tract as an effective administration route for gene delivery. This review focuses on the main advantages and challenges of vaginal gene therapy, and on the most used nucleic acid delivery systems, including viral and non-viral vectors. Additionally, the advances in the application of vaginal gene therapy for the treatment and/or prevention of infectious diseases such as the human immunodeficiency virus (HIV), the human papillomavirus (HPV) or the herpes simplex virus (HSV) are presented. PMID:26189799

  16. "Bad genes" & criminal responsibility.

    PubMed

    González-Tapia, María Isabel; Obsuth, Ingrid

    2015-01-01

    The genetics of the accused is trying to break into the courts. To date several candidate genes have been put forward and their links to antisocial behavior have been examined and documented with some consistency. In this paper, we focus on the so called "warrior gene", or the low-activity allele of the MAOA gene, which has been most consistently related to human behavior and specifically to violence and antisocial behavior. In preparing this paper we had two objectives. First, to summarize and analyze the current scientific evidence, in order to gain an in depth understanding of the state of the issue and determine whether a dominant line of generally accepted scientific knowledge in this field can be asserted. Second, to derive conclusions and put forward recommendations related to the use of genetic information, specifically the presence of the low-activity genotype of the MAOA gene, in modulation of criminal responsibility in European and US courts. PMID:25708001

  17. Fibrinogen gene regulation.

    PubMed

    Fish, Richard J; Neerman-Arbez, Marguerite

    2012-09-01

    The Aα, Bβ and γ polypeptide chains of fibrinogen are encoded by a three gene cluster on human chromosome four. The fibrinogen genes (FGB-FGA-FGG) are expressed almost exclusively in hepatocytes where their output is coordinated to ensure a sufficient mRNA pool for each chain and maintain an abundant plasma fibrinogen protein level. Fibrinogen gene expression is controlled by the activity of proximal promoters which contain binding sites for hepatocyte transcription factors, including proteins which influence fibrinogen transcription in response to acute-phase inflammatory stimuli. The fibrinogen gene cluster also contains cis regulatory elements; enhancer sequences with liver activities identified by sequence conservation and functional genomics. While the transcriptional control of this gene cluster is fascinating biology, the medical impetus to understand fibrinogen gene regulation stems from the association of cardiovascular disease risk with high level circulating fibrinogen. In the general population this level varies from about 1.5 to 3.5 g/l. This variation between individuals is influenced by genotype, suggesting there are genetic variants contributing to fibrinogen levels which reside in fibrinogen regulatory loci. A complete picture of how fibrinogen genes are regulated will therefore point towards novel sources of regulatory variants. In this review we discuss regulation of the fibrinogen genes from proximal promoters and enhancers, the influence of acute-phase stimulation, post-transcriptional regulation by miRNAs and functional regulatory variants identified in genetic studies. Finally, we discuss the fibrinogen locus in light of recent advances in understanding chromosomal architecture and suggest future directions for researching the mechanisms that control fibrinogen expression. PMID:22836683

  18. Gene therapy in epilepsy

    PubMed Central

    Riban, Véronique; Fitzsimons, Helen L.; During, Matthew J.

    2009-01-01

    SUMMARY Results from animal models suggest gene therapy is a promising new approach for the treatment of epilepsy. Several candidate genes such as neuropeptide Y and galanin have been demonstrated in preclinical studies to have a positive effect on seizure activity. For a successful gene therapy-based treatment, efficient delivery of a transgene to target neurons is also essential. To this end, advances have been made in the areas of cell transplantation and in the development of recombinant viral vectors for gene delivery. Recombinant adeno-associated viral (rAAV) vectors in particular show promise for gene therapy of neurological disorders due to their neuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene. rAAV vectors have been recently used in phase I clinical trials of Parkinson’s disease with an excellent safety profile. Prior to commencement of phase I trials for gene therapy of epilepsy, further preclinical studies are ongoing including evaluation of the therapeutic benefit in chronicmodels of epileptogenesis, as well as assessment of safety intoxicological studies. PMID:18717707

  19. Evidence for homosexuality gene

    SciTech Connect

    Pool, R.

    1993-07-16

    A genetic analysis of 40 pairs of homosexual brothers has uncovered a region on the X chromosome that appears to contain a gene or genes for homosexuality. When analyzing the pedigrees of homosexual males, the researcheres found evidence that the trait has a higher likelihood of being passed through maternal genes. This led them to search the X chromosome for genes predisposing to homosexuality. The researchers examined the X chromosomes of pairs of homosexual brothers for regions of DNA that most or all had in common. Of the 40 sets of brothers, 33 shared a set of five markers in the q28 region of the long arm of the X chromosome. The linkage has a LOD score of 4.0, which translates into a 99.5% certainty that there is a gene or genes in this area that predispose males to homosexuality. The chief researcher warns, however, that this one site cannot explain all instances of homosexuality, since there were some cases where the trait seemed to be passed paternally. And even among those brothers where there was no evidence that the trait was passed paternally, seven sets of brothers did not share the Xq28 markers. It seems likely that homosexuality arises from a variety of causes.

  20. Identification of four soybean reference genes for gene expression normalization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene expression analysis requires the use of reference genes stably expressed independently of specific tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin and elongation factor 1-alpha) are commonly used as reference genes with the assumption tha...

  1. 5. OVERHEAD VIEW OF GENE CAMP LOOKING SOUTH. GENE PUMP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. OVERHEAD VIEW OF GENE CAMP LOOKING SOUTH. GENE PUMP PLANT IS AT CENTER WITH ADMINISTRATIVE COMPLEX IN FOREGROUND AND RESIDENTIAL AREA BEYOND PLANT. - Gene Pump Plant, South of Gene Wash Reservoir, 2 miles west of Whitsett Pump Plant, Parker Dam, San Bernardino County, CA

  2. Prospects for gene therapy.

    PubMed

    Ali, Robin R

    2004-01-01

    Inherited retinal disease, which includes conditions such as retinitis pigmentosa (RP), affects about 1/3000 of the population in the Western world. It is characterized by gradual loss of vision and results from mutations in any one of 60 or so different genes. There are currently no effective treatments, but many of the genes have now been identified and their functions elucidated, providing a major impetus to develop gene-based treatments. Many of the disease genes are photoreceptor- or retinal pigment epithelium (RPE) cell specific. Since adeno-associated viral (AAV) vectors can be used for efficient gene transfer to these two cell types, we are developing AAV-mediated gene therapy approaches for inherited retinal degeneration using animal models that have defects in these cells. The retinal degeneration slow (rds or Prph2Rd2/Rd) mouse, a model of recessive RP, lacks a functional gene encoding peripherin 2, which is a photoreceptor-specific protein required for the formation of outer segment discs. We have previously demonstrated restoration of photoreceptor ultrastructure and function by AAV-mediated gene transfer of peripherin 2. We have now extended our assessment to central visual neuronal responses in order to show an improvement of central visual function. The Royal College of Surgeons (RCS) rat, provides another model of recessive RP. Here the defect is due to a defect in Mertk, a gene that is expressed in the RPE and encodes a receptor tyrosine kinase that is thought to be involved in the recognition and binding of outer segment debris. The gene defect results in the inability of the RPE to phagocytose the shed outer segments from photoreceptor cells. The resulting accumulation of debris between the RPE and the neuroretina leads to progressive loss of photoreceptor cells. AAV-mediated delivery of Mertk to the RPE results in reduction of debris indicating that the phagocytosing function of the RPE is restored and delays the degeneration of the

  3. Classification of genes based on gene expression analysis

    NASA Astrophysics Data System (ADS)

    Angelova, M.; Myers, C.; Faith, J.

    2008-05-01

    Systems biology and bioinformatics are now major fields for productive research. DNA microarrays and other array technologies and genome sequencing have advanced to the point that it is now possible to monitor gene expression on a genomic scale. Gene expression analysis is discussed and some important clustering techniques are considered. The patterns identified in the data suggest similarities in the gene behavior, which provides useful information for the gene functionalities. We discuss measures for investigating the homogeneity of gene expression data in order to optimize the clustering process. We contribute to the knowledge of functional roles and regulation of E. coli genes by proposing a classification of these genes based on consistently correlated genes in expression data and similarities of gene expression patterns. A new visualization tool for targeted projection pursuit and dimensionality reduction of gene expression data is demonstrated.

  4. Classification of genes based on gene expression analysis

    SciTech Connect

    Angelova, M. Myers, C. Faith, J.

    2008-05-15

    Systems biology and bioinformatics are now major fields for productive research. DNA microarrays and other array technologies and genome sequencing have advanced to the point that it is now possible to monitor gene expression on a genomic scale. Gene expression analysis is discussed and some important clustering techniques are considered. The patterns identified in the data suggest similarities in the gene behavior, which provides useful information for the gene functionalities. We discuss measures for investigating the homogeneity of gene expression data in order to optimize the clustering process. We contribute to the knowledge of functional roles and regulation of E. coli genes by proposing a classification of these genes based on consistently correlated genes in expression data and similarities of gene expression patterns. A new visualization tool for targeted projection pursuit and dimensionality reduction of gene expression data is demonstrated.

  5. GeneCards Version 3: the human gene integrator.

    PubMed

    Safran, Marilyn; Dalah, Irina; Alexander, Justin; Rosen, Naomi; Iny Stein, Tsippi; Shmoish, Michael; Nativ, Noam; Bahir, Iris; Doniger, Tirza; Krug, Hagit; Sirota-Madi, Alexandra; Olender, Tsviya; Golan, Yaron; Stelzer, Gil; Harel, Arye; Lancet, Doron

    2010-01-01

    GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73,000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a speedy and sophisticated search engine and a revamped, technologically enabling infrastructure, catering to the expanding needs of biomedical researchers. A key focus is on gene-set analyses, which leverage GeneCards' unique wealth of combinatorial annotations. These include the GeneALaCart batch query facility, which tabulates user-selected annotations for multiple genes and GeneDecks, which identifies similar genes with shared annotations, and finds set-shared annotations by descriptor enrichment analysis. Such set-centric features address a host of applications, including microarray data analysis, cross-database annotation mapping and gene-disorder associations for drug targeting. We highlight the new Version 3 database architecture, its multi-faceted search engine, and its semi-automated quality assurance system. Data enhancements include an expanded visualization of gene expression patterns in normal and cancer tissues, an integrated alternative splicing pattern display, and augmented multi-source SNPs and pathways sections. GeneCards now provides direct links to gene-related research reagents such as antibodies, recombinant proteins, DNA clones and inhibitory RNAs and features gene-related drugs and compounds lists. We also portray the GeneCards Inferred Functionality Score annotation landscape tool for scoring a gene's functional information status. Finally, we delineate examples of applications and collaborations that have benefited from the GeneCards suite. Database

  6. GH1-family 6-P-β-glucosidases from human microbiome lactic acid bacteria

    PubMed Central

    Michalska, Karolina; Tan, Kemin; Li, Hui; Hatzos-Skintges, Catherine; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej

    2013-01-01

    In lactic acid bacteria and other bacteria, carbohydrate uptake is mostly governed by phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). PTS-dependent translocation through the cell membrane is coupled with phosphorylation of the incoming sugar. After translocation through the bacterial membrane, the β-glycosidic bond in 6′-­P-­β-glucoside is cleaved, releasing 6-P-β-glucose and the respective aglycon. This reaction is catalyzed by 6-P-β-glucosidases, which belong to two glycoside hydrolase (GH) families: GH1 and GH4. Here, the high-resolution crystal structures of GH1 6-P-β-glucosidases from Lactobacillus plantarum (LpPbg1) and Streptococcus mutans (SmBgl) and their complexes with ligands are reported. Both enzymes show hydrolytic activity towards 6′-P-β-glucosides. The LpPbg1 structure has been determined in an apo form as well as in a complex with phosphate and a glucose molecule corresponding to the aglycon molecule. The S. mutans homolog contains a sulfate ion in the phosphate-dedicated subcavity. SmBgl was also crystallized in the presence of the reaction product 6-P-β-glucose. For a mutated variant of the S. mutans enzyme (E375Q), the structure of a 6′-P-salicin complex has also been determined. The presence of natural ligands enabled the definition of the structural elements that are responsible for substrate recognition during catalysis. PMID:23519420

  7. Physiological implications of class IIa bacteriocin resistance in Listeria monocytogenes strains.

    PubMed

    Vadyvaloo, Viveka; Snoep, Jacky L; Hastings, John W; Rautenbach, Marina

    2004-02-01

    High-level resistance to class IIa bacteriocins has been directly associated with the absent EIIAB(Man) (MptA) subunit of the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (EIIt(MAN)) in Listeria monocytogenes strains. Class IIa bacteriocin-resistant strains used in this study were a spontaneous resistant, L. monocytogenes B73-MR1, and a defined mutant, L. monocytogenes EGDe-mptA. Both strains were previously reported to have the EIIAB(Man) PTS component missing. This study shows that these class IIa bacteriocin-resistant strains have significantly decreased specific growth and glucose consumption rates, but they also have a significantly higher growth yield than their corresponding wild-type strains, L. monocytogenes B73 and L. monocytogenes EGDe, respectively. In the presence of glucose, the strains showed a shift from a predominantly lactic-acid to a mixed-acid fermentation. It is here proposed that elimination of the EIIAB(Man) in the resistant strains has caused a reduced glucose consumption rate and a reduced specific growth rate. The lower glucose consumption rate can be correlated to a shift in metabolism to a more efficient pathway with respect to ATP production per glucose, leading to a higher biomass yield. Thus, the cost involved in obtaining bacteriocin resistance, i.e. losing substrate transport capacity leading to a lower growth rate, is compensated for by a higher biomass yield. PMID:14766911

  8. Dephosphorylated NPr is involved in an envelope stress response of Escherichia coli

    PubMed Central

    Lee, Jaeseop; Park, Young-Ha; Kim, Yeon-Ran; Seok, Yeong-Jae

    2015-01-01

    Besides the canonical phosphoenolpyruvate-dependent phosphotransferase system (PTS) for carbohydrate transport, most Proteobacteria possess the so-called nitrogen PTS (PTSNtr) that transfers a phosphate group from phosphoenolpyruvate (PEP) over enzyme INtr (EINtr) and NPr to enzyme IIANtr (EIIANtr). The PTSNtr lacks membrane-bound components and functions exclusively in a regulatory capacity. While EIIANtr has been implicated in a variety of cellular processes such as potassium homeostasis, phosphate starvation, nitrogen metabolism, carbon metabolism, regulation of ABC transporters and poly-β-hydroxybutyrate accumulation in many Proteobacteria, the only identified role of NPr is the regulation of biosynthesis of the lipopolysaccharide (LPS) layer by direct interaction with LpxD in Escherichia coli. In this study, we provide another phenotype related to NPr. Several lines of evidence demonstrate that E. coli strains with increased levels of dephosphorylated NPr are sensitive to envelope stresses, such as osmotic, ethanol and SDS stresses, and these phenotypes are independent of LpxD. The C-terminal region of NPr plays an important role in sensitivity to envelope stresses. Thus, our data suggest that the dephospho-form of NPr affects adaptation to envelope stresses through a C-terminus-dependent mechanism. PMID:25701731

  9. How old is my gene?

    PubMed Central

    Capra, John A.; Stolzer, Maureen; Durand, Dannie; Pollard, Katherine S.

    2013-01-01

    Gene functions, interactions, disease associations, and ecological distributions are all correlated with gene age. However, it is challenging to estimate the intricate series of evolutionary events leading to a modern day gene and then reduce this history to a single age estimate. Focusing on eukaryotic gene families, we introduce a framework in which to compare current strategies for quantifying gene age, discuss key differences between these methods, and highlight several common problems. We argue that genes with complex evolutionary histories do not have a single well-defined age. As a result, care must be taken to articulate the goals and assumptions of any analysis that uses gene age estimates. Recent algorithmic advances offer the promise of gene age estimates that are fast, accurate, and consistent across gene families. This will enable a shift to integrated genome-wide analyses of all events in gene evolutionary histories in the near future. PMID:23915718

  10. Saporin suicide gene therapy.

    PubMed

    Zarovni, Natasa; Vago, Riccardo; Fabbrini, Maria Serena

    2009-01-01

    New genes useful in suicide gene therapy are those encoding toxins such as plant ribosome-inactivating proteins (RIPs), which can irreversibly block protein synthesis, triggering apoptotic cell death. Plasmids expressing a cytosolic saporin (SAP) gene from common soapwort (Saponaria officinalis) are generated by placing the region encoding the mature plant toxin under the control of strong viral promoters and may be placed under tumor-specific promoters. The ability of the resulting constructs to inhibit protein synthesis is tested in cultured tumor cells co-transfected with a luciferase reporter gene. SAP expression driven by the cytomegalovirus (CMV) promoter (pCI-SAP) demonstrates that only 10 ng ofplasmid DNA per 1.6 x 10(4) B16 melanoma cells drastically reduces luciferase reporter activity to 18% of that in control cells (1). Direct intratumoral injections are performed in an aggressive melanoma model. B16 melanoma-bearing mice injected with pCI-SAP complexed with lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) show a noteworthy attenuation in tumor growth, and this effect is significantly augmented by repeated administrations of the DNA complexes. Here, we describe in detail this cost-effective and safe suicide gene approach. PMID:19565907

  11. Hox genes and evolution

    PubMed Central

    Hrycaj, Steven M.; Wellik, Deneen M.

    2016-01-01

    Hox proteins are a deeply conserved group of transcription factors originally defined for their critical roles in governing segmental identity along the antero-posterior (AP) axis in Drosophila. Over the last 30 years, numerous data generated in evolutionarily diverse taxa have clearly shown that changes in the expression patterns of these genes are closely associated with the regionalization of the AP axis, suggesting that Hox genes have played a critical role in the evolution of novel body plans within Bilateria. Despite this deep functional conservation and the importance of these genes in AP patterning, key questions remain regarding many aspects of Hox biology. In this commentary, we highlight recent reports that have provided novel insight into the origins of the mammalian Hox cluster, the role of Hox genes in the generation of a limbless body plan, and a novel putative mechanism in which Hox genes may encode specificity along the AP axis. Although the data discussed here offer a fresh perspective, it is clear that there is still much to learn about Hox biology and the roles it has played in the evolution of the Bilaterian body plan. PMID:27239281

  12. LQTS gene LOVD database.

    PubMed

    Zhang, Tao; Moss, Arthur; Cong, Peikuan; Pan, Min; Chang, Bingxi; Zheng, Liangrong; Fang, Quan; Zareba, Wojciech; Robinson, Jennifer; Lin, Changsong; Li, Zhongxiang; Wei, Junfang; Zeng, Qiang; Qi, Ming

    2010-11-01

    The Long QT Syndrome (LQTS) is a group of genetically heterogeneous disorders that predisposes young individuals to ventricular arrhythmias and sudden death. LQTS is mainly caused by mutations in genes encoding subunits of cardiac ion channels (KCNQ1, KCNH2,SCN5A, KCNE1, and KCNE2). Many other genes involved in LQTS have been described recently(KCNJ2, AKAP9, ANK2, CACNA1C, SCNA4B, SNTA1, and CAV3). We created an online database(http://www.genomed.org/LOVD/introduction.html) that provides information on variants in LQTS-associated genes. As of February 2010, the database contains 1738 unique variants in 12 genes. A total of 950 variants are considered pathogenic, 265 are possible pathogenic, 131 are unknown/unclassified, and 292 have no known pathogenicity. In addition to these mutations collected from published literature, we also submitted information on gene variants, including one possible novel pathogenic mutation in the KCNH2 splice site found in ten Chinese families with documented arrhythmias. The remote user is able to search the data and is encouraged to submit new mutations into the database. The LQTS database will become a powerful tool for both researchers and clinicians. PMID:20809527

  13. Engineered Gene Circuits

    NASA Astrophysics Data System (ADS)

    Hasty, Jeff

    2003-03-01

    Uncovering the structure and function of gene regulatory networks has become one of the central challenges of the post-genomic era. Theoretical models of protein-DNA feedback loops and gene regulatory networks have long been proposed, and recently, certain qualitative features of such models have been experimentally corroborated. This talk will focus on model and experimental results that demonstrate how a naturally occurring gene network can be used as a ``parts list'' for synthetic network design. The model formulation leads to computational and analytical approaches relevant to nonlinear dynamics and statistical physics, and the utility of such a formulation will be demonstrated through the consideration of specific design criteria for several novel genetic devices. Fluctuations originating from small molecule-number effects will be discussed in the context of model predictions, and the experimental validation of these stochastic effects underscores the importance of internal noise in gene expression. Potential biotech applications will be highlighted within the framework of cellular control schemes. Specifically, the coupling of an oscillating cellular process to a synthetic oscillator will be considered, and the resulting model behavior will be analyzed in the context of synchronization. The underlying methodology highlights the utility of engineering-based methods in the design of synthetic gene regulatory networks.

  14. FunGene: the functional gene pipeline and repository

    PubMed Central

    Fish, Jordan A.; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C. Titus; Tiedje, James M.; Cole, James R.

    2013-01-01

    Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes. PMID:24101916

  15. GeneCards Version 3: the human gene integrator

    PubMed Central

    Safran, Marilyn; Dalah, Irina; Alexander, Justin; Rosen, Naomi; Iny Stein, Tsippi; Shmoish, Michael; Nativ, Noam; Bahir, Iris; Doniger, Tirza; Krug, Hagit; Sirota-Madi, Alexandra; Olender, Tsviya; Golan, Yaron; Stelzer, Gil; Harel, Arye; Lancet, Doron

    2010-01-01

    GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73 000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a speedy and sophisticated search engine and a revamped, technologically enabling infrastructure, catering to the expanding needs of biomedical researchers. A key focus is on gene-set analyses, which leverage GeneCards’ unique wealth of combinatorial annotations. These include the GeneALaCart batch query facility, which tabulates user-selected annotations for multiple genes and GeneDecks, which identifies similar genes with shared annotations, and finds set-shared annotations by descriptor enrichment analysis. Such set-centric features address a host of applications, including microarray data analysis, cross-database annotation mapping and gene-disorder associations for drug targeting. We highlight the new Version 3 database architecture, its multi-faceted search engine, and its semi-automated quality assurance system. Data enhancements include an expanded visualization of gene expression patterns in normal and cancer tissues, an integrated alternative splicing pattern display, and augmented multi-source SNPs and pathways sections. GeneCards now provides direct links to gene-related research reagents such as antibodies, recombinant proteins, DNA clones and inhibitory RNAs and features gene-related drugs and compounds lists. We also portray the GeneCards Inferred Functionality Score annotation landscape tool for scoring a gene’s functional information status. Finally, we delineate examples of applications and collaborations that have benefited from the GeneCards suite

  16. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  17. Virus induced gene silencing of Arabidopsis gene homologues in wheat identify genes conferring improved drought tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a non-model staple crop like wheat, functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for wheat breeding. Virus induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited tra...

  18. Neighboring Genes Show Correlated Evolution in Gene Expression

    PubMed Central

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  19. Prokaryotic gene prediction using GeneMark and GeneMark.hmm.

    PubMed

    Borodovsky, Mark; Mills, Ryan; Besemer, John; Lomsadze, Alex

    2003-05-01

    In this unit, the GeneMark and GeneMark.hmm programs are presented as two different methods for the in silico prediction of genes in prokaryotes. GeneMark can be used for whole genome analysis as well as for the local analysis of a particular gene and its surrounding regions. GeneMark.hmm makes use of Hidden Markov models to find the transition points (boundaries) between protein coding states and noncoding states and can be efficiently used for larger genome sequences. These methods can be used in conjunction with each other for a higher sensitivity of gene detection. PMID:18428700

  20. Genes and Vocal Learning

    PubMed Central

    White, Stephanie A.

    2009-01-01

    Could a mutation in a single gene be the evolutionary lynchpin supporting the development of human language? A rare mutation in the molecule known as FOXP2 discovered in a human family seemed to suggest so, and its sequence phylogeny reinforced a Chomskian view that language emerged wholesale in humans. Spurred by this discovery, research in primates, rodents and birds suggests that FoxP2 and other language-related genes are interactors in the neuromolecular networks that underlie subsystems of language, such symbolic understanding, vocal learning and theory of mind. The whole picture will only come together through comparative and integrative study into how the human language singularity evolved. PMID:19913899

  1. The gene tree delusion.

    PubMed

    Springer, Mark S; Gatesy, John

    2016-01-01

    Higher-level relationships among placental mammals are mostly resolved, but several polytomies remain contentious. Song et al. (2012) claimed to have resolved three of these using shortcut coalescence methods (MP-EST, STAR) and further concluded that these methods, which assume no within-locus recombination, are required to unravel deep-level phylogenetic problems that have stymied concatenation. Here, we reanalyze Song et al.'s (2012) data and leverage these re-analyses to explore key issues in systematics including the recombination ratchet, gene tree stoichiometry, the proportion of gene tree incongruence that results from deep coalescence versus other factors, and simulations that compare the performance of coalescence and concatenation methods in species tree estimation. Song et al. (2012) reported an average locus length of 3.1 kb for the 447 protein-coding genes in their phylogenomic dataset, but the true mean length of these loci (start codon to stop codon) is 139.6 kb. Empirical estimates of recombination breakpoints in primates, coupled with consideration of the recombination ratchet, suggest that individual coalescence genes (c-genes) approach ∼12 bp or less for Song et al.'s (2012) dataset, three to four orders of magnitude shorter than the c-genes reported by these authors. This result has general implications for the application of coalescence methods in species tree estimation. We contend that it is illogical to apply coalescence methods to complete protein-coding sequences. Such analyses amalgamate c-genes with different evolutionary histories (i.e., exons separated by >100,000 bp), distort true gene tree stoichiometry that is required for accurate species tree inference, and contradict the central rationale for applying coalescence methods to difficult phylogenetic problems. In addition, Song et al.'s (2012) dataset of 447 genes includes 21 loci with switched taxonomic names, eight duplicated loci, 26 loci with non-homologous sequences that are

  2. XLMR genes: Update 1994

    SciTech Connect

    Neri, G.; Chiurazzi, P.; Arena, J.F.; Lubs, H.A.

    1994-07-15

    We provide a comprehensive list of all known forms of X-linked mental retardation. It comprises 127 entries, subdivided into 5 categories (syndromes, dominant disorders, and nonspecific mental retardation). Map location of 69 putative loci demonstrates several overlaps, which will only be resolved by more refined mapping or cloning of the respective genes. The ultimate goal of identifying all the genes on the X chromosome whose mutations cause mental retardation will require a concerted effort between clinical and molecular investigators. 74 refs., 2 figs., 5 tabs.

  3. Gene therapy: progress and predictions

    PubMed Central

    Collins, Mary; Thrasher, Adrian

    2015-01-01

    The first clinical gene delivery, which involved insertion of a marker gene into lymphocytes from cancer patients, was published 25 years ago. In this review, we describe progress since then in gene therapy. Patients with some inherited single-gene defects can now be treated with their own bone marrow stem cells that have been engineered with a viral vector carrying the missing gene. Patients with inherited retinopathies and haemophilia B can also be treated by local or systemic injection of viral vectors. There are also a number of promising gene therapy approaches for cancer and infectious disease. We predict that the next 25 years will see improvements in safety, efficacy and manufacture of gene delivery vectors and introduction of gene-editing technologies to the clinic. Gene delivery may also prove a cost-effective method for the delivery of biological medicines. PMID:26702034

  4. Multidimensional gene search with Genehopper

    PubMed Central

    Munz, Matthias; Tönnies, Sascha; Balke, Wolf-Tilo; Simon, Eric

    2015-01-01

    The high abundance of genetic information enables researchers to gain new insights from the comparison of human genes according to their similarities. However, existing tools that allow the exploration of such gene-to-gene relationships, apply each similarity independently. To make use of multidimensional scoring, we developed a new search engine named Genehopper. It can handle two query types: (i) the typical use case starts with a term-to-gene search, i.e. an optimized full-text search for an anchor gene of interest. The web-interface can handle one or more terms including gene symbols and identifiers of Ensembl, UniProt, EntrezGene and RefSeq. (ii) When the anchor gene is defined, the user can explore its neighborhood by a gene-to-gene search as the weighted sum of nine normalized gene similarities based on sequence homology, protein domains, mRNA expression profiles, Gene Ontology Annotation, gene symbols and other features. Each weight can be adjusted by the user, allowing flexible customization of the gene search. All implemented similarities have a low pairwise correlation (max r2 = 0.4) implying a low linear dependency, i.e. any change in a single weight has an effect on the ranking. Thus, we treated them as separate dimensions in the search space. Genehopper is freely available at http://genehopper.ifis.cs.tu-bs.de. PMID:25990726

  5. Multidimensional gene search with Genehopper.

    PubMed

    Munz, Matthias; Tönnies, Sascha; Balke, Wolf-Tilo; Simon, Eric

    2015-07-01

    The high abundance of genetic information enables researchers to gain new insights from the comparison of human genes according to their similarities. However, existing tools that allow the exploration of such gene-to-gene relationships, apply each similarity independently. To make use of multidimensional scoring, we developed a new search engine named Genehopper. It can handle two query types: (i) the typical use case starts with a term-to-gene search, i.e. an optimized full-text search for an anchor gene of interest. The web-interface can handle one or more terms including gene symbols and identifiers of Ensembl, UniProt, EntrezGene and RefSeq. (ii) When the anchor gene is defined, the user can explore its neighborhood by a gene-to-gene search as the weighted sum of nine normalized gene similarities based on sequence homology, protein domains, mRNA expression profiles, Gene Ontology Annotation, gene symbols and other features. Each weight can be adjusted by the user, allowing flexible customization of the gene search. All implemented similarities have a low pairwise correlation (max r(2) = 0.4) implying a low linear dependency, i.e. any change in a single weight has an effect on the ranking. Thus, we treated them as separate dimensions in the search space. Genehopper is freely available at http://genehopper.ifis.cs.tu-bs.de. PMID:25990726

  6. Gene therapy in pancreatic cancer

    PubMed Central

    Liu, Si-Xue; Xia, Zhong-Sheng; Zhong, Ying-Qiang

    2014-01-01

    Pancreatic cancer (PC) is a highly lethal disease and notoriously difficult to treat. Only a small proportion of PC patients are eligible for surgical resection, whilst conventional chemoradiotherapy only has a modest effect with substantial toxicity. Gene therapy has become a new widely investigated therapeutic approach for PC. This article reviews the basic rationale, gene delivery methods, therapeutic targets and developments of laboratory research and clinical trials in gene therapy of PC by searching the literature published in English using the PubMed database and analyzing clinical trials registered on the Gene Therapy Clinical Trials Worldwide website (http://www. wiley.co.uk/genmed/ clinical). Viral vectors are main gene delivery tools in gene therapy of cancer, and especially, oncolytic virus shows brighter prospect due to its tumor-targeting property. Efficient therapeutic targets for gene therapy include tumor suppressor gene p53, mutant oncogene K-ras, anti-angiogenesis gene VEGFR, suicide gene HSK-TK, cytosine deaminase and cytochrome p450, multiple cytokine genes and so on. Combining different targets or combination strategies with traditional chemoradiotherapy may be a more effective approach to improve the efficacy of cancer gene therapy. Cancer gene therapy is not yet applied in clinical practice, but basic and clinical studies have demonstrated its safety and clinical benefits. Gene therapy will be a new and promising field for the treatment of PC. PMID:25309069

  7. Association Between a Prognostic Gene Signature and Functional Gene Sets

    PubMed Central

    Hummel, Manuela; Metzeler, Klaus H.; Buske, Christian; Bohlander, Stefan K.; Mansmann, Ulrich

    2008-01-01

    Background The development of expression-based gene signatures for predicting prognosis or class membership is a popular and challenging task. Besides their stringent validation, signatures need a functional interpretation and must be placed in a biological context. Popular tools such as Gene Set Enrichment have drawbacks because they are restricted to annotated genes and are unable to capture the information hidden in the signature’s non-annotated genes. Methodology We propose concepts to relate a signature with functional gene sets like pathways or Gene Ontology categories. The connection between single signature genes and a specific pathway is explored by hierarchical variable selection and gene association networks. The risk score derived from an individual patient’s signature is related to expression patterns of pathways and Gene Ontology categories. Global tests are useful for these tasks, and they adjust for other factors. GlobalAncova is used to explore the effect on gene expression in specific functional groups from the interaction of the score and selected mutations in the patient’s genome. Results We apply the proposed methods to an expression data set and a corresponding gene signature for predicting survival in Acute Myeloid Leukemia (AML). The example demonstrates strong relations between the signature and cancer-related pathways. The signature-based risk score was found to be associated with development-related biological processes. Conclusions Many authors interpret the functional aspects of a gene signature by linking signature genes to pathways or relevant functional gene groups. The method of gene set enrichment is preferred to annotating signature genes to specific Gene Ontology categories. The strategies proposed in this paper go beyond the restriction of annotation and deepen the insights into the biological mechanisms reflected in the information given by a signature. PMID:19812786

  8. Old genes experience stronger translational selection than young genes.

    PubMed

    Yin, Hongyan; Ma, Lina; Wang, Guangyu; Li, Mengwei; Zhang, Zhang

    2016-09-15

    Selection on synonymous codon usage for translation efficiency and/or accuracy has been identified as a widespread mechanism in many living organisms. However, it remains unknown whether translational selection associates closely with gene age and acts differentially on genes with different evolutionary ages. To address this issue, here we investigate the strength of translational selection acting on different aged genes in human. Our results show that old genes present stronger translational selection than young genes, demonstrating that translational selection correlates positively with gene age. We further explore the difference of translational selection in duplicates vs. singletons and in housekeeping vs. tissue-specific genes. We find that translational selection acts comparably in old singletons and old duplicates and stronger translational selection in old genes is contributed primarily by housekeeping genes. For young genes, contrastingly, singletons experience stronger translational selection than duplicates, presumably due to redundant function of duplicated genes during their early evolutionary stage. Taken together, our results indicate that translational selection acting on a gene would not be constant during all stages of evolution, associating closely with gene age. PMID:27259662

  9. Genes2FANs: connecting genes through functional association networks

    PubMed Central

    2012-01-01

    Background Protein-protein, cell signaling, metabolic, and transcriptional interaction networks are useful for identifying connections between lists of experimentally identified genes/proteins. However, besides physical or co-expression interactions there are many ways in which pairs of genes, or their protein products, can be associated. By systematically incorporating knowledge on shared properties of genes from diverse sources to build functional association networks (FANs), researchers may be able to identify additional functional interactions between groups of genes that are not readily apparent. Results Genes2FANs is a web based tool and a database that utilizes 14 carefully constructed FANs and a large-scale protein-protein interaction (PPI) network to build subnetworks that connect lists of human and mouse genes. The FANs are created from mammalian gene set libraries where mouse genes are converted to their human orthologs. The tool takes as input a list of human or mouse Entrez gene symbols to produce a subnetwork and a ranked list of intermediate genes that are used to connect the query input list. In addition, users can enter any PubMed search term and then the system automatically converts the returned results to gene lists using GeneRIF. This gene list is then used as input to generate a subnetwork from the user’s PubMed query. As a case study, we applied Genes2FANs to connect disease genes from 90 well-studied disorders. We find an inverse correlation between the counts of links connecting disease genes through PPI and links connecting diseases genes through FANs, separating diseases into two categories. Conclusions Genes2FANs is a useful tool for interpreting the relationships between gene/protein lists in the context of their various functions and networks. Combining functional association interactions with physical PPIs can be useful for revealing new biology and help form hypotheses for further experimentation. Our finding that disease genes in

  10. Gene Manipulation In Cereals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum, the most abundant metal on earth, is detrimental to plant growth and agricultural production. There are about 2.5 billion hectares of acid soils high in aluminum around the world. Molecular markers linked to aluminum tolerance gene complexes in rye would be of value in marker-mediated ge...

  11. Ultrasound mediated gene transfection

    NASA Astrophysics Data System (ADS)

    Williamson, Rene G.; Apfel, Robert E.; Brandsma, Janet L.

    2002-05-01

    Gene therapy is a promising modality for the treatment of a variety of human diseases both inherited and acquired, such as cystic fibrosis and cancer. The lack of an effective, safe method for the delivery of foreign genes into the cells, a process known as transfection, limits this effort. Ultrasound mediated gene transfection is an attractive method for gene delivery since it is a noninvasive technique, does not introduce any viral particles into the host and can offer very good temporal and spatial control. Previous investigators have shown that sonication increases transfection efficiency with and without ultrasound contrast agents. The mechanism is believed to be via a cavitation process where collapsing bubble nuclei permeabilize the cell membrane leading to increased DNA transfer. The research is focused on the use of pulsed wave high frequency focused ultrasound to transfect DNA into mammalian cells in vitro and in vivo. A better understanding of the mechanism behind the transfection process is also sought. A summary of some in vitro results to date will be presented, which includes the design of a sonication chamber that allows us to model the in vivo case more accurately.

  12. Resistance gene capture.

    PubMed

    Rowe-Magnus, D A; Mazel, D

    1999-10-01

    Integrons are the primary mechanism for antibiotic-resistance gene capture and dissemination among Gram-negative bacteria. The recent finding of super-integron structures in the genomes of several bacterial species has expanded their role in genome evolution and suggests that they are the source of mobile multi-resistant integrons. PMID:10508722

  13. Naming genes beyond Caenorhabditis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nomenclature of genes in Caenorhabditis elegans is based on long-standing, successful guidelines established in the late 1970s. Over time these guidelines have matured into a comprehensive, systematic nomenclature that is easy to apply, descriptive and therefore highly informative. Recently, a f...

  14. Gene stacking by recombinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits into diverse crops grown in a variety of environments. Over two decades of research has identified several site-specific recombinases that carry out efficient cis and trans recombination betw...

  15. Genes and Vocal Learning

    ERIC Educational Resources Information Center

    White, Stephanie A.

    2010-01-01

    Could a mutation in a single gene be the evolutionary lynchpin supporting the development of human language? A rare mutation in the molecule known as FOXP2 discovered in a human family seemed to suggest so, and its sequence phylogeny reinforced a Chomskian view that language emerged wholesale in humans. Spurred by this discovery, research in…

  16. Entrez Gene: gene-centered information at NCBI.

    PubMed

    Maglott, Donna; Ostell, Jim; Pruitt, Kim D; Tatusova, Tatiana

    2007-01-01

    Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) is NCBI's database for gene-specific information. Entrez Gene includes records from genomes that have been completely sequenced, that have an active research community to contribute gene-specific information or that are scheduled for intense sequence analysis. The content of Entrez Gene represents the result of both curation and automated integration of data from NCBI's Reference Sequence project (RefSeq), from collaborating model organism databases and from other databases within NCBI. Records in Entrez Gene are assigned unique, stable and tracked integers as identifiers. The content (nomenclature, map location, gene products and their attributes, markers, phenotypes and links to citations, sequences, variation details, maps, expression, homologs, protein domains and external databases) is provided via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programing utilities (E-Utilities), and for bulk transfer by ftp. PMID:17148475

  17. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

    PubMed Central

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  18. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    PubMed

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  19. Magnetic nanoparticles: Applications in gene delivery and gene therapy.

    PubMed

    Majidi, Sima; Zeinali Sehrig, Fatemeh; Samiei, Mohammad; Milani, Morteza; Abbasi, Elham; Dadashzadeh, Kianoosh; Akbarzadeh, Abolfazl

    2016-06-01

    Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs. PMID:25727710

  20. Dominance from the perspective of gene-gene and gene-chemical interactions.

    PubMed

    Gladki, Arkadiusz; Zielenkiewicz, Piotr; Kaczanowski, Szymon

    2016-02-01

    In this study, we used genetic interaction (GI) and gene-chemical interaction (GCI) data to compare mutations with different dominance phenotypes. Our analysis focused primarily on Saccharomyces cerevisiae, where haploinsufficient genes (HI; genes with dominant loss-of-function mutations) were found to be participating in gene expression processes, namely, the translation and regulation of gene transcription. Non-ribosomal HI genes (mainly regulators of gene transcription) were found to have more GIs and GCIs than haplosufficient (HS) genes. Several properties seem to lead to the enrichment of interactions, most notably, the following: importance, pleiotropy, gene expression level and gene expression variation. Importantly, after these properties were appropriately considered in the analysis, the correlation between dominance and GI/GCI degrees was still observed. Strikingly, for the GCIs of heterozygous strains, haploinsufficiency was the only property significantly correlated with the number of GCIs. We found ribosomal HI genes to be depleted in GIs/GCIs. This finding can be explained by their high variation in gene expression under different genetic backgrounds and environmental conditions. We observed the same distributions of GIs among non-ribosomal HI, ribosomal HI and HS genes in three other species: Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens. One potentially interesting exception was the lack of significant differences in the degree of GIs between non-ribosomal HI and HS genes in Schizosaccharomyces pombe. PMID:26613610

  1. Avirulence Genes in Cereal Powdery Mildews: The Gene-for-Gene Hypothesis 2.0

    PubMed Central

    Bourras, Salim; McNally, Kaitlin E.; Müller, Marion C.; Wicker, Thomas; Keller, Beat

    2016-01-01

    The gene-for-gene hypothesis states that for each gene controlling resistance in the host, there is a corresponding, specific gene controlling avirulence in the pathogen. Allelic series of the cereal mildew resistance genes Pm3 and Mla provide an excellent system for genetic and molecular analysis of resistance specificity. Despite this opportunity for molecular research, avirulence genes in mildews remain underexplored. Earlier work in barley powdery mildew (B.g. hordei) has shown that the reaction to some Mla resistance alleles is controlled by multiple genes. Similarly, several genes are involved in the specific interaction of wheat mildew (B.g. tritici) with the Pm3 allelic series. We found that two mildew genes control avirulence on Pm3f: one gene is involved in recognition by the resistance protein as demonstrated by functional studies in wheat and the heterologous host Nicotiana benthamiana. A second gene is a suppressor, and resistance is only observed in mildew genotypes combining the inactive suppressor and the recognized Avr. We propose that such suppressor/avirulence gene combinations provide the basis of specificity in mildews. Depending on the particular gene combinations in a mildew race, different genes will be genetically identified as the “avirulence” gene. Additionally, the observation of two LINE retrotransposon-encoded avirulence genes in B.g. hordei further suggests that the control of avirulence in mildew is more complex than a canonical gene-for-gene interaction. To fully understand the mildew–cereal interactions, more knowledge on avirulence determinants is needed and we propose ways how this can be achieved based on recent advances in the field. PMID:26973683

  2. Gene prediction and gene classes in Arabidopsis thaliana.

    PubMed

    Mathé, C; Déhais, P; Pavy, N; Rombauts, S; Van Montagu, M; Rouzé, P

    2000-03-31

    Gene prediction methods for eukaryotic genomes still are not fully satisfying. One way to improve gene prediction accuracy, proven to be relevant for prokaryotes, is to consider more than one model of genes. Thus, we used our classification of Arabidopsis thaliana genes in two classes (CU(1) and CU(2)), previously delineated according to statistical features, in the GeneMark gene identification program. For each gene class, as well as for the two classes combined, a Markov model was developed (respectively, GM-CU(1), GM-CU(2) and GM-all) and then used on a test set of 168 genes to compare their respective efficiency. We concluded from this analysis that GM-CU(1) is more sensitive than GM-CU(2) which seems to be more specific to a gene type. Besides, GM-all does not give better results than GM-CU(1) and combining results from GM-CU(1) and GM-CU(2) greatly improve prediction efficiency in comparison with predictions made with GM-all only. Thus, this work confirms the necessity to consider more than one gene model for gene prediction in eukaryotic genomes, and to look for gene classes in order to build these models. PMID:10751690

  3. GENE METHYLATION CHANGES IN TUMOR SUPPRESSOR GENES INDUCED BY ARSENIC

    EPA Science Inventory

    The choice of a dose-response model used for extrapolation can be influenced by knowledge of mechanism of action. We have already showed that arsenic affects methylation of the human p53 gene promoter. Evidence that genes other than the p53 tumor suppressor gene are affected woul...

  4. Eukaryotic gene prediction using GeneMark.hmm-E and GeneMark-ES.

    PubMed

    Borodovsky, Mark; Lomsadze, Alex

    2011-09-01

    This unit describes how to use the gene-finding programs GeneMark.hmm-E and GeneMark-ES for finding protein-coding genes in the genomic DNA of eukaryotic organisms. These bioinformatics tools have been demonstrated to have state-of-the-art accuracy for many fungal, plant, and animal genomes, and have frequently been used for gene annotation in novel genomic sequences. An additional advantage of GeneMark-ES is that the problem of algorithm parameterization is solved automatically, with parameters estimated by iterative self-training (unsupervised training). PMID:21901742

  5. Chapter 15: Disease Gene Prioritization

    PubMed Central

    Bromberg, Yana

    2013-01-01

    Disease-causing aberrations in the normal function of a gene define that gene as a disease gene. Proving a causal link between a gene and a disease experimentally is expensive and time-consuming. Comprehensive prioritization of candidate genes prior to experimental testing drastically reduces the associated costs. Computational gene prioritization is based on various pieces of correlative evidence that associate each gene with the given disease and suggest possible causal links. A fair amount of this evidence comes from high-throughput experimentation. Thus, well-developed methods are necessary to reliably deal with the quantity of information at hand. Existing gene prioritization techniques already significantly improve the outcomes of targeted experimental studies. Faster and more reliable techniques that account for novel data types are necessary for the development of new diagnostics, treatments, and cure for many diseases. PMID:23633938

  6. SOX genes: architects of development.

    PubMed Central

    Prior, H. M.; Walter, M. A.

    1996-01-01

    Development in higher organisms involves complex genetic regulation at the molecular level. The emerging picture of development control includes several families of master regulatory genes which can affect the expression of down-stream target genes in developmental cascade pathways. One new family of such development regulators is the SOX gene family. The SOX genes are named for a shared motif called the SRY box a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene. Like SRY, SOX genes play important roles in chordate development. At least a dozen human SOX genes have been identified and partially characterized (Tables 1 and 2). Mutations in SOX9 have recently been linked to campomelic dysplasia and autosomal sex reversal, and other SOX genes may also be associated with human disease. Images FIG. 1 FIG. 2 PMID:8827711

  7. Gene Therapy for Lung Cancer.

    PubMed

    Lara-Guerra, Humberto; Roth, Jack A

    2016-01-01

    Gene therapy was originally conceived to treat monogenic diseases. The replacement of a defective gene with a functional gene can theoretically cure the disease. In cancer, multiple genetic defects are present and the molecular profile changes during the course of the disease, making the replacement of all defective genes impossible. To overcome these difficulties, various gene therapy strategies have been adopted, including immune stimulation, transfer of suicide genes, inhibition of driver oncogenes, replacement of tumor-suppressor genes that could mediate apoptosis or anti-angiogenesis, and transfer of genes that enhance conventional treatments such as radiotherapy and chemotherapy. Some of these strategies have been tested successfully in non-small-cell lung cancer patients and the results of laboratory studies and clinical trials are reviewed herein. PMID:27481008

  8. On atavisms and atavistic genes.

    PubMed

    Cantú, J M; Ruiz, C

    1985-01-01

    The authors propose the term atavistic to designate a gene producing an ancestral phenotype (atavism). Several examples are presented, and the possible origin of atavistic genes, as well as their pathological implications discussed. PMID:3879145

  9. Gene Testing for Hereditary Ataxia

    MedlinePlus

    ... have a family history of ataxia, but diagnostic tests for known ataxia genes cannot explain the ataxia in their family. In recent years, scientists have developed technologies to sequence thousands of genes at the same ...

  10. Gene therapy in keratoconus

    PubMed Central

    Farjadnia, Mahgol; Naderan, Mohammad; Mohammadpour, Mehrdad

    2015-01-01

    Keratoconus (KC) is the most common ectasia of the cornea and is a common reason for corneal transplant. Therapeutic strategies that can arrest the progression of this disease and modify the underlying pathogenesis are getting more and more popularity among scientists. Cumulating data represent strong evidence of a genetic role in the pathogenesis of KC. Different loci have been identified, and certain mutations have also been mapped for this disease. Moreover, Biophysical properties of the cornea create an appropriate candidate of this tissue for gene therapy. Immune privilege, transparency and ex vivo stability are among these properties. Recent advantage in vectors, besides the ability to modulate the corneal milieu for accepting the target gene for a longer period and fruitful translation, make a big hope for stupendous results reasonable. PMID:25709266

  11. Graphene based gene transfection

    NASA Astrophysics Data System (ADS)

    Feng, Liangzhu; Zhang, Shuai; Liu, Zhuang

    2011-03-01

    Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI

  12. Brains, Genes and Primates

    PubMed Central

    Belmonte, Juan Carlos Izpisua; Callaway, Edward M.; Churchland, Patricia; Caddick, Sarah J.; Feng, Guoping; Homanics, Gregg E.; Lee, Kuo-Fen; Leopold, David A.; Miller, Cory T.; Mitchell, Jude F.; Mitalipov, Shoukhrat; Moutri, Alysson R.; Movshon, J. Anthony; Okano, Hideyuki; Reynolds, John H.; Ringach, Dario; Sejnowski, Terrence J.; Silva, Afonso C.; Strick, Peter L.; Wu, Jun; Zhang, Feng

    2015-01-01

    One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward. PMID:25950631

  13. Gene expression technology

    SciTech Connect

    Goeddel, D.V. )

    1990-01-01

    The articles in this volume were assemble to enable the reader to design effective strategies for the expression of cloned genes and cDNAs. More than a compilation of papers describing the multitude of techniques now available for expressing cloned genes, this volume provides a manual that should prove useful for solving the majority of expression problems one likely to encounter. The four major expression systems commonly available to most investigators are stressed: Escherichia coli, Bacillus subtilis, yeast, and mammalian cells. Each of these system has its advantages and disadvantages, details of which are found in Chapter 1 and the strategic overviews for the four major sections of the volume. The papers in each of these sections provide many suggestions on how to proceed if initial expression levels are not sufficient.

  14. Eukaryotic gene prediction using GeneMark.hmm.

    PubMed

    Borodovsky, Mark; Lomsadze, Alex; Ivanov, Nikolai; Mills, Ryan

    2003-05-01

    In this unit, eukaryotic GeneMark.hmm is presented as a method for detecting genes in eukaryotic DNA sequences. The eukaryotic GeneMark.hmm uses Markov models of protein coding and noncoding sequences, as well as positional nucleotide frequency matrices for prediction of the translational start, translational termination and splice sites. All these models along with length distributions of exons, introns and intergenic regions are integrated into one Hidden Markov model. The unit describes running the program over the Internet and locally on a Unix machine. It also discusses GeneMarkS EV, which can be used to detect genes in eukaryotic viruses. PMID:18428701

  15. Independent Gene Discovery and Testing

    ERIC Educational Resources Information Center

    Palsule, Vrushalee; Coric, Dijana; Delancy, Russell; Dunham, Heather; Melancon, Caleb; Thompson, Dennis; Toms, Jamie; White, Ashley; Shultz, Jeffry

    2010-01-01

    A clear understanding of basic gene structure is critical when teaching molecular genetics, the central dogma and the biological sciences. We sought to create a gene-based teaching project to improve students' understanding of gene structure and to integrate this into a research project that can be implemented by instructors at the secondary level…

  16. Time ordering of gene coexpression.

    PubMed

    Leng, Xiaoyan; Müller, Hans-Georg

    2006-10-01

    Temporal microarray gene expression profiles allow characterization of gene function through time dynamics of gene coexpression within the same genetic pathway. In this paper, we define and estimate a global time shift characteristic for each gene via least squares, inferred from pairwise curve alignments. These time shift characteristics of individual genes reflect a time ordering that is derived from ob- served temporal gene expression profiles. Once these time shift characteristics are obtained for each gene, they can be entered into further analyses, such as clustering. We illustrate the proposed methodology using Drosophila embryonic development and yeast cell-cycle gene expression profiles, as well as simulations. Feasibility is demonstrated through the successful recovery of time ordering. Estimated time shifts for Drosophila maternal and zygotic genes provide excellent discrimination between these two categories and confirm known genetic pathways through the time order of gene expression. The application to yeast cell-cycle data establishes a natural time order of genes that is in line with cell-cycle phases. The method does not require periodicity of gene expression profiles. Asymptotic justifications are also provided. PMID:16495429

  17. Gene Porter Bridwell

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Gene Porter Bridwell served as the director of the Marshall Space Flight Center from January 6, 1994 until February 3, 1996, when he retired from NASA after thirty-four years service. Bridwell, a Marshall employee since 1962, had been Marshall's Space Shuttle Projects Office Director and Space Station Redesign Team deputy manager. Under Bridwell, Marshall worked to develop its role as a Center of Excellence for propulsion and for providing access to space.

  18. Genes and athletes.

    PubMed

    Patel, Dilip R; Greydanus, Donald E

    2002-06-01

    Genetics plays an important role in determining characteristics desired for success in a given sport. Advances in biotechnology pose interesting and perplexing dilemmas for athletes, parents, health care providers, and society at large. Gene therapy holds great prospects for disease prevention and treatment. The same techniques also can be misused for genetic manipulation to enhance athletic prowess. This chapter reviews selective aspects of genotype influence on sport performance, uses and misuses of genetic technology, and ethical as well as legal dilemmas. PMID:11986034

  19. nanosheets for gene therapy

    NASA Astrophysics Data System (ADS)

    Kou, Zhongyang; Wang, Xin; Yuan, Renshun; Chen, Huabin; Zhi, Qiaoming; Gao, Ling; Wang, Bin; Guo, Zhaoji; Xue, Xiaofeng; Cao, Wei; Guo, Liang

    2014-10-01

    A new class of two-dimensional (2D) nanomaterial, transition metal dichalcogenides (TMDCs) such as MoS2, MoSe2, WS2, and WSe2 which have fantastic physical and chemical properties, has drawn tremendous attention in different fields recently. Herein, we for the first time take advantage of the great potential of MoS2 with well-engineered surface as a novel type of 2D nanocarriers for gene delivery and therapy of cancer. In our system, positively charged MoS2-PEG-PEI is synthesized with lipoic acid-modified polyethylene glycol (LA-PEG) and branched polyethylenimine (PEI). The amino end of positively charged nanomaterials can bind to the negatively charged small interfering RNA (siRNA). After detection of physical and chemical characteristics of the nanomaterial, cell toxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Polo-like kinase 1 (PLK1) was investigated as a well-known oncogene, which was a critical regulator of cell cycle transmission at multiple levels. Through knockdown of PLK1 with siRNA carried by novel nanovector, qPCR and Western blot were used to measure the interfering efficiency; apoptosis assay was used to detect the transfection effect of PLK1. All results showed that the novel nanocarrier revealed good biocompatibility, reduced cytotoxicity, as well as high gene-carrying ability without serum interference, thus would have great potential for gene delivery and therapy.

  20. Extracting gene-gene interactions through curve fitting.

    PubMed

    Das, Ranajit; Mitra, Sushmita; Murthy, C A

    2012-12-01

    This paper presents a simple and novel curve fitting approach for generating simple gene regulatory subnetworks from time series gene expression data. Microarray experiments simultaneously generate massive data sets and help immensely in the large-scale study of gene expression patterns. Initial biclustering reduces the search space in the high-dimensional microarray data. The least-squares error between fitting of gene pairs is minimized to extract a set of gene-gene interactions, involving transcriptional regulation of genes. The higher error values are eliminated to retain only the strong interacting gene pairs in the resultant gene regulatory subnetwork. Next the algorithm is extended to a generalized framework to enhance its capability. The methodology takes care of the higher-order dependencies involving multiple genes co-regulating a single gene, while eliminating the need for user-defined parameters. It has been applied to the time-series Yeast data, and the experimental results biologically validated using standard databases and literature. PMID:22997274

  1. From SNPs to Genes: Disease Association at the Gene Level

    PubMed Central

    Lehne, Benjamin; Lewis, Cathryn M.; Schlitt, Thomas

    2011-01-01

    Interpreting Genome-Wide Association Studies (GWAS) at a gene level is an important step towards understanding the molecular processes that lead to disease. In order to incorporate prior biological knowledge such as pathways and protein interactions in the analysis of GWAS data it is necessary to derive one measure of association for each gene. We compare three different methods to obtain gene-wide test statistics from Single Nucleotide Polymorphism (SNP) based association data: choosing the test statistic from the most significant SNP; the mean test statistics of all SNPs; and the mean of the top quartile of all test statistics. We demonstrate that the gene-wide test statistics can be controlled for the number of SNPs within each gene and show that all three methods perform considerably better than expected by chance at identifying genes with confirmed associations. By applying each method to GWAS data for Crohn's Disease and Type 1 Diabetes we identified new potential disease genes. PMID:21738570

  2. Advances in Gene Delivery Systems

    PubMed Central

    Kamimura, Kenya; Suda, Takeshi; Zhang, Guisheng; Liu, Dexi

    2011-01-01

    The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retro-virus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral- and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives. PMID:22200988

  3. The Gene Wiki: community intelligence applied to human gene annotation.

    PubMed

    Huss, Jon W; Lindenbaum, Pierre; Martone, Michael; Roberts, Donabel; Pizarro, Angel; Valafar, Faramarz; Hogenesch, John B; Su, Andrew I

    2010-01-01

    Annotating the function of all human genes is a critical, yet formidable, challenge. Current gene annotation efforts focus on centralized curation resources, but it is increasingly clear that this approach does not scale with the rapid growth of the biomedical literature. The Gene Wiki utilizes an alternative and complementary model based on the principle of community intelligence. Directly integrated within the online encyclopedia, Wikipedia, the goal of this effort is to build a gene-specific review article for every gene in the human genome, where each article is collaboratively written, continuously updated and community reviewed. Previously, we described the creation of Gene Wiki 'stubs' for approximately 9000 human genes. Here, we describe ongoing systematic improvements to these articles to increase their utility. Moreover, we retrospectively examine the community usage and improvement of the Gene Wiki, providing evidence of a critical mass of users and editors. Gene Wiki articles are freely accessible within the Wikipedia web site, and additional links and information are available at http://en.wikipedia.org/wiki/Portal:Gene_Wiki. PMID:19755503

  4. Identification of genes and gene products necessary for bacterial bioluminescence.

    PubMed

    Engebrecht, J; Silverman, M

    1984-07-01

    Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation analysis and the minicell programing experiments. These genes, in the order of their position on a linear map, and the apparent molecular weights of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli containing mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light production and luxR and luxI encode regulatory functions. PMID:6377310

  5. Progress in gene targeting and gene therapy for retinitis pigmentosa

    SciTech Connect

    Farrar, G.J.; Humphries, M.M.; Erven, A.

    1994-09-01

    Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectors for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.

  6. Gene Circuit Analysis of the Terminal Gap Gene huckebein

    PubMed Central

    Ashyraliyev, Maksat; Siggens, Ken; Janssens, Hilde; Blom, Joke; Akam, Michael; Jaeger, Johannes

    2009-01-01

    The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network. PMID:19876378

  7. Identifying Gene Interaction Networks

    PubMed Central

    Bebek, Gurkan

    2016-01-01

    In this chapter, we introduce interaction networks by describing how they are generated, where they are stored, and how they are shared. We focus on publicly available interaction networks and describe a simple way of utilizing these resources. As a case study, we used Cytoscape, an open source and easy-to-use network visualization and analysis tool to first gather and visualize a small network. We have analyzed this network’s topological features and have looked at functional enrichment of the network nodes by integrating the gene ontology database. The methods described are applicable to larger networks that can be collected from various resources. PMID:22307715

  8. Computation in gene networks

    NASA Astrophysics Data System (ADS)

    Ben-Hur, Asa; Siegelmann, Hava T.

    2004-03-01

    Genetic regulatory networks have the complex task of controlling all aspects of life. Using a model of gene expression by piecewise linear differential equations we show that this process can be considered as a process of computation. This is demonstrated by showing that this model can simulate memory bounded Turing machines. The simulation is robust with respect to perturbations of the system, an important property for both analog computers and biological systems. Robustness is achieved using a condition that ensures that the model equations, that are generally chaotic, follow a predictable dynamics.

  9. Genes for sexual behavior.

    PubMed

    Yamamoto, D; Nakano, Y

    1998-05-01

    The mating behavior of Drosophila melanogaster is a stereotyped sequence of fixed action patterns, composed of orientation, tapping, singing, licking, attempted copulation and copulation. Mutations that block a unique aspect of mating behavior were isolated and analyzed at the cellular and molecular levels. The wild-type counterparts of the mutated genes were shown to rescue the phenotypes by their ubiquitous or targeted expression in some of the mutants. This strategy of artificial control of fly behavior opens up an avenue for studies to identify the neural center for individual behavioral actions. PMID:9600058

  10. Gene transfer: transduction.

    PubMed

    Frangipani, Emanuela

    2014-01-01

    Bacteriophages able to propagate on Pseudomonas strains are very common and can be easily isolated from natural environments or lysogenic strains. The development of transducing systems has allowed bacterial geneticists to perform chromosome analyses and mutation mapping. Moreover, these systems have also been proved to be a successful tool for molecular microbiologists to introduce a foreign gene or a mutation into the chromosome of a bacterial cell. This chapter provides a description of the phage methodology illustrated by Adams in 1959 and applicable to strain PAO1 derivatives. PMID:24818891

  11. Alternative Gene Form Discovery and Candidate Gene Selection from Gene Indexing Projects

    PubMed Central

    Burke, John; Wang, Hui; Hide, Winston; Davison, Daniel B.

    1998-01-01

    Several efforts are under way to partition single-read expressed sequence tag (EST), as well as full-length transcript data, into large-scale gene indices, where transcripts are in common index classes if and only if they share a common progenitor gene. Accurate gene indexing facilitates gene expression studies, as well as inexpensive and early gene sequence discovery through assembly of ESTs that are derived from genes that have not been sequenced by classical methods. We extend, correct, and enhance the information obtained from index groups by splitting index classes into subclasses based on sequence dissimilarity (diversity). Two applications of this are highlighted in this report. First it is shown that our method can ameliorate the damage that artifacts, such as chimerism, inflict on index integrity. Additionally, we demonstrate how the organization imposed by an effective subpartition can greatly increase the sensitivity of gene expression studies by accounting for the existence and tissue- or pathology-specific regulation of novel gene isoforms and polymorphisms. We apply our subpartitioning treatment to the UniGene gene indexing project to measure a marked increase in information quality and abundance (in terms of assembly length and insertion/deletion error) after treatment and demonstrate cases where new levels of information concerning differential expression of alternate gene forms, such as regulated alternative splicing, are discovered. [Tables 2 and 3 can be viewed in their entirety as Online Supplements at http://www.genome.org.] PMID:9521931

  12. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function.

    PubMed

    Warde-Farley, David; Donaldson, Sylva L; Comes, Ovi; Zuberi, Khalid; Badrawi, Rashad; Chao, Pauline; Franz, Max; Grouios, Chris; Kazi, Farzana; Lopes, Christian Tannus; Maitland, Anson; Mostafavi, Sara; Montojo, Jason; Shao, Quentin; Wright, George; Bader, Gary D; Morris, Quaid

    2010-07-01

    GeneMANIA (http://www.genemania.org) is a flexible, user-friendly web interface for generating hypotheses about gene function, analyzing gene lists and prioritizing genes for functional assays. Given a query list, GeneMANIA extends the list with functionally similar genes that it identifies using available genomics and proteomics data. GeneMANIA also reports weights that indicate the predictive value of each selected data set for the query. Six organisms are currently supported (Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Homo sapiens and Saccharomyces cerevisiae) and hundreds of data sets have been collected from GEO, BioGRID, Pathway Commons and I2D, as well as organism-specific functional genomics data sets. Users can select arbitrary subsets of the data sets associated with an organism to perform their analyses and can upload their own data sets to analyze. The GeneMANIA algorithm performs as well or better than other gene function prediction methods on yeast and mouse benchmarks. The high accuracy of the GeneMANIA prediction algorithm, an intuitive user interface and large database make GeneMANIA a useful tool for any biologist. PMID:20576703

  13. Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

    PubMed Central

    Wold, William S.M.; Toth, Karoly

    2015-01-01

    Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vectors are engineered to replicate preferentially in cancer cells and to destroy cancer cells through the natural process of lytic virus replication. Many clinical trials indicate that replication-defective and replication-competent adenovirus vectors are safe and have therapeutic activity. PMID:24279313

  14. Gene function prediction with knowledge from gene ontology.

    PubMed

    Shen, Ying; Zhang, Lin

    2015-01-01

    Gene function prediction is an important problem in bioinformatics. Due to the inherent noise existing in the gene expression data, the attempt to improve the prediction accuracy resorting to new classification techniques is limited. With the emergence of Gene Ontology (GO), extra knowledge about the gene products can be extracted from GO and facilitates solving the gene function prediction problem. In this paper, we propose a new method which utilises GO information to improve the classifiers' performance in gene function prediction. Specifically, our method learns a distance metric under the supervision of the GO knowledge using the distance learning technique. Compared with the traditional distance metrics, the learned one produces a better performance and consequently classification accuracy can be improved. The effectiveness of our proposed method has been corroborated by the extensive experimental results. PMID:26529907

  15. Human AZU-1 gene, variants thereof and expressed gene products

    DOEpatents

    Chen, Huei-Mei; Bissell, Mina

    2004-06-22

    A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

  16. Endocrine regulation of HOX genes.

    PubMed

    Daftary, Gaurang S; Taylor, Hugh S

    2006-06-01

    Hox genes have a well-characterized role in embryonic development, where they determine identity along the anteroposterior body axis. Hox genes are expressed not only during embryogenesis but also in the adult, where they are necessary for functional differentiation. Despite the known function of these genes as transcription factors, few regulatory mechanisms that drive Hox expression are known. Recently, several hormones and their cognate receptors have been shown to regulate Hox gene expression and thereby mediate development in the embryo as well as functional differentiation in the adult organism. Estradiol, progesterone, testosterone, retinoic acid, and vitamin D have been shown to regulate Hox gene expression. In the embryo, the endocrine system directs axial Hox gene expression; aberrant Hox gene expression due to exposure to endocrine disruptors contributes to the teratogenicity of these compounds. In the adult, endocrine regulation of Hox genes is necessary to enable such diverse functions as hematopoiesis and reproduction; endocrinopathies can result in dysregulated HOX gene expression affecting physiology. By regulating HOX genes, hormonal signals utilize a conserved mechanism that allows generation of structural and functional diversity in both developing and adult tissues. This review discusses endocrine Hox regulation and its impact on physiology and human pathology. PMID:16632680

  17. Imprinting genes associated with endometriosis

    PubMed Central

    Kobayashi, Hiroshi

    2014-01-01

    Purpose: Much work has been carried out to investigate the genetic and epigenetic basis of endometriosis and proposed that endometriosis has been described as an epigenetic disease. The purpose of this study was to extract the imprinting genes that are associated with endometriosis development. Methods: The information on the imprinting genes can be accessed publicly from a web-based interface at http://www.geneimprint.com/site/genes-by-species. Results: In the current version, the database contains 150 human imprinted genes derived from the literature. We searched gene functions and their roles in particular biological processes or events, such as development and pathogenesis of endometriosis. From the genomic imprinting database, we picked 10 genes that were highly associated with female reproduction; prominent among them were paternally expressed genes (DIRAS3, BMP8B, CYP1B1, ZFAT, IGF2, MIMT1, or MIR296) and maternally expressed genes (DVL1, FGFRL1, or CDKN1C). These imprinted genes may be associated with reproductive biology such as endometriosis, pregnancy loss, decidualization process and preeclampsia. Discussion: This study supports the possibility that aberrant epigenetic dysregulation of specific imprinting genes may contribute to endometriosis predisposition. PMID:26417259

  18. Vectors for cancer gene therapy.

    PubMed

    Zhang, J; Russell, S J

    1996-09-01

    Many viral and non-viral vector systems have now been developed for gene therapy applications. In this article, the pros and cons of these vector systems are discussed in relation to the different cancer gene therapy strategies. The protocols used in cancer gene therapy can be broadly divided into six categories including gene transfer to explanted cells for use as cell-based cancer vaccines; gene transfer to a small number of tumour cells in situ to achieve a vaccine effect; gene transfer to vascular endothelial cells (VECs) lining the blood vessels of the tumour to interfere with tumour angiogenesis; gene transfer to T lymphocytes to enhance their antitumour effector capability; gene transfer to haemopoietic stem cells (HSCs) to enhance their resistance to cytotoxic drugs and gene transfer to a large number of tumour cells in situ to achieve nonimmune tumour reduction with or without bystander effect. Each of the six strategies makes unique demands on the vector system and these are discussed with reference to currently available vectors. Aspects of vector biology that are in need of further development are discussed in some detail. The final section points to the potential use of replicating viruses as delivery vehicles for efficient in vivo gene transfer to disseminated cancers. PMID:9034598

  19. Aberrant Gene Expression in Humans

    PubMed Central

    Yang, Ence; Ji, Guoli; Brinkmeyer-Langford, Candice L.; Cai, James J.

    2015-01-01

    Gene expression as an intermediate molecular phenotype has been a focus of research interest. In particular, studies of expression quantitative trait loci (eQTL) have offered promise for understanding gene regulation through the discovery of genetic variants that explain variation in gene expression levels. Existing eQTL methods are designed for assessing the effects of common variants, but not rare variants. Here, we address the problem by establishing a novel analytical framework for evaluating the effects of rare or private variants on gene expression. Our method starts from the identification of outlier individuals that show markedly different gene expression from the majority of a population, and then reveals the contributions of private SNPs to the aberrant gene expression in these outliers. Using population-scale mRNA sequencing data, we identify outlier individuals using a multivariate approach. We find that outlier individuals are more readily detected with respect to gene sets that include genes involved in cellular regulation and signal transduction, and less likely to be detected with respect to the gene sets with genes involved in metabolic pathways and other fundamental molecular functions. Analysis of polymorphic data suggests that private SNPs of outlier individuals are enriched in the enhancer and promoter regions of corresponding aberrantly-expressed genes, suggesting a specific regulatory role of private SNPs, while the commonly-occurring regulatory genetic variants (i.e., eQTL SNPs) show little evidence of involvement. Additional data suggest that non-genetic factors may also underlie aberrant gene expression. Taken together, our findings advance a novel viewpoint relevant to situations wherein common eQTLs fail to predict gene expression when heritable, rare inter-individual variation exists. The analytical framework we describe, taking into consideration the reality of differential phenotypic robustness, may be valuable for investigating

  20. The Zebrafish Annexin Gene Family

    PubMed Central

    Farber, Steven A.; De Rose, Robert A.; Olson, Eric S.; Halpern, Marnie E.

    2003-01-01

    The Annexins (ANXs) are a family of calcium- and phospholipid-binding proteins that have been implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport, and regulation of phospholipase A2 activity. As a first step toward understanding in vivo function, we have cloned 11 zebrafish anx genes. Four genes (anx1a, anx2a, anx5,and anx11a) were identified by screening a zebrafish cDNA library with a Xenopus anx2 fragment. For these genes, full-length cDNA sequences were used to cluster 212 EST sequences generated by the Zebrafish Genome Resources Project. The EST analysis revealed seven additional anx genes that were subsequently cloned. The genetic map positions of all 11 genes were determined by using a zebrafish radiation hybrid panel. Sequence and syntenic relationships between zebrafish and human genes indicate that the 11 genes represent orthologs of human anx1,2,4,5,6,11,13,and suggest that several zebrafish anx genes resulted from duplications that arose after divergence of the zebrafish and mammalian genomes. Zebrafish anx genes are expressed in a wide range of tissues during embryonic and larval stages. Analysis of the expression patterns of duplicated genes revealed both redundancy and divergence, with the most similar genes having almost identical tissue-specific patterns of expression and with less similar duplicates showing no overlap. The differences in gene expression of recently duplicated anx genes could explain why highly related paralogs were maintained in the genome and did not rapidly become pseudogenes. PMID:12799347

  1. Gene-gene interaction between tuberculosis candidate genes in a South African population.

    PubMed

    de Wit, Erika; van der Merwe, Lize; van Helden, Paul D; Hoal, Eileen G

    2011-02-01

    In a complex disease such as tuberculosis (TB) it is increasingly evident that gene-gene interactions play a far more important role in an individual's susceptibility to develop the disease than single polymorphisms on their own, as one gene can enhance or hinder the expression of another gene. Gene-gene interaction analysis is a new approach to elucidate susceptibility to TB. The possibility of gene-gene interactions was assessed, focusing on 11 polymorphisms in nine genes (DC-SIGN, IFN-γ, IFNGR1, IL-8, IL-1Ra, MBL, NRAMP1, RANTES, and SP-D) that have been associated with TB, some repeatedly. An optimal model, which best describes and predicts TB case-control status, was constructed. Significant interactions were detected between eight pairs of variants. The models fitted the observed data extremely well, with p < 0.0001 for all eight models. A highly significant interaction was detected between INFGR1 and NRAMP1, which is not surprising because macrophage activation is greatly enhanced by IFN-γ and IFN-γ response elements that are present in the human NRAMP1 promoter region, providing further evidence for their interaction. This study enabled us to test the theory that disease outcome may be due to interaction of several gene effects. With eight instances of statistically significant gene-gene interactions, the importance of epistasis is clearly identifiable in this study. Methods for studying gene-gene interactions are based on a multilocus and multigene approach, consistent with the nature of complex-trait diseases, and may provide the paradigm for future genetic studies of TB. PMID:20799037

  2. Ancient origins of axial patterning genes: Hox genes and ParaHox genes in the Cnidaria.

    PubMed

    Finnerty, J R; Martindale, M Q

    1999-01-01

    Among the bilaterally symmetrical, triploblastic animals (the Bilateria), a conserved set of developmental regulatory genes are known to function in patterning the anterior-posterior (AP) axis. This set includes the well-studied Hox cluster genes, and the recently described genes of the ParaHox cluster, which is believed to be the evolutionary sister of the Hox cluster (Brooke et al. 1998). The conserved role of these axial patterning genes in animals as diverse as frogs and flies is believed to reflect an underlying homology (i.e., all bilaterians derive from a common ancestor which possessed an AP axis and the developmental mechanisms responsible for patterning the axis). However, the origin and early evolution of Hox genes and ParaHox genes remain obscure. Repeated attempts have been made to reconstruct the early evolution of Hox genes by analyzing data from the triphoblastic animals, the Bilateria (Schubert et al. 1993; Zhang and Nei 1996). A more precise dating of Hox origins has been elusive due to a lack of sufficient information from outgroup taxa such as the phylum Cnidaria (corals, hydras, jellyfishes, and sea anemones). In combination with outgroup taxa, another potential source of information about Hox origins is outgroup genes (e.g., the genes of the ParaHox cluster). In this article, we present cDNA sequences of two Hox-like genes (anthox2 and anthox6) from the sea anemone, Nematostella vectensis. Phylogenetic analysis indicates that anthox2 (= Cnox2) is homologous to the GSX class of ParaHox genes, and anthox6 is homologous to the anterior class of Hox genes. Therefore, the origin of Hox genes and ParaHox genes occurred prior to the evolutionary split between the Cnidaria and the Bilateria and predated the evolution of the anterior-posterior axis of bilaterian animals. Our analysis also suggests that the central Hox class was invented in the bilaterian lineage, subsequent to their split from the Cnidaria. PMID:11324016

  3. Identifying Driver Genes in Cancer by Triangulating Gene Expression, Gene Location, and Survival Data

    PubMed Central

    Rouam, Sigrid; Miller, Lance D; Karuturi, R Krishna Murthy

    2014-01-01

    Driver genes are directly responsible for oncogenesis and identifying them is essential in order to fully understand the mechanisms of cancer. However, it is difficult to delineate them from the larger pool of genes that are deregulated in cancer (ie, passenger genes). In order to address this problem, we developed an approach called TRIAngulating Gene Expression (TRIAGE through clinico-genomic intersects). Here, we present a refinement of this approach incorporating a new scoring methodology to identify putative driver genes that are deregulated in cancer. TRIAGE triangulates – or integrates – three levels of information: gene expression, gene location, and patient survival. First, TRIAGE identifies regions of deregulated expression (ie, expression footprints) by deriving a newly established measure called the Local Singular Value Decomposition (LSVD) score for each locus. Driver genes are then distinguished from passenger genes using dual survival analyses. Incorporating measurements of gene expression and weighting them according to the LSVD weight of each tumor, these analyses are performed using the genes located in significant expression footprints. Here, we first use simulated data to characterize the newly established LSVD score. We then present the results of our application of this refined version of TRIAGE to gene expression data from five cancer types. This refined version of TRIAGE not only allowed us to identify known prominent driver genes, such as MMP1, IL8, and COL1A2, but it also led us to identify several novel ones. These results illustrate that TRIAGE complements existing tools, allows for the identification of genes that drive cancer and could perhaps elucidate potential future targets of novel anticancer therapeutics. PMID:25949096

  4. Introns in gene evolution.

    PubMed

    Fedorova, Larisa; Fedorov, Alexei

    2003-07-01

    Introns are integral elements of eukaryotic genomes that perform various important functions and actively participate in gene evolution. We review six distinct roles of spliceosomal introns: (1) sources of non-coding RNA; (2) carriers of transcription regulatory elements; (3) actors in alternative and trans-splicing; (4) enhancers of meiotic crossing over within coding sequences; (5) substrates for exon shuffling; and (6) signals for mRNA export from the nucleus and nonsense-mediated decay. We consider transposable capacities of introns and the current state of the long-lasting debate on the 'early-or-late' origin of introns. Cumulative data on known types of contemporary exon shuffling and the estimation of the size of the underlying exon universe are also discussed. We argue that the processes central to introns-early (exon shuffling) and introns-late (intron insertion) theories are entirely compatible. Each has provided insight: the latter through elucidating the transposon capabilities of introns, and the former through understanding the importance of introns in genomic recombination leading to gene rearrangements and evolution. PMID:12868603

  5. Conotoxin Gene Superfamilies

    PubMed Central

    Robinson, Samuel D.; Norton, Raymond S.

    2014-01-01

    Conotoxins are the peptidic components of the venoms of marine cone snails (genus Conus). They are remarkably diverse in terms of structure and function. Unique potency and selectivity profiles for a range of neuronal targets have made several conotoxins valuable as research tools, drug leads and even therapeutics, and has resulted in a concerted and increasing drive to identify and characterise new conotoxins. Conotoxins are translated from mRNA as peptide precursors, and cDNA sequencing is now the primary method for identification of new conotoxin sequences. As a result, gene superfamily, a classification based on precursor signal peptide identity, has become the most convenient method of conotoxin classification. Here we review each of the described conotoxin gene superfamilies, with a focus on the structural and functional diversity present in each. This review is intended to serve as a practical guide to conotoxin superfamilies and to facilitate interpretation of the increasing number of conotoxin precursor sequences being identified by targeted-cDNA sequencing and more recently high-throughput transcriptome sequencing. PMID:25522317

  6. GeneMark.hmm: new solutions for gene finding.

    PubMed

    Lukashin, A V; Borodovsky, M

    1998-02-15

    The number of completely sequenced bacterial genomes has been growing fast. There are computer methods available for finding genes but yet there is a need for more accurate algorithms. The GeneMark. hmm algorithm presented here was designed to improve the gene prediction quality in terms of finding exact gene boundaries. The idea was to embed the GeneMark models into naturally derived hidden Markov model framework with gene boundaries modeled as transitions between hidden states. We also used the specially derived ribosome binding site pattern to refine predictions of translation initiation codons. The algorithm was evaluated on several test sets including 10 complete bacterial genomes. It was shown that the new algorithm is significantly more accurate than GeneMark in exact gene prediction. Interestingly, the high gene finding accuracy was observed even in the case when Markov models of order zero, one and two were used. We present the analysis of false positive and false negative predictions with the caution that these categories are not precisely defined if the public database annotation is used as a control. PMID:9461475

  7. Gene: a gene-centered information resource at NCBI

    PubMed Central

    Brown, Garth R.; Hem, Vichet; Katz, Kenneth S.; Ovetsky, Michael; Wallin, Craig; Ermolaeva, Olga; Tolstoy, Igor; Tatusova, Tatiana; Pruitt, Kim D.; Maglott, Donna R.; Murphy, Terence D.

    2015-01-01

    The National Center for Biotechnology Information's (NCBI) Gene database (www.ncbi.nlm.nih.gov/gene) integrates gene-specific information from multiple data sources. NCBI Reference Sequence (RefSeq) genomes for viruses, prokaryotes and eukaryotes are the primary foundation for Gene records in that they form the critical association between sequence and a tracked gene upon which additional functional and descriptive content is anchored. Additional content is integrated based on the genomic location and RefSeq transcript and protein sequence data. The content of a Gene record represents the integration of curation and automated processing from RefSeq, collaborating model organism databases, consortia such as Gene Ontology, and other databases within NCBI. Records in Gene are assigned unique, tracked integers as identifiers. The content (citations, nomenclature, genomic location, gene products and their attributes, phenotypes, sequences, interactions, variation details, maps, expression, homologs, protein domains and external databases) is available via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programming utilities (E-Utilities and Entrez Direct) and for bulk transfer by FTP. PMID:25355515

  8. Bacteriophage phiX174: gene A overlaps gene B.

    PubMed Central

    Weisbeek, P J; Borrias, W E; Langeveld, S A; Baas, P D; Van Arkel, G A

    1977-01-01

    The map position of several phiX174 mutations in the genes A and B was determined by marker rescue with DNA fragments produced by the restriction enzymes Hha I, HindII, Hae III, and Alu I. All the gene B mutants were found to be located within gene A. Genetic complementation and analysis of phage-specific protein synthesis show that, under restrictive conditions, nonsense mutants in gene A do not block the synthesis and activity of the B protein and nonsense mutants in gene B do not affect the gene A function. The map position of the COOH-terminal end of gene A was determined using an amber mutant that synthesizes slightly shortened A and A proteins. It is concluded from these experiments that gene A overlaps gene B completely (or almost completely) and that the overlap region can be translated in two ways with different reading frames: one frame for the synthesis of the A and A proteins and another for the synthesis of the B protein. Images PMID:267943

  9. Immunity-related genes and gene families in Anopheles gambiae.

    PubMed

    Christophides, George K; Zdobnov, Evgeny; Barillas-Mury, Carolina; Birney, Ewan; Blandin, Stephanie; Blass, Claudia; Brey, Paul T; Collins, Frank H; Danielli, Alberto; Dimopoulos, George; Hetru, Charles; Hoa, Ngo T; Hoffmann, Jules A; Kanzok, Stefan M; Letunic, Ivica; Levashina, Elena A; Loukeris, Thanasis G; Lycett, Gareth; Meister, Stephan; Michel, Kristin; Moita, Luis F; Müller, Hans-Michael; Osta, Mike A; Paskewitz, Susan M; Reichhart, Jean-Marc; Rzhetsky, Andrey; Troxler, Laurent; Vernick, Kenneth D; Vlachou, Dina; Volz, Jennifer; von Mering, Christian; Xu, Jiannong; Zheng, Liangbiao; Bork, Peer; Kafatos, Fotis C

    2002-10-01

    We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire. PMID:12364793

  10. Gene-targeting pharmaceuticals for single-gene disorders.

    PubMed

    Beaudet, Arthur L; Meng, Linyan

    2016-04-15

    The concept of orphan drugs for treatment of orphan genetic diseases is perceived enthusiastically at present, and this is leading to research investment on the part of governments, disease-specific foundations and industry. This review attempts to survey the potential to use traditional pharmaceuticals as opposed to biopharmaceuticals to treat single-gene disorders. The available strategies include the use of antisense oligonucleotides (ASOs) to alter splicing or knock-down expression of a transcript, siRNAs to knock-down gene expression and drugs for nonsense mutation read-through. There is an approved drug for biallelic knock-down of the APOB gene as treatment for familial hypercholesterolemia. Both ASOs and siRNAs are being explored to knock-down the transthyretin gene to prevent the related form of amyloidosis. The use of ASOs to alter gene-splicing to treat spinal muscular atrophy is in phase 3 clinical trials. Work is progressing on the use of ASOs to activate the normally silent paternal copy of the imprinted UBE3A gene in neurons as a treatment for Angelman syndrome. A gene-activation or gene-specific ramp-up strategy would be generally helpful if such could be developed. There is exciting theoretical potential for converting biopharmaceutical strategies such gene correction and CRISPR-Cas9 editing to a synthetic pharmaceutical approach. PMID:26628634

  11. Sexually antagonistic genes: experimental evidence.

    PubMed

    Rice, W R

    1992-06-01

    When selection differs between the sexes, a mutation beneficial to one sex may be harmful to the other (sexually antagonistic). Because the sexes share a common gene pool, selection in one sex can interfere with the other's adaptive evolution. Theory predicts that sexually antagonistic mutations should accumulate in tight linkage with a new sex-determining gene, even when the harm to benefit ratio is high. Genetic markers and artificial selection were used to make a pair of autosomal genes segregate like a new pair of sex-determining genes in a Drosophila melanogaster model system. A 29-generation study provides experimental evidence that sexually antagonistic genes may be common in nature and will accumulate in response to a new sex-determining gene. PMID:1604317

  12. Gene Therapy for Cartilage Repair

    PubMed Central

    Madry, Henning; Orth, Patrick; Cucchiarini, Magali

    2011-01-01

    The concept of using gene transfer strategies for cartilage repair originates from the idea of transferring genes encoding therapeutic factors into the repair tissue, resulting in a temporarily and spatially defined delivery of therapeutic molecules to sites of cartilage damage. This review focuses on the potential benefits of using gene therapy approaches for the repair of articular cartilage and meniscal fibrocartilage, including articular cartilage defects resulting from acute trauma, osteochondritis dissecans, osteonecrosis, and osteoarthritis. Possible applications for meniscal repair comprise meniscal lesions, meniscal sutures, and meniscal transplantation. Recent studies in both small and large animal models have demonstrated the applicability of gene-based approaches for cartilage repair. Chondrogenic pathways were stimulated in the repair tissue and in osteoarthritic cartilage using genes for polypeptide growth factors and transcription factors. Although encouraging data have been generated, a successful translation of gene therapy for cartilage repair will require an ongoing combined effort of orthopedic surgeons and of basic scientists. PMID:26069580

  13. Gene targeting with retroviral vectors

    SciTech Connect

    Ellis, J.; Bernstein, A. )

    1989-04-01

    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

  14. The Perils of Gene Patents

    PubMed Central

    Salzberg, SL

    2013-01-01

    I argue here that gene patents, and patented genetic tests based on them, are a very bad idea. First, I discuss whether genes can reasonably be the subject of patents in the first place; I maintain that the answer is no. Second, I explain how gene patents interfere with scientific progress, slowing down the development of new cures and treatments for genetic diseases. PMID:22609909

  15. Combinatorial approaches to gene recognition.

    PubMed

    Roytberg, M A; Astakhova, T V; Gelfand, M S

    1997-01-01

    Recognition of genes via exon assembly approaches leads naturally to the use of dynamic programming. We consider the general graph-theoretical formulation of the exon assembly problem and analyze in detail some specific variants: multicriterial optimization in the case of non-linear gene-scoring functions; context-dependent schemes for scoring exons and related procedures for exon filtering; and highly specific recognition of arbitrary gene segments, oligonucleotide probes and polymerase chain reaction (PCR) primers. PMID:9440930

  16. Symmetry and Stochastic Gene Regulation

    NASA Astrophysics Data System (ADS)

    Ramos, Alexandre F.; Hornos, José E. M.

    2007-09-01

    Lorentz-like noncompact Lie symmetry SO(2,1) is found in a spin-boson stochastic model for gene expression. The invariant of the algebra characterizes the switch decay to equilibrium. The azimuthal eigenvalue describes the affinity between the regulatory protein and the gene operator site. Raising and lowering operators are constructed and their actions increase or decrease the affinity parameter. The classification of the noise regime of the gene arises from the group theoretical numbers.

  17. Serial analysis of gene expression.

    PubMed

    Velculescu, V E; Zhang, L; Vogelstein, B; Kinzler, K W

    1995-10-20

    The characteristics of an organism are determined by the genes expressed within it. A method was developed, called serial analysis of gene expression (SAGE), that allows the quantitative and simultaneous analysis of a large number of transcripts. To demonstrate this strategy, short diagnostic sequence tags were isolated from pancreas, concatenated, and cloned. Manual sequencing of 1000 tags revealed a gene expression pattern characteristic of pancreatic function. New pancreatic transcripts corresponding to novel tags were identified. SAGE should provide a broadly applicable means for the quantitative cataloging and comparison of expressed genes in a variety of normal, developmental, and disease states. PMID:7570003

  18. Gene therapy for Parkinson's disease.

    PubMed

    Lawlor, Patricia A; During, Matthew J

    2004-03-01

    Parkinson's disease (PD) is a debilitating neurodegenerative disorder arising from loss of dopaminergic neurons in the substantia nigra pars compacta and subsequent depletion of striatal dopamine levels, which results in distressing motor symptoms. The current standard pharmacological treatment for PD is direct replacement of dopamine by treatment with its precursor, levodopa (L-dopa). However, this does not significantly alter disease progression and might contribute to the ongoing pathology. Several features of PD make this disease one of the most promising targets for clinical gene therapy of any neurological disease. The confinement of the major pathology to a compact, localised neuronal population and the anatomy of the basal ganglia circuitry mean that global gene transfer is not required and there are well-defined sites for gene transfer. The multifactorial aetiology of idiopathic PD means that it is unlikely any single gene will cure the disease, and as a result at least three separate gene-transfer strategies are currently being pursued: transfer of genes for enzymes involved in dopamine production; transfer of genes for growth factors involved in dopaminergic cell survival and regeneration; and transfer of genes to reset neuronal circuitry by switching cellular phenotype. The merits of these strategies are discussed here, along with remaining hurdles that might impede transfer of gene therapy technology to the clinic as a treatment for PD. PMID:15000692

  19. ERGDB: Estrogen Responsive Genes Database.

    PubMed

    Tang, Suisheng; Han, Hao; Bajic, Vladimir B

    2004-01-01

    ERGDB is an integrated knowledge database dedicated to genes responsive to estrogen. Genes included in ERGDB are those whose expression levels are experimentally proven to be either up-regulated or down-regulated by estrogen. Genes included are identified based on publications from the PubMed database and each record has been manually examined, evaluated and selected for inclusion by biologists. ERGDB aims to be a unified gateway to store, search, retrieve and update information about estrogen responsive genes. Each record contains links to relevant databases, such as GenBank, LocusLink, Refseq, PubMed and ATCC. The unique feature of ERGDB is that it contains information on the dependence of gene reactions on experimental conditions. In addition to basic information about the genes, information for each record includes gene functional description, experimental methods used, tissue or cell type, gene reaction, estrogen exposure time and the summary of putative estrogen response elements if the gene's promoter sequence was available. Through a web interface at http://sdmc.i2r.a-star.edu.sg/ergdb/ cgi-bin/explore.pl users can either browse or query ERGDB. Access is free for academic and non-profit users. PMID:14681475

  20. Cardiac Gene Therapy

    PubMed Central

    Chaanine, Antoine H.; Kalman, Jill; Hajjar, Roger J.

    2010-01-01

    Heart failure is a chronic progressive disorder where frequent and recurrent hospitalizations are associated with high mortality and morbidity. The incidence and the prevalence of this disease will increase with the increase in the number of the aging population of the United States. Understanding the molecular pathology and pathophysiology of this disease will uncover novel targets and therapies that can restore the function or attenuate the damage of malfunctioning cardiomyocytes by gene therapy that becomes an interesting and a promising field for the treatment of heart failure as well as other diseases in the future. Of equal importance is developing vectors and delivery methods that can efficiently transduce the majority of the cardiomyocytes, that can offer a long term expression and that can escape the host immune response. Recombinant adeno-associated virus vectors have the potential to become a promising novel therapeutic vehicles for molecular medicine in the future. PMID:21092890

  1. Taste Receptor Genes

    PubMed Central

    Bachmanov, Alexander A.; Beauchamp, Gary K.

    2009-01-01

    In the past several years, tremendous progress has been achieved with the discovery and characterization of vertebrate taste receptors from the T1R and T2R families, which are involved in recognition of bitter, sweet, and umami taste stimuli. Individual differences in taste, at least in some cases, can be attributed to allelic variants of the T1R and T2R genes. Progress with understanding how T1R and T2R receptors interact with taste stimuli and with identifying their patterns of expression in taste cells sheds light on coding of taste information by the nervous system. Candidate mechanisms for detection of salts, acids, fat, complex carbohydrates, and water have also been proposed, but further studies are needed to prove their identity. PMID:17444812

  2. New genes for boys

    SciTech Connect

    Sinclair, A.H.

    1995-11-01

    Sex is a fascinating topic, particularly at the level of molecular genetics, since it represents a wonderful paradigm for mammalian organ development. Recently, interest in the molecular basis for mammalian sex determination has been heating up as new pieces are added to the jigsaw puzzle of testis development. In mammals, the Y chromosome is male determining and encodes a gene referred to as TDF (testis-determining factor), which induces the indifferent embryonic gonad to develop as a testis. Subsequent male sexual differentiation is largely a consequence of hormonal secretion from the testis. In the absence of the Y chromosome, the testis-determining pathway fails to be initiated, and the embryonic gonad develops as an ovary, resulting in female development. 32 refs.

  3. Gene Express Inc.

    PubMed

    Saccomanno, Colette F

    2006-07-01

    Gene Express, Inc. is a technology-licensing company and provider of Standardized Reverse Transcription Polymerase Chain Reaction (StaRT-PCR) services. Designed by and for clinical researchers involved in pharmaceutical, biomarker and molecular diagnostic product development, StaRT-PCR is a unique quantitative and standardized multigene expression measurement platform. StaRT-PCR meets all of the performance characteristics defined by the US FDA as required to support regulatory submissions [101,102] , and by the Clinical Laboratory Improvement Act of 1988 (CLIA) as necessary to support diagnostic testing [1] . A standardized mixture of internal standards (SMIS), manufactured in bulk, provides integrated quality control wherein each native template target gene is measured relative to a competitive template internal standard. Bulk production enables the compilation of a comprehensive standardized database from across multiple experiments, across collaborating laboratories and across the entire clinical development lifecycle of a given compound or diagnostic product. For the first time, all these data are able to be directly compared. Access to such a database can dramatically shorten the time from investigational new drug (IND) to new drug application (NDA), or save time and money by hastening a substantiated 'no-go' decision. High-throughput StaRT-PCR is conducted at the company's automated Standardized Expression Measurement (SEM) Center. Currently optimized for detection on a microcapillary electrophoretic platform, StaRT-PCR products also may be analyzed on microarray, high-performance liquid chromatography (HPLC), or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) platforms. SEM Center services deliver standardized genomic data--data that will accelerate the application of pharmacogenomic technology to new drug and diagnostic test development and facilitate personalized medicine. PMID:16886903

  4. DIFFERENTIAL GENE EXPRESSION OF PUTATIVE VIRULENCE GENES IN Flavobacterium columnare

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A shot-gun genomic library of the Flavobacterium columnare ALG-530 virulent strain has been constructed and more than 3,000 clones have been sequenced to date (800 contigs). Based on sequence identity with putative known virulence genes from related species, seven genes were selected for differentia...

  5. Candidate reference genes for gene expression studies in water lily.

    PubMed

    Luo, Huolin; Chen, Sumei; Wan, Hongjian; Chen, Fadi; Gu, Chunsun; Liu, Zhaolei

    2010-09-01

    The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1alpha) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11. PMID:20452325

  6. Determining Semantically Related Significant Genes.

    PubMed

    Taha, Kamal

    2014-01-01

    GO relation embodies some aspects of existence dependency. If GO term xis existence-dependent on GO term y, the presence of y implies the presence of x. Therefore, the genes annotated with the function of the GO term y are usually functionally and semantically related to the genes annotated with the function of the GO term x. A large number of gene set enrichment analysis methods have been developed in recent years for analyzing gene sets enrichment. However, most of these methods overlook the structural dependencies between GO terms in GO graph by not considering the concept of existence dependency. We propose in this paper a biological search engine called RSGSearch that identifies enriched sets of genes annotated with different functions using the concept of existence dependency. We observe that GO term xcannot be existence-dependent on GO term y, if x- and y- have the same specificity (biological characteristics). After encoding into a numeric format the contributions of GO terms annotating target genes to the semantics of their lowest common ancestors (LCAs), RSGSearch uses microarray experiment to identify the most significant LCA that annotates the result genes. We evaluated RSGSearch experimentally and compared it with five gene set enrichment systems. Results showed marked improvement. PMID:26357049

  7. Candidate gene prioritization with Endeavour.

    PubMed

    Tranchevent, Léon-Charles; Ardeshirdavani, Amin; ElShal, Sarah; Alcaide, Daniel; Aerts, Jan; Auboeuf, Didier; Moreau, Yves

    2016-07-01

    Genomic studies and high-throughput experiments often produce large lists of candidate genes among which only a small fraction are truly relevant to the disease, phenotype or biological process of interest. Gene prioritization tackles this problem by ranking candidate genes by profiling candidates across multiple genomic data sources and integrating this heterogeneous information into a global ranking. We describe an extended version of our gene prioritization method, Endeavour, now available for six species and integrating 75 data sources. The performance (Area Under the Curve) of Endeavour on cross-validation benchmarks using 'gold standard' gene sets varies from 88% (for human phenotypes) to 95% (for worm gene function). In addition, we have also validated our approach using a time-stamped benchmark derived from the Human Phenotype Ontology, which provides a setting close to prospective validation. With this benchmark, using 3854 novel gene-phenotype associations, we observe a performance of 82%. Altogether, our results indicate that this extended version of Endeavour efficiently prioritizes candidate genes. The Endeavour web server is freely available at https://endeavour.esat.kuleuven.be/. PMID:27131783

  8. Using Genes to Guide Prescriptions

    MedlinePlus

    ... Science > Using Genes to Guide Prescriptions Inside Life Science View All Articles | Inside Life Science Home Page Using Genes to Guide Prescriptions By ... to Zoloft: Ways Medicines Work This Inside Life Science article also appears on LiveScience . Learn about related ...

  9. Gene therapy on the move

    PubMed Central

    Kaufmann, Kerstin B; Büning, Hildegard; Galy, Anne; Schambach, Axel; Grez, Manuel

    2013-01-01

    The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders. PMID:24106209

  10. Multifunctional nanorods for gene delivery

    NASA Astrophysics Data System (ADS)

    Salem, Aliasger K.; Searson, Peter C.; Leong, Kam W.

    2003-10-01

    The goal of gene therapy is to introduce foreign genes into somatic cells to supplement defective genes or provide additional biological functions, and can be achieved using either viral or synthetic non-viral delivery systems. Compared with viral vectors, synthetic gene-delivery systems, such as liposomes and polymers, offer several advantages including ease of production and reduced risk of cytotoxicity and immunogenicity, but their use has been limited by the relatively low transfection efficiency. This problem mainly stems from the difficulty in controlling their properties at the nanoscale. Synthetic inorganic gene carriers have received limited attention in the gene-therapy community, the only notable example being gold nanoparticles with surface-immobilized DNA applied to intradermal genetic immunization by particle bombardment. Here we present a non-viral gene-delivery system based on multisegment bimetallic nanorods that can simultaneously bind compacted DNA plasmids and targeting ligands in a spatially defined manner. This approach allows precise control of composition, size and multifunctionality of the gene-delivery system. Transfection experiments performed in vitro and in vivo provide promising results that suggest potential in genetic vaccination applications.

  11. Gene Expression in Oligodendroglial Tumors

    PubMed Central

    Shaw, Elisabeth J.; Haylock, Brian; Husband, David; du Plessis, Daniel; Sibson, D. Ross; Warnke, Peter C.; Walker, Carol

    2010-01-01

    Background: Oligodendroglial tumors with 1p/19q loss are more likely to be chemosensitive and have longer survival than those with intact 1p/19q, but not all respond to chemotherapy, warranting investigation of the biological basis of chemosensitivity. Methods: Gene expression profiling was performed using amplified antisense RNA from 28 oligodendroglial tumors treated with chemotherapy (26 serial stereotactic biopsy, 2 resection). Expression of differentially expressed genes was validated by real-time PCR. Results: Unsupervised hierarchical clustering showed clustering of multiple samples from the same case in 14/17 cases and identified subgroups associated with tumor grade and 1p/19q status. 176 genes were differentially expressed, 164 being associated with 1p/19q loss (86% not on 1p or 19q). 94 genes differed between responders and non-responders to chemotherapy; 12 were not associated with 1p/19q loss. Significant differential expression was confirmed in 11/13 selected genes. Novel genes associated with response to therapy included SSBP2, GFRA1, FAP and RASD1. IQGAP1, INA, TGIF1, NR2F2 and MYCBP were differentially expressed in oligodendroglial tumors with 1p/19q loss. Conclusion: Gene expression profiling using serial stereotactic biopsies indicated greater homogeneity within tumors than between tumors. Genes associated with 1p/19q status or response were identified warranting further elucidation of their role in oligodendroglial tumors. PMID:20966545

  12. From genes to genome biology

    SciTech Connect

    Pennisi, E.

    1996-06-21

    This article describes a change in the approach to mapping genomes, from looking at one gene at a time, to other approaches. Strategies include everything from lab techniques to computer programs designed to analyze whole batches of genes at once. Also included is a update on the work on the human genome.

  13. Method of controlling gene expression

    DOEpatents

    Peters, Norman K.; Frost, John W.; Long, Sharon R.

    1991-12-03

    A method of controlling expression of a DNA segment under the control of a nod gene promoter which comprises administering to a host containing a nod gene promoter an amount sufficient to control expression of the DNA segment of a compound of the formula: ##STR1## in which each R is independently H or OH, is described.

  14. Susceptibility Genes in Thyroid Autoimmunity

    PubMed Central

    Ban, Yoshiyuki; Tomer, Yaron

    2005-01-01

    The autoimmune thyroid diseases (AITD) are complex diseases which are caused by an interaction between susceptibility genes and environmental triggers. Genetic susceptibility in combination with external factors (e.g. dietary iodine) is believed to initiate the autoimmune response to thyroid antigens. Abundant epidemiological data, including family and twin studies, point to a strong genetic influence on the development of AITD. Various techniques have been employed to identify the genes contributing to the etiology of AITD, including candidate gene analysis and whole genome screening. These studies have enabled the identification of several loci (genetic regions) that are linked with AITD, and in some of these loci, putative AITD susceptibility genes have been identified. Some of these genes/loci are unique to Graves' disease (GD) and Hashimoto's thyroiditis (HT) and some are common to both the diseases, indicating that there is a shared genetic susceptibility to GD and HT. The putative GD and HT susceptibility genes include both immune modifying genes (e.g. HLA, CTLA-4) and thyroid specific genes (e.g. TSHR, Tg). Most likely, these loci interact and their interactions may influence disease phenotype and severity. PMID:15712599

  15. Uncovering trends in gene naming

    PubMed Central

    Seringhaus, Michael R; Cayting, Philip D; Gerstein, Mark B

    2008-01-01

    We take stock of current genetic nomenclature and attempt to organize strange and notable gene names. We categorize, for instance, those that involve a naming system transferred from another context (for example, Pavlov’s dogs). We hope this analysis provides clues to better steer gene naming in the future. PMID:18254929

  16. Nonviral Vectors for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  17. Gene Therapy for Retinal Diseases

    PubMed Central

    Samiy, Nasrollah

    2014-01-01

    Gene therapy has a growing research potential particularly in the field of ophthalmic and retinal diseases owing to three main characteristics of the eye; accessibility in terms of injections and surgical interventions, its immune-privileged status facilitating the accommodation to the antigenicity of a viral vector, and tight blood-ocular barriers which save other organs from unwanted contamination. Gene therapy has tremendous potential for different ocular diseases. In fact, the perspective of gene therapy in the field of eye research does not confine to exclusive monogenic ophthalmic problems and it has the potential to include gene based pharmacotherapies for non-monogenic problems such as age related macular disease and diabetic retinopathy. The present article has focused on how gene transfer into the eye has been developed and used to treat retinal disorders with no available therapy at present. PMID:25709778

  18. Fast parsers for Entrez Gene.

    PubMed

    Liu, Mingyi; Grigoriev, Andrei

    2005-07-15

    NCBI completed the transition of its main genome annotation database from Locuslink to Entrez Gene in Spring 2005. However, to this date few parsers exist for the Entrez Gene annotation file. Owing to the widespread use of Locuslink and the popularity of Perl programming language in bioinformatics, a publicly available high performance Entrez Gene parser in Perl is urgently needed. We present four such parsers that were developed using several parsing approaches (Parse::RecDescent, Parse::Yapp, Perl-byacc and Perl 5 regular expressions) and provide the first in-depth comparison of these sophisticated Perl tools. Our fastest parser processes the entire human Entrez Gene annotation file in under 12 min on one Intel Xeon 2.4 GHz CPU and can be of help to the bioinformatics community during and after the transition from Locuslink to Entrez Gene. PMID:15879451

  19. The Gene Network Underlying Hypodontia.

    PubMed

    Yin, W; Bian, Z

    2015-07-01

    Mammalian tooth development is a precise and complicated procedure. Several signaling pathways, such as nuclear factor (NF)-κB and WNT, are key regulators of tooth development. Any disturbance of these signaling pathways can potentially affect or block normal tooth development, and presently, there are more than 150 syndromes and 80 genes known to be related to tooth agenesis. Clarifying the interaction and crosstalk among these genes will provide important information regarding the mechanisms underlying missing teeth. In the current review, we summarize recently published findings on genes related to isolated and syndromic tooth agenesis; most of these genes function as positive regulators of cell proliferation or negative regulators of cell differentiation and apoptosis. Furthermore, we explore the corresponding networks involving these genes in addition to their implications for the clinical management of tooth agenesis. We conclude that this requires further study to improve patients' quality of life in the future. PMID:25910507

  20. Genes and gene networks implicated in aggression related behaviour.

    PubMed

    Malki, Karim; Pain, Oliver; Du Rietz, Ebba; Tosto, Maria Grazia; Paya-Cano, Jose; Sandnabba, Kenneth N; de Boer, Sietse; Schalkwyk, Leonard C; Sluyter, Frans

    2014-10-01

    Aggressive behaviour is a major cause of mortality and morbidity. Despite of moderate heritability estimates, progress in identifying the genetic factors underlying aggressive behaviour has been limited. There are currently three genetic mouse models of high and low aggression created using selective breeding. This is the first study to offer a global transcriptomic characterization of the prefrontal cortex across all three genetic mouse models of aggression. A systems biology approach has been applied to transcriptomic data across the three pairs of selected inbred mouse strains (Turku Aggressive (TA) and Turku Non-Aggressive (TNA), Short Attack Latency (SAL) and Long Attack Latency (LAL) mice and North Carolina Aggressive (NC900) and North Carolina Non-Aggressive (NC100)), providing novel insight into the neurobiological mechanisms and genetics underlying aggression. First, weighted gene co-expression network analysis (WGCNA) was performed to identify modules of highly correlated genes associated with aggression. Probe sets belonging to gene modules uncovered by WGCNA were carried forward for network analysis using ingenuity pathway analysis (IPA). The RankProd non-parametric algorithm was then used to statistically evaluate expression differences across the genes belonging to modules significantly associated with aggression. IPA uncovered two pathways, involving NF-kB and MAPKs. The secondary RankProd analysis yielded 14 differentially expressed genes, some of which have previously been implicated in pathways associated with aggressive behaviour, such as Adrbk2. The results highlighted plausible candidate genes and gene networks implicated in aggression-related behaviour. PMID:25142712

  1. Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

    DOE PAGESBeta

    Berg, Jörg; Mcdowell, Mindy; Jäck, Hans-Martin; Wabl, Matthias

    1990-01-01

    Imore » mmunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ .It has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ -chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ -chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.« less

  2. Gene therapy progress and prospects: gene therapy for diabetes mellitus.

    PubMed

    Yechoor, V; Chan, L

    2005-01-01

    Diabetes mellitus has long been targeted, as yet unsuccessfully, as being curable with gene therapy. The main hurdles have not only been vector-related toxicity but also the lack of physiological regulation of the expressed insulin. Recent advances in understanding the developmental biology of beta-cells and the transcriptional cascade that drives it have enabled both in vivo and ex vivo gene therapy combined with cell therapy to be used in animal models of diabetes with success. The associated developments in the stem cell biology and immunology have opened up further opportunities for gene therapy to be applied to target autoimmune diabetes. PMID:15496957

  3. Genes, Economics, and Happiness.

    PubMed

    De Neve, Jan-Emmanuel; Christakis, Nicholas A; Fowler, James H; Frey, Bruno S

    2012-11-01

    We explore the influence of genetic variation on subjective well-being by employing a twin design and genetic association study. In a nationally-representative twin sample, we first show that about 33% of the variation in life satisfaction is explained by genetic variation. Although previous studies have shown that baseline happiness is significantly heritable, little research has considered molecular genetic associations with subjective well-being. We study the relationship between a functional polymorphism on the serotonin transporter gene (5-HTTLPR) and life satisfaction. We initially find that individuals with the longer, transcriptionally more efficient variant of this genotype report greater life satisfaction (n=2,545, p=0.012). However, our replication attempts on independent samples produce mixed results indicating that more work needs to be done to better understand the relationship between this genotype and subjective well-being. This work has implications for how economists think about the determinants of utility, and the extent to which exogenous shocks might affect individual well-being. PMID:24349601

  4. Environment, genes, and cancer

    SciTech Connect

    Manuel, J.

    1996-03-01

    In January, comedian George Burns turned 100 years old. In recent appearances in the media, he still seems sharp as a tack, and is still seen smoking his trademark cigars. Others of us, however, were never very funny, and would die of cancer at age 60 if we continuously smoked cigars or cigarettes. Burns presents a common but perplexing paradox; some people are able to tolerate at least moderate exposure to toxins such as cigarette smoke with little adverse affect, while others develop cancer, emphysema, or heart disease. New studies support the idea that there is an interaction between genes and the environment, and that this interaction may be an important determinant of cancer risk. To understand such risks, it is essential to look at both an individual`s genetic makeup and environmental exposures. Such studies require the collaboration of molecular epidemiologists and molecular biologists. At the NIEHS, Jack A. Taylor, a lead clinical investigator in the Epidemiology Branch, and Douglas A. Bell, an investigator with the Genetic Risk Group of the Laboratory of Biochemical Risk Analysis, have worked together and with other scientists to uncover new information in this area.

  5. Genes and equality.

    PubMed

    Farrelly, C

    2004-12-01

    The way people think about equality as a value will influence how they think genetic interventions should be regulated. In this paper the author uses the taxonomy of equality put forth by Derek Parfit and applies this to the issue of genetic interventions. It is argued that telic egalitarianism is untenable and that deontic egalitarianism collapses into prioritarianism. The priority view maintains that it is morally more important to benefit the people who are worse off. Once this precision has been given to the concerns egalitarians have, a number of diverse issues must be considered before determining what the just regulation of genetic interventions would be. Consideration must be given to the current situation of the least advantaged, the fiscal realities behind genetic interventions, the budget constraints on other social programmes egalitarians believe should receive scarce public funds, and the interconnected nature of genetic information. These considerations might lead egalitarians to abandon what they take to be the obvious policy recommendations for them to endorse regarding the regulation of gene therapies and enhancements. PMID:15574450

  6. Genes, Economics, and Happiness *

    PubMed Central

    De Neve, Jan-Emmanuel; Christakis, Nicholas A.; Fowler, James H.; Frey, Bruno S.

    2012-01-01

    We explore the influence of genetic variation on subjective well-being by employing a twin design and genetic association study. In a nationally-representative twin sample, we first show that about 33% of the variation in life satisfaction is explained by genetic variation. Although previous studies have shown that baseline happiness is significantly heritable, little research has considered molecular genetic associations with subjective well-being. We study the relationship between a functional polymorphism on the serotonin transporter gene (5-HTTLPR) and life satisfaction. We initially find that individuals with the longer, transcriptionally more efficient variant of this genotype report greater life satisfaction (n=2,545, p=0.012). However, our replication attempts on independent samples produce mixed results indicating that more work needs to be done to better understand the relationship between this genotype and subjective well-being. This work has implications for how economists think about the determinants of utility, and the extent to which exogenous shocks might affect individual well-being. PMID:24349601

  7. GenePRIMP: A GENE PRediction IMprovement Pipeline for Prokaryotic genomes

    SciTech Connect

    Pati, Amrita; Ivanova, Natalia N.; Mikhailova, Natalia; Ovchinnikova, Galina; Hooper, Sean D.; Lykidis, Athanasios; Kyrpides, Nikos C.

    2010-04-01

    We present 'gene prediction improvement pipeline' (GenePRIMP; http://geneprimp.jgi-psf.org/), a computational process that performs evidence-based evaluation of gene models in prokaryotic genomes and reports anomalies including inconsistent start sites, missed genes and split genes. We found that manual curation of gene models using the anomaly reports generated by GenePRIMP improved their quality, and demonstrate the applicability of GenePRIMP in improving finishing quality and comparing different genome-sequencing and annotation technologies.

  8. Simulating evolution by gene duplication.

    PubMed

    Ohta, T

    1987-01-01

    By considering the recent finding that unequal crossing over and other molecular interactions are contributing to the evolution of multigene families, a model of the origin of repetitive genes was studied by Monte Carlo simulations. Starting from a single gene copy, how genetic systems evolve was examined under unequal crossing over, random drift and natural selection. Both beneficial and deteriorating mutations were incorporated, and the latter were assumed to occur ten times more frequently than the former. Positive natural selection favors those chromosomes with more beneficial mutations in redundant copies than others in the population, but accumulation of deteriorating mutations (pseudogenes) have no effect on fitness so long as there remains a functional gene. The results imply the following: Positive natural selection is needed in order to acquire gene families with new functions. Without it, too many pseudogenes accumulate before attaining a functional gene family. There is a large fluctuation in the outcome even if parameters are the same. When unequal crossing over occurs more frequently, the system evolves more rapidly. It was also shown, under realistic values of parameters, that the genetic load for acquiring a new gene is not as large as J.B.S. Haldane suggested, but not so small as in a model in which a system for selection started from already redundant genes. PMID:3557113

  9. GENES IN SPORT AND DOPING

    PubMed Central

    Kaliszewski, P.; Majorczyk, E.; Zembroń-Łacny, A.

    2013-01-01

    Genes control biological processes such as muscle production of energy, mitochondria biogenesis, bone formation, erythropoiesis, angiogenesis, vasodilation, neurogenesis, etc. DNA profiling for athletes reveals genetic variations that may be associated with endurance ability, muscle performance and power exercise, tendon susceptibility to injuries and psychological aptitude. Already, over 200 genes relating to physical performance have been identified by several research groups. Athletes’ genotyping is developing as a tool for the formulation of personalized training and nutritional programmes to optimize sport training as well as for the prediction of exercise-related injuries. On the other hand, development of molecular technology and gene therapy creates a risk of non-therapeutic use of cells, genes and genetic elements to improve athletic performance. Therefore, the World Anti-Doping Agency decided to include prohibition of gene doping within their World Anti-Doping Code in 2003. In this review article, we will provide a current overview of genes for use in athletes’ genotyping and gene doping possibilities, including their development and detection techniques. PMID:24744482

  10. Gene Electrotransfer: A Mechanistic Perspective.

    PubMed

    Rosazza, Christelle; Meglic, Sasa Haberl; Zumbusch, Andreas; Rols, Marie-Pierre; Miklavcic, Damijan

    2016-01-01

    Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer. PMID:27029943

  11. Linking Genes to Cardiovascular Diseases: Gene Action and Gene-Environment Interactions.

    PubMed

    Pasipoularides, Ares

    2015-12-01

    A unique myocardial characteristic is its ability to grow/remodel in order to adapt; this is determined partly by genes and partly by the environment and the milieu intérieur. In the "post-genomic" era, a need is emerging to elucidate the physiologic functions of myocardial genes, as well as potential adaptive and maladaptive modulations induced by environmental/epigenetic factors. Genome sequencing and analysis advances have become exponential lately, with escalation of our knowledge concerning sometimes controversial genetic underpinnings of cardiovascular diseases. Current technologies can identify candidate genes variously involved in diverse normal/abnormal morphomechanical phenotypes, and offer insights into multiple genetic factors implicated in complex cardiovascular syndromes. The expression profiles of thousands of genes are regularly ascertained under diverse conditions. Global analyses of gene expression levels are useful for cataloging genes and correlated phenotypes, and for elucidating the role of genes in maladies. Comparative expression of gene networks coupled to complex disorders can contribute insights as to how "modifier genes" influence the expressed phenotypes. Increasingly, a more comprehensive and detailed systematic understanding of genetic abnormalities underlying, for example, various genetic cardiomyopathies is emerging. Implementing genomic findings in cardiology practice may well lead directly to better diagnosing and therapeutics. There is currently evolving a strong appreciation for the value of studying gene anomalies, and doing so in a non-disjointed, cohesive manner. However, it is challenging for many-practitioners and investigators-to comprehend, interpret, and utilize the clinically increasingly accessible and affordable cardiovascular genomics studies. This survey addresses the need for fundamental understanding in this vital area. PMID:26545598

  12. Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy.

    PubMed

    Papapetrou, Eirini P; Schambach, Axel

    2016-04-01

    Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy. PMID:26867951

  13. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

    The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. omparisons of transcription initiation and termination site...

  14. BRCA1 and BRCA2 gene testing

    MedlinePlus

    ... gov/ency/patientinstructions/000690.htm BRCA1 and BRCA2 gene testing To use the sharing features on this ... br east ca ncer. What is the BRCA Gene Mutation? BRCA1 and BRCA2 are genes that suppress ...

  15. Genomics screens for metastasis genes

    PubMed Central

    Yan, Jinchun; Huang, Qihong

    2014-01-01

    Metastasis is responsible for most cancer mortality. The process of metastasis is complex, requiring the coordinated expression and fine regulation of many genes in multiple pathways in both the tumor and host tissues. Identification and characterization of the genetic programs that regulate metastasis is critical to understanding the metastatic process and discovering molecular targets for the prevention and treatment of metastasis. Genomic approaches and functional genomic analyses can systemically discover metastasis genes. In this review, we summarize the genetic tools and methods that have been used to identify and characterize the genes that play critical roles in metastasis. PMID:22684367

  16. The design of synthetic genes.

    PubMed Central

    Presnell, S R; Benner, S A

    1988-01-01

    Computer programs are described that aid in the design of synthetic genes coding for proteins that are targets of a research program in site directed mutagenesis. These programs "reverse-translate" protein sequences into general nucleic acid sequences (those where codons have not yet been selected), map restriction sites into general DNA sequences, identify points in the synthetic gene where unique restriction sites can be introduced, and assist in the design of genes coding for hybrids and evolutionary intermediates between homologous proteins. Application of these programs therefore facilitates the use of modular mutagenesis to create variants of proteins, and the implementation of evolutionary guidance as a strategy for selecting mutants. PMID:2451218

  17. Gene networks controlling petal organogenesis.

    PubMed

    Huang, Tengbo; Irish, Vivian F

    2016-01-01

    One of the biggest unanswered questions in developmental biology is how growth is controlled. Petals are an excellent organ system for investigating growth control in plants: petals are dispensable, have a simple structure, and are largely refractory to environmental perturbations that can alter their size and shape. In recent studies, a number of genes controlling petal growth have been identified. The overall picture of how such genes function in petal organogenesis is beginning to be elucidated. This review will focus on studies using petals as a model system to explore the underlying gene networks that control organ initiation, growth, and final organ morphology. PMID:26428062

  18. The Ensembl gene annotation system.

    PubMed

    Aken, Bronwen L; Ayling, Sarah; Barrell, Daniel; Clarke, Laura; Curwen, Valery; Fairley, Susan; Fernandez Banet, Julio; Billis, Konstantinos; García Girón, Carlos; Hourlier, Thibaut; Howe, Kevin; Kähäri, Andreas; Kokocinski, Felix; Martin, Fergal J; Murphy, Daniel N; Nag, Rishi; Ruffier, Magali; Schuster, Michael; Tang, Y Amy; Vogel, Jan-Hinnerk; White, Simon; Zadissa, Amonida; Flicek, Paul; Searle, Stephen M J

    2016-01-01

    The Ensembl gene annotation system has been used to annotate over 70 different vertebrate species across a wide range of genome projects. Furthermore, it generates the automatic alignment-based annotation for the human and mouse GENCODE gene sets. The system is based on the alignment of biological sequences, including cDNAs, proteins and RNA-seq reads, to the target genome in order to construct candidate transcript models. Careful assessment and filtering of these candidate transcripts ultimately leads to the final gene set, which is made available on the Ensembl website. Here, we describe the annotation process in detail.Database URL: http://www.ensembl.org/index.html. PMID:27337980

  19. The Ensembl gene annotation system

    PubMed Central

    Aken, Bronwen L.; Ayling, Sarah; Barrell, Daniel; Clarke, Laura; Curwen, Valery; Fairley, Susan; Fernandez Banet, Julio; Billis, Konstantinos; García Girón, Carlos; Hourlier, Thibaut; Howe, Kevin; Kähäri, Andreas; Kokocinski, Felix; Martin, Fergal J.; Murphy, Daniel N.; Nag, Rishi; Ruffier, Magali; Schuster, Michael; Tang, Y. Amy; Vogel, Jan-Hinnerk; White, Simon; Zadissa, Amonida; Flicek, Paul

    2016-01-01

    The Ensembl gene annotation system has been used to annotate over 70 different vertebrate species across a wide range of genome projects. Furthermore, it generates the automatic alignment-based annotation for the human and mouse GENCODE gene sets. The system is based on the alignment of biological sequences, including cDNAs, proteins and RNA-seq reads, to the target genome in order to construct candidate transcript models. Careful assessment and filtering of these candidate transcripts ultimately leads to the final gene set, which is made available on the Ensembl website. Here, we describe the annotation process in detail. Database URL: http://www.ensembl.org/index.html PMID:27337980

  20. Saporin as a novel suicide gene in anticancer gene therapy.

    PubMed

    Zarovni, N; Vago, R; Soldà, T; Monaco, L; Fabbrini, M S

    2007-02-01

    We used a non-viral gene delivery approach to explore the potential of the plant saporin (SAP) gene as an alternative to the currently employed suicide genes in cancer therapy. Plasmids expressing cytosolic SAP were generated by placing the region encoding the mature plant ribosome-inactivating protein under the control of cytomegalovirus (CMV) or simian virus 40 (SV40) promoters. Their ability to inhibit protein synthesis was first tested in cultured tumor cells co-transfected with a luciferase reporter gene. In particular, SAP expression driven by CMV promoter (pCI-SAP) demonstrated that only 10 ng of plasmid per 1.6 x 10(4) B16 cells drastically reduced luciferase activity to 18% of that in control cells. Direct intratumoral injection of pCI-SAP complexed with either lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) in B16 melanoma-bearing mice resulted in a noteworthy attenuation of tumor growth. This antitumor effect was increased in mice that received repeated intratumoral injections. A SAP catalytic inactive mutant (SAP-KQ) failed to exert any antitumor effect demonstrating that this was specifically owing to the SAP N-glycosidase activity. Our overall data strongly suggest that the gene encoding SAP, owing to its rapid and effective action and its independence from the proliferative state of target cells might become a suitable candidate suicide gene for oncologic applications. PMID:17008932

  1. Gene Cernan on Apollo 17

    NASA Video Gallery

    Apollo 17 Commander Gene Cernan recalls fixing a lunar rover problem with duct tape during his December 1972 mission. Cernan's interview was part of the commemoration of NASA's 50th anniversary in ...

  2. How eukaryotic genes are transcribed

    PubMed Central

    Venters, Bryan J.; Pugh, B. Franklin

    2009-01-01

    Summary Regulation of eukaryotic gene expression is far more complex than one might have imagined thirty years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: 1) a well-defined organization of nucleosomes and modification states at most genes, 2) regulatory networks of sequence-specific transcription factors, 3) chromatin remodeling coupled to promoter assembly of the general transcription factors and RNA polymerase II, and 4) phosphorylation states of RNA polymerase II coupled to chromatin modification states during transcription. The wealth of new insights arising from the tools of biochemistry, genomics, cell biology, and genetics is providing a remarkable view into the mechanics of gene regulation. PMID:19514890

  3. [Genes Responsible for Epileptic Syndromes].

    PubMed

    Kato, Mitsuhiro

    2016-02-01

    The first causative gene for epileptic syndrome was revealed 20 years ago. Since then, many genes responsible for epileptic syndrome, particularly sporadic epileptic encephalopathies, such as Ohtahara syndrome, West syndrome, and focal cortical dysplasia, have been identified. Although epilepsy was recognized as a channelopathy in the beginning stages of gene discovery, other molecular mechanisms for epileptic syndromes, such as interneuronopathy, synaptic vesicle release, and mTOR signal transduction, are emerging. A new technique for gene analysis using the next-generation sequencer is now available for clinical purpose abroad and precision medicine based on the molecular mechanisms has started. Infrastructural development of the official framework, from molecular diagnosis to personalized therapy, is urgently required in Japan. PMID:26873236

  4. Why genes are like lemons.

    PubMed

    Boem, F; Ratti, E; Andreoletti, M; Boniolo, G

    2016-06-01

    In the last few years, the lack of a unitary notion of gene across biological sciences has troubled the philosophy of biology community. However, the debate on this concept has remained largely historical or focused on particular cases presented by the scientific empirical advancements. Moreover, in the literature there are no explicit and reasonable arguments about why a philosophical clarification of the concept of gene is needed. In our paper, we claim that a philosophical clarification of the concept of gene does not contribute to biology. Unlike the question, for example, "What is a biological function?", we argue that the question "What is a gene?" could be answered by means of empirical research, in the sense that biologists' labour is enough to shed light on it. PMID:27155220

  5. Gene Therapy for Cardiovascular Disease

    PubMed Central

    2003-01-01

    The last decade has seen substantial advances in the development of gene therapy strategies and vector technology for the treatment of a diverse number of diseases, with a view to translating the successes observed in animal models into the clinic. Perhaps the overwhelming drive for the increase in vascular gene transfer studies is the current lack of successful long-term pharmacological treatments for complex cardiovascular diseases. The increase in cardiovascular disease to epidemic proportions has also led many to conclude that drug therapy may have reached a plateau in its efficacy and that gene therapy may represent a realistic solution to a long-term problem. Here, we discuss gene delivery approaches and target diseases. PMID:12721517

  6. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  7. [Genes for extreme violent behaviour?].

    PubMed

    Jordan, Bertrand

    2015-01-01

    A new genetic study focussing on the degree of violence in criminals and using both candidate gene and GWAS approaches finds statistically significant associations of extreme violent behaviour with low activity alleles of monoamine oxydase A (MAOA) and with the CD13 gene. However, the alleles implicated are common in the general population, thus they cannot be causal, and only represent potential indicators of increased risk. PMID:25658738

  8. Gene Transfer into Cardiac Myocytes

    PubMed Central

    Lang, Sarah E.; Westfall, Margaret V.

    2016-01-01

    Traditional methods for DNA transfection are often inefficient and toxic for terminally differentiated cells, such as cardiac myocytes. Vector-based gene transfer is an efficient approach for introducing exogenous cDNA into these types of primary cell cultures. In this chapter, separate protocols for adult rat cardiac myocyte isolation and gene transfer with recombinant adenovirus are provided and are routinely utilized for studying the effects of sarcomeric proteins on myofilament function. PMID:25836585

  9. Gene therapy for paediatric leukaemia.

    PubMed

    Rousseau, R F; Bollard, C M; Heslop, H E

    2001-07-01

    Improvements in the chemotherapeutic and transplant regimens have had a significant impact in improving survival rates for paediatric leukaemia. However, there are still important problems to address including what options are available for patients with chemoresistant disease and what strategies are available to avoid the concerns regarding the toxicity associated with highly cytotoxic treatment regimens. Gene therapy and immunotherapy protocols hold great promise. Using gene transfer of a marker gene, a number of biological issues in the therapy of leukaemia have been addressed. For example, by gene marking autologous bone marrow grafts it has been possible to demonstrate that infused marrow contributes to relapse in acute and chronic myeloid leukaemias. In the allogeneic transplant setting, genetically modified T-cells have proven valuable for the prophylaxis and treatment of viral diseases and may have an important role in preventing or treating disease relapse. Gene transfer is also being used to modify tumour function, enhance immunogenicity, and confer drug-resistance to normal haematopoietic stem cells. With the continued scientific advancements in this field, gene therapy will almost certainly have a major impact on the treatment of paediatric leukaemia in the future. PMID:11727502

  10. Homologous gene replacement in Physarum

    SciTech Connect

    Burland, T.G.; Pallotta, D.

    1995-01-01

    The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD{Delta}1, of the ardD actin gene; ardD{Delta}1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD{sup +} with ardD{Delta}1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD{Delta}1 in addition to ardD{sup +}, but in two (5%), ardD{sup +} was replaced by a single copy of ardD{Delta}1. This is the first example of homologous gene replacement in Physarum. ardD{Delta}1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD{Delta}l on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene. 38 refs., 6 figs., 1 tab.

  11. Gene-gene, gene-environment, gene-nutrient interactions and single nucleotide polymorphisms of inflammatory cytokines.

    PubMed

    Nadeem, Amina; Mumtaz, Sadaf; Naveed, Abdul Khaliq; Aslam, Muhammad; Siddiqui, Arif; Lodhi, Ghulam Mustafa; Ahmad, Tausif

    2015-05-15

    Inflammation plays a significant role in the etiology of type 2 diabetes mellitus (T2DM). The rise in the pro-inflammatory cytokines is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress and beta cell apoptosis in T2DM. Among the recognized markers are interleukin (IL)-6, IL-1, IL-10, IL-18, tissue necrosis factor-alpha (TNF-α), C-reactive protein, resistin, adiponectin, tissue plasminogen activator, fibrinogen and heptoglobins. Diabetes mellitus has firm genetic and very strong environmental influence; exhibiting a polygenic mode of inheritance. Many single nucleotide polymorphisms (SNPs) in various genes including those of pro and anti-inflammatory cytokines have been reported as a risk for T2DM. Not all the SNPs have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups. The inter-ethnic variations can be explained by the fact that gene expression may be regulated by gene-gene, gene-environment and gene-nutrient interactions. This review highlights the impact of these interactions on determining the role of single nucleotide polymorphism of IL-6, TNF-α, resistin and adiponectin in pathogenesis of T2DM. PMID:25987962

  12. Transcriptional gene silencing in humans.

    PubMed

    Weinberg, Marc S; Morris, Kevin V

    2016-08-19

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  13. Building Developmental Gene Regulatory Networks

    PubMed Central

    Li, Enhu; Davidson, Eric H.

    2009-01-01

    Animal development is an elaborate process programmed by genomic regulatory instructions. Regulatory genes encode transcription factors and signal molecules, and their expression is under the control of cis-regulatory modules that define the logic of transcriptional responses to the inputs of other regulatory genes. The functional linkages amongst regulatory genes constitute the gene regulatory networks (GRNs) that govern cell specification and patterning in development. Constructing such networks requires identification of the regulatory genes involved and characterization of their temporal and spatial expression patterns. Interactions (activation/repression) among transcription factors or signals can be investigated by large-scale perturbation analysis, in which the function of each gene is specifically blocked. Resultant expression changes are then integrated to identify direct linkages, and to reveal the structure of the GRN. Predicted GRN linkages can be tested and verified by cis-regulatory analysis. The explanatory power of the GRN was shown in the lineage specification of sea urchin endomesoderm. Acquiring such networks is essential for a systematic and mechanistic understanding of the developmental process. PMID:19530131

  14. Gene-gene, gene-environment, gene-nutrient interactions and single nucleotide polymorphisms of inflammatory cytokines

    PubMed Central

    Nadeem, Amina; Mumtaz, Sadaf; Naveed, Abdul Khaliq; Aslam, Muhammad; Siddiqui, Arif; Lodhi, Ghulam Mustafa; Ahmad, Tausif

    2015-01-01

    Inflammation plays a significant role in the etiology of type 2 diabetes mellitus (T2DM). The rise in the pro-inflammatory cytokines is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress and beta cell apoptosis in T2DM. Among the recognized markers are interleukin (IL)-6, IL-1, IL-10, IL-18, tissue necrosis factor-alpha (TNF-α), C-reactive protein, resistin, adiponectin, tissue plasminogen activator, fibrinogen and heptoglobins. Diabetes mellitus has firm genetic and very strong environmental influence; exhibiting a polygenic mode of inheritance. Many single nucleotide polymorphisms (SNPs) in various genes including those of pro and anti-inflammatory cytokines have been reported as a risk for T2DM. Not all the SNPs have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups. The inter-ethnic variations can be explained by the fact that gene expression may be regulated by gene-gene, gene-environment and gene-nutrient interactions. This review highlights the impact of these interactions on determining the role of single nucleotide polymorphism of IL-6, TNF-α, resistin and adiponectin in pathogenesis of T2DM. PMID:25987962

  15. Vectors for airway gene delivery.

    PubMed

    Davis, Pamela B; Cooper, Mark J

    2007-01-01

    Delivery of genes to the airway epithelium for therapeutic purposes seemed easy at first, because the epithelial cells interface with the environment and are therefore accessible. However, problems encountered were more substantial than were originally expected. Nonviral systems may be preferred for long-term gene expression, for they can be dosed repeatedly. Two nonviral gene transfer systems have been in clinical trials, lipid-mediated gene transfer and DNA nanoparticles. Both have sufficient efficiency to be candidates for correction of the cystic fibrosis defect, and both can be dosed repeatedly. However, lipid-mediated gene transfer in the first generation provokes significant inflammatory toxicity, which may be engineered out by adjustments of the lipids, the plasmid CpG content, or both. Both lipid-mediated gene transfer and DNA nanoparticles in the first generation have short duration of expression, but reengineering of the plasmid DNA to contain mostly eukaryotic sequences may address this problem. Considerable advances in the understanding of the cellular uptake and expression of these agents and in their practical utility have occurred in the last few years; these advances are reviewed here. PMID:17408235

  16. Cationic Bolaamphiphiles for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Tan, Amelia Li Min; Lim, Alisa Xue Ling; Zhu, Yiting; Yang, Yi Yan; Khan, Majad

    2014-05-01

    Advances in medical research have shed light on the genetic cause of many human diseases. Gene therapy is a promising approach which can be used to deliver therapeutic genes to treat genetic diseases at its most fundamental level. In general, nonviral vectors are preferred due to reduced risk of immune response, but they are also commonly associated with low transfection efficiency and high cytotoxicity. In contrast to viral vectors, nonviral vectors do not have a natural mechanism to overcome extra- and intracellular barriers when delivering the therapeutic gene into cell. Hence, its design has been increasingly complex to meet challenges faced in targeting of, penetration of and expression in a specific host cell in achieving more satisfactory transfection efficiency. Flexibility in design of the vector is desirable, to enable a careful and controlled manipulation of its properties and functions. This can be met by the use of bolaamphiphile, a special class of lipid. Unlike conventional lipids, bolaamphiphiles can form asymmetric complexes with the therapeutic gene. The advantage of having an asymmetric complex lies in the different purposes served by the interior and exterior of the complex. More effective gene encapsulation within the interior of the complex can be achieved without triggering greater aggregation of serum proteins with the exterior, potentially overcoming one of the great hurdles faced by conventional single-head cationic lipids. In this review, we will look into the physiochemical considerations as well as the biological aspects of a bolaamphiphile-based gene delivery system.

  17. PET genes of Saccharomyces cerevisiae.

    PubMed Central

    Tzagoloff, A; Dieckmann, C L

    1990-01-01

    We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae. PMID:2215420

  18. Gene Therapy in Heart Failure

    PubMed Central

    Vinge, Leif Erik; Raake, Philip W.; Koch, Walter J.

    2008-01-01

    With increasing knowledge of basic molecular mechanisms governing the development of heart failure (HF), the possibility of specifically targeting key pathological players is evolving. Technology allowing for efficient in vivo transduction of myocardial tissue with long-term expression of a transgene enables translation of basic mechanistic knowledge into potential gene therapy approaches. Gene therapy in HF is in its infancy clinically with the predominant amount of experience being from animal models. Nevertheless, this challenging and promising field is gaining momentum as recent preclinical studies in larger animals have been carried out and, importantly, there are 2 newly initiated phase I clinical trials for HF gene therapy. To put it simply, 2 parameters are needed for achieving success with HF gene therapy: (1) clearly identified detrimental/beneficial molecular targets; and (2) the means to manipulate these targets at a molecular level in a sufficient number of cardiac cells. However, several obstacles do exist on our way to efficient and safe gene transfer to human myocardium. Some of these obstacles are discussed in this review; however, it primarily focuses on the molecular target systems that have been subjected to intense investigation over the last decade in an attempt to make gene therapy for human HF a reality. PMID:18566312

  19. Homology-dependent Gene Silencing in Paramecium

    PubMed Central

    Ruiz, Françoise; Vayssié, Laurence; Klotz, Catherine; Sperling, Linda; Madeddu, Luisa

    1998-01-01

    Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant. PMID:9529389

  20. PoplarGene: poplar gene network and resource for mining functional information for genes from woody plants

    PubMed Central

    Liu, Qi; Ding, Changjun; Chu, Yanguang; Chen, Jiafei; Zhang, Weixi; Zhang, Bingyu; Huang, Qinjun; Su, Xiaohua

    2016-01-01

    Poplar is not only an important resource for the production of paper, timber and other wood-based products, but it has also emerged as an ideal model system for studying woody plants. To better understand the biological processes underlying various traits in poplar, e.g., wood development, a comprehensive functional gene interaction network is highly needed. Here, we constructed a genome-wide functional gene network for poplar (covering ~70% of the 41,335 poplar genes) and created the network web service PoplarGene, offering comprehensive functional interactions and extensive poplar gene functional annotations. PoplarGene incorporates two network-based gene prioritization algorithms, neighborhood-based prioritization and context-based prioritization, which can be used to perform gene prioritization in a complementary manner. Furthermore, the co-functional information in PoplarGene can be applied to other woody plant proteomes with high efficiency via orthology transfer. In addition to poplar gene sequences, the webserver also accepts Arabidopsis reference gene as input to guide the search for novel candidate functional genes in PoplarGene. We believe that PoplarGene (http://bioinformatics.caf.ac.cn/PoplarGene and http://124.127.201.25/PoplarGene) will greatly benefit the research community, facilitating studies of poplar and other woody plants. PMID:27515999

  1. [Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

    PubMed

    Wang, Guozhong; Hu, Shenjiang; Zheng, Zhelan; Sun, Jian; Zheng, Xia; Zhu, Zhaohui; Li, Jiang; Yao, Yumei

    2006-08-01

    To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle. PMID:17002125

  2. PoplarGene: poplar gene network and resource for mining functional information for genes from woody plants.

    PubMed

    Liu, Qi; Ding, Changjun; Chu, Yanguang; Chen, Jiafei; Zhang, Weixi; Zhang, Bingyu; Huang, Qinjun; Su, Xiaohua

    2016-01-01

    Poplar is not only an important resource for the production of paper, timber and other wood-based products, but it has also emerged as an ideal model system for studying woody plants. To better understand the biological processes underlying various traits in poplar, e.g., wood development, a comprehensive functional gene interaction network is highly needed. Here, we constructed a genome-wide functional gene network for poplar (covering ~70% of the 41,335 poplar genes) and created the network web service PoplarGene, offering comprehensive functional interactions and extensive poplar gene functional annotations. PoplarGene incorporates two network-based gene prioritization algorithms, neighborhood-based prioritization and context-based prioritization, which can be used to perform gene prioritization in a complementary manner. Furthermore, the co-functional information in PoplarGene can be applied to other woody plant proteomes with high efficiency via orthology transfer. In addition to poplar gene sequences, the webserver also accepts Arabidopsis reference gene as input to guide the search for novel candidate functional genes in PoplarGene. We believe that PoplarGene (http://bioinformatics.caf.ac.cn/PoplarGene and http://124.127.201.25/PoplarGene) will greatly benefit the research community, facilitating studies of poplar and other woody plants. PMID:27515999

  3. Newer Gene Editing Technologies toward HIV Gene Therapy

    PubMed Central

    Manjunath, N.; Yi, Guohua; Dang, Ying; Shankar, Premlata

    2013-01-01

    Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy. PMID:24284874

  4. Noninvasive Tracking of Gene Transcript and Neuroprotection after Gene Therapy

    PubMed Central

    Ren, Jiaqian; Chen, Y. Iris; Liu, Christina H.; Chen, Po-Chih; Prentice, Howard; Wu, Jang-Yen; Liu, Philip K.

    2015-01-01

    Gene therapy holds exceptional potential for translational medicine by improving the products of defective genes in diseases and/or providing necessary biologics from endogenous sources during recovery processes. However, validating methods for the delivery, distribution and expression of the exogenous genes from such therapy can generally not be applicable to monitor effects over the long term because they are invasive. We report here that human granulocyte colony-stimulating factor (hG-CSF) cDNA encoded in scAAV-type 2 adeno-associated virus, as delivered through eye drops at multiple time points after cerebral ischemia using bilateral carotid occlusion for 60 min (BCAO-60) led to significant reduction in mortality rates, cerebral atrophy, and neurological deficits in C57black6 mice. Most importantly, we validated hG-CSF cDNA expression using translatable magnetic resonance imaging (MRI) in living brains. This noninvasive approach for monitoring exogenous gene expression in the brains has potential for great impact in the area of experimental gene therapy in animal models of heart attack, stroke, Alzheimer’s dementia, Parkinson’s disorder and amyotrophic lateral sclerosis, and the translation of such techniques to emergency medicine. PMID:26207935

  5. Noninvasive tracking of gene transcript and neuroprotection after gene therapy.

    PubMed

    Ren, J; Chen, Y I; Liu, C H; Chen, P-C; Prentice, H; Wu, J-Y; Liu, P K

    2016-01-01

    Gene therapy holds exceptional potential for translational medicine by improving the products of defective genes in diseases and/or providing necessary biologics from endogenous sources during recovery processes. However, validating methods for the delivery, distribution and expression of the exogenous genes from such therapy can generally not be applicable to monitor effects over the long term because they are invasive. We report here that human granulocyte colony-stimulating factor (hG-CSF) complimentary DNA (cDNA) encoded in self-complementary adeno-associated virus-type 2 adeno-associated virus, as delivered through eye drops at multiple time points after cerebral ischemia using bilateral carotid occlusion for 60 min (BCAO-60) led to significant reduction in mortality rates, cerebral atrophy and neurological deficits in C57black6 mice. Most importantly, we validated hG-CSF cDNA expression using translatable magnetic resonance imaging (MRI) in living brains. This noninvasive approach for monitoring exogenous gene expression in the brains has potential for great impact in the area of experimental gene therapy in animal models of heart attack, stroke, Alzheimer's dementia, Parkinson's disorder and amyotrophic lateral sclerosis, and the translation of such techniques to emergency medicine. PMID:26207935

  6. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  7. Combining Hierarchical and Associative Gene Ontology Relations with Textual Evidence in Estimating Gene and Gene Product Similarity

    SciTech Connect

    Sanfilippo, Antonio P.; Posse, Christian; Gopalan, Banu; Riensche, Roderick M.; Beagley, Nathaniel; Baddeley, Bob L.; Tratz, Stephen C.; Gregory, Michelle L.

    2007-03-01

    Gene and gene product similarity is a fundamental diagnostic measure in analyzing biological data and constructing predictive models for functional genomics. With the rising influence of the Gene Ontology, two complementary approaches have emerged where the similarity between two genes or gene products is obtained by comparing Gene Ontology (GO) annotations associated with the genes or gene products. One approach captures GO-based similarity in terms of hierarchical relations within each gene subontology. The other approach identifies GO-based similarity in terms of associative relations across the three gene subontologies. We propose a novel methodology where the two approaches can be merged with ensuing benefits in coverage and accuracy, and demonstrate that further improvements can be obtained by integrating textual evidence extracted from relevant biomedical literature.

  8. Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: The role of extra copies of glpK, glpX, and tktA genes

    PubMed Central

    2014-01-01

    Background For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported. Results L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM CPhe/CGlucose and a maximal space-time-yield (STY) of 0.13 g l−1 h−1 was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM CPhe/CGlycerol), but the maximal STY was higher (0.21 g l−1 h−1). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpKG232D) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM CPhe/CGlycerol; 0.25 g l−1 h−1 and 0.21 mM CPhe/CGlycerol; 0.23 g l−1 h−1, respectively), the combination of extra genes of glpX and tktA together led

  9. The Influence of Gene-Gene and Gene-Environment Interactions on the Risk of Asbestosis

    PubMed Central

    Franko, A.; Dolžan, V.; Arnerić, N.; Dodič-Fikfak, M.

    2013-01-01

    This study investigated the influence of gene-gene and gene-environment interactions on the risk of developing asbestosis. The study comprised 262 cases with asbestosis and 265 controls with no asbestos-related disease previously studied for MnSOD, ECSOD, CAT, GSTT1, GSTM1, GSTP1, and iNOS polymorphisms. Data on cumulative asbestos and smoking were available for all subjects. To assess gene-gene and gene-environmental interactions, logistic regression was used. The associations between MnSOD Ala −9Val polymorphism and the risk of asbestosis and between iNOS genotypes and asbestosis were modified by CAT –262 C > T polymorphism (P = 0.038; P = 0.031). A strong interaction was found between GSTM1-null polymorphism and smoking (P = 0.007), iNOS (CCTTT)n polymorphism and smoking (P = 0.054), and between iNOS (CCTTT)n polymorphism and cumulative asbestos exposure (P = 0.037). The findings of this study suggest that the interactions between different genotypes, genotypes and smoking, and between genotypes and asbestos exposure have an important influence on the development of asbestosis and should be seriously considered in future research on occupational/environmental asbestos-related diseases. PMID:23984360

  10. [Review of cancer gene therapy].

    PubMed

    Tani, K

    2000-09-01

    Since the first introduction of gene-marking technology to the clinical field in 1989 by Rosenberg et al, more than 4,000 patients have participated gene therapy clinical trials worldwide. Most of those patients had malignancies. Nearly 90% of clinical trials, however, are still in phase I-II stage, and only 3 protocols are in the phase III stage in early 2000. As current clinical gene therapy protocols are intended essentially to examine the safety and feasibility of the new strategy, more careful and steady steps may be required before these clinical trials really produce clinical benefits. Focused on cancer gene therapy, direct and indirect approaches are undertaken. In the direct approach, HSV-TK, HLA-B7, or p53 tumor suppressor gene therapies are the three major approaches historically. In for the indirect approach, cytokine or adhesion molecule gene-transferred tumor cells or immunocompetent cells are considered to be promising to enhance patients' antitumor immunity. In particular, we have concentrated on developing immuno gene therapy using GM-CSF-transduced autologous tumor cells. We have already recruited three patients with stage IV renal cell cancer. In all patients, peripheral blood T cells were mobilized after vaccination with GM-CSF-transduced tumor cells, and two of the three patients showed the persistence of cytotoxic T cells against autologous tumor cells. Clinically, one patient has been followed up with stable disease for more than one year since the start of vaccination. Further clinical studies are required to obtain conclusive results. PMID:11022677

  11. Genes and Abdominal Aortic Aneurysm

    PubMed Central

    Hinterseher, Irene; Tromp, Gerard; Kuivaniemi, Helena

    2010-01-01

    Abdominal aortic aneurysm (AAA) is a multifactorial disease with a strong genetic component. Since first candidate gene studies were published 20 years ago, nearly 100 genetic association studies using single nucleotide polymorphisms (SNPs) in biologically relevant genes have been reported on AAA. The studies investigated SNPs in genes of the extracellular matrix, the cardiovascular system, the immune system, and signaling pathways. Very few studies were large enough to draw firm conclusions and very few results could be replicated in another sample set. The more recent unbiased approaches are family-based DNA linkage studies and genome-wide genetic association studies, which have the potential of identifying the genetic basis for AAA, if appropriately powered and well-characterized large AAA cohorts are used. SNPs associated with AAA have already been identified in these large multicenter studies. One significant association was of a variant in a gene called CNTN3 which is located on chromosome 3p12.3. Two follow-up studies, however, could not replicate the association. Two other SNPs, which are located on chromosome 9p21 and 9q33 were replicated in other samples. The two genes with the strongest supporting evidence of contribution to the genetic risk for AAA are the CDKN2BAS gene, also known as ANRIL, which encodes an antisense RNA that regulates expression of the cyclin-dependent kinase inhibitors CDKN2A and CDKN2B, and DAB2IP, which encodes an inhibitor of cell growth and survival. Functional studies are now needed to establish the mechanisms by which these genes contribute to AAA pathogenesis. PMID:21146954

  12. Gene expression during memory formation.

    PubMed

    Igaz, Lionel Muller; Bekinschtein, Pedro; Vianna, Monica M R; Izquierdo, Ivan; Medina, Jorge H

    2004-01-01

    For several decades, neuroscientists have provided many clues that point out the involvement of de novo gene expression during the formation of long-lasting forms of memory. However, information regarding the transcriptional response networks involved in memory formation has been scarce and fragmented. With the advent of genome-based technologies, combined with more classical approaches (i.e., pharmacology and biochemistry), it is now feasible to address those relevant questions--which gene products are modulated, and when that processes are necessary for the proper storage of memories--with unprecedented resolution and scale. Using one-trial inhibitory (passive) avoidance training of rats, one of the most studied tasks so far, we found two time windows of sensitivity to transcriptional and translational inhibitors infused into the hippocampus: around the time of training and 3-6 h after training. Remarkably, these periods perfectly overlap with the involvement of hippocampal cAMP/PKA (protein kinase A) signaling pathways in memory consolidation. Given the complexity of transcriptional responses in the brain, particularly those related to processing of behavioral information, it was clearly necessary to address this issue with a multi-variable, parallel-oriented approach. We used cDNA arrays to screen for candidate inhibitory avoidance learning-related genes and analyze the dynamic pattern of gene expression that emerges during memory consolidation. These include genes involved in intracellular kinase networks, synaptic function, DNA-binding and chromatin modification, transcriptional activation and repression, translation, membrane receptors, and oncogenes, among others. Our findings suggest that differential and orchestrated hippocampal gene expression is necessary in both early and late periods of long-term memory consolidation. Additionally, this kind of studies may lead to the identification and characterization of genes that are relevant for the pathogenesis

  13. Integrating heterogeneous gene expression data for gene regulatory network modelling.

    PubMed

    Sîrbu, Alina; Ruskin, Heather J; Crane, Martin

    2012-06-01

    Gene regulatory networks (GRNs) are complex biological systems that have a large impact on protein levels, so that discovering network interactions is a major objective of systems biology. Quantitative GRN models have been inferred, to date, from time series measurements of gene expression, but at small scale, and with limited application to real data. Time series experiments are typically short (number of time points of the order of ten), whereas regulatory networks can be very large (containing hundreds of genes). This creates an under-determination problem, which negatively influences the results of any inferential algorithm. Presented here is an integrative approach to model inference, which has not been previously discussed to the authors' knowledge. Multiple heterogeneous expression time series are used to infer the same model, and results are shown to be more robust to noise and parameter perturbation. Additionally, a wavelet analysis shows that these models display limited noise over-fitting within the individual datasets. PMID:21948152

  14. A Hybrid Approach of Gene Sets and Single Genes for the Prediction of Survival Risks with Gene Expression Data

    PubMed Central

    Seok, Junhee; Davis, Ronald W.; Xiao, Wenzhong

    2015-01-01

    Accumulated biological knowledge is often encoded as gene sets, collections of genes associated with similar biological functions or pathways. The use of gene sets in the analyses of high-throughput gene expression data has been intensively studied and applied in clinical research. However, the main interest remains in finding modules of biological knowledge, or corresponding gene sets, significantly associated with disease conditions. Risk prediction from censored survival times using gene sets hasn’t been well studied. In this work, we propose a hybrid method that uses both single gene and gene set information together to predict patient survival risks from gene expression profiles. In the proposed method, gene sets provide context-level information that is poorly reflected by single genes. Complementarily, single genes help to supplement incomplete information of gene sets due to our imperfect biomedical knowledge. Through the tests over multiple data sets of cancer and trauma injury, the proposed method showed robust and improved performance compared with the conventional approaches with only single genes or gene sets solely. Additionally, we examined the prediction result in the trauma injury data, and showed that the modules of biological knowledge used in the prediction by the proposed method were highly interpretable in biology. A wide range of survival prediction problems in clinical genomics is expected to benefit from the use of biological knowledge. PMID:25933378

  15. Consensus gene regulatory networks: combining multiple microarray gene expression datasets

    NASA Astrophysics Data System (ADS)

    Peeling, Emma; Tucker, Allan

    2007-09-01

    In this paper we present a method for modelling gene regulatory networks by forming a consensus Bayesian network model from multiple microarray gene expression datasets. Our method is based on combining Bayesian network graph topologies and does not require any special pre-processing of the datasets, such as re-normalisation. We evaluate our method on a synthetic regulatory network and part of the yeast heat-shock response regulatory network using publicly available yeast microarray datasets. Results are promising; the consensus networks formed provide a broader view of the potential underlying network, obtaining an increased true positive rate over networks constructed from a single data source.

  16. Gene methylation in gastric cancer.

    PubMed

    Qu, Yiping; Dang, Siwen; Hou, Peng

    2013-09-23

    Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. Over 70% of new cases and deaths occur in developing countries. In the early years of the molecular biology revolution, cancer research mainly focuses on genetic alterations, including gastric cancer. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs, and microRNAs. Aberrant DNA methylation in the promoter regions of gene, which leads to inactivation of tumor suppressor and other cancer-related genes in cancer cells, is the most well-defined epigenetic hallmark in gastric cancer. The advantages of gene methylation as a target for detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as serum and gastric washes have led to many studies of application in gastric cancer. This review focuses on the most common and important phenomenon of epigenetics, DNA methylation, in gastric cancer and illustrates the impact epigenetics has had on this field. PMID:23669186

  17. Gene amplification during myogenic differentiation

    PubMed Central

    Fischer, Ulrike; Ludwig, Nicole; Raslan, Abdulrahman; Meier, Carola; Meese, Eckart

    2016-01-01

    Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians. PMID:26760505

  18. Gene Ontology Consortium: going forward.

    PubMed

    2015-01-01

    The Gene Ontology (GO; http://www.geneontology.org) is a community-based bioinformatics resource that supplies information about gene product function using ontologies to represent biological knowledge. Here we describe improvements and expansions to several branches of the ontology, as well as updates that have allowed us to more efficiently disseminate the GO and capture feedback from the research community. The Gene Ontology Consortium (GOC) has expanded areas of the ontology such as cilia-related terms, cell-cycle terms and multicellular organism processes. We have also implemented new tools for generating ontology terms based on a set of logical rules making use of templates, and we have made efforts to increase our use of logical definitions. The GOC has a new and improved web site summarizing new developments and documentation, serving as a portal to GO data. Users can perform GO enrichment analysis, and search the GO for terms, annotations to gene products, and associated metadata across multiple species using the all-new AmiGO 2 browser. We encourage and welcome the input of the research community in all biological areas in our continued effort to improve the Gene Ontology. PMID:25428369

  19. Gene Ontology Consortium: going forward

    PubMed Central

    2015-01-01

    The Gene Ontology (GO; http://www.geneontology.org) is a community-based bioinformatics resource that supplies information about gene product function using ontologies to represent biological knowledge. Here we describe improvements and expansions to several branches of the ontology, as well as updates that have allowed us to more efficiently disseminate the GO and capture feedback from the research community. The Gene Ontology Consortium (GOC) has expanded areas of the ontology such as cilia-related terms, cell-cycle terms and multicellular organism processes. We have also implemented new tools for generating ontology terms based on a set of logical rules making use of templates, and we have made efforts to increase our use of logical definitions. The GOC has a new and improved web site summarizing new developments and documentation, serving as a portal to GO data. Users can perform GO enrichment analysis, and search the GO for terms, annotations to gene products, and associated metadata across multiple species using the all-new AmiGO 2 browser. We encourage and welcome the input of the research community in all biological areas in our continued effort to improve the Gene Ontology. PMID:25428369

  20. Metazoan Gene Families from Metazome

    DOE Data Explorer

    Metazome is a joint project of the Department of Energy's Joint Genome Institute and the Center for Integrative Genomics to facilitate comparative genomic studies amongst metazoans. Clusters of orthologous and paralogous genes that represent the modern descendents of ancestral gene sets are constructed at key phylogenetic nodes. These clusters allow easy access to clade specific orthology/paralogy relationships as well as clade specific genes and gene expansions. As of version 2.0.4, Metazome provides access to twenty-four sequenced and annotated metazoan genomes, clustered at nine evolutionarily significant nodes. Where possible, each gene has been annotated with PFAM, KOG, KEGG, and PANTHER assignments, and publicly available annotations from RefSeq, UniProt, Ensembl, and JGI are hyper-linked and searchable. The included organisms (by common name) are: Human, Mouse, Rat, Dog, Opossum, Chicken, Frog, Stickleback, Medaka, Fugu pufferfish; Zebrafish, Seasquirt - savignyi, Seasquirt - intestinalis, Amphioxus, Sea Urchin, Fruitfly, Mosquite, Yellow Fever Mosquito, Silkworm, Red Flour Beetle, Worm, Briggsae Worm, Owl limpet (snail), and Sea anemone. [Copied from Metazome Overview at http://www.metazome.net/Metazome_info.php

  1. Systems Biophysics of Gene Expression

    PubMed Central

    Vilar, Jose M.G.; Saiz, Leonor

    2013-01-01

    Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365

  2. Control of Renin Gene Expression

    PubMed Central

    Glenn, Sean T.; Jones, Craig A.; Gross, Kenneth W.; Pan, Li

    2015-01-01

    Renin, as part of the renin-angiotensin system, plays a critical role in the regulation of blood pressure, electrolyte homeostasis, mammalian renal development and progression of fibrotic/hypertrophic diseases. Renin gene transcription is subject to complex developmental and tissue-specific regulation. Initial studies using the mouse As4.1 cell line, which has many characteristics of the renin-expressing juxtaglomerular cells of the kidney, have identified a proximal promoter region (−197 to −50 bp) and an enhancer (−2866 to −2625 bp) upstream of the Ren-1c gene, which are critical for renin gene expression. The proximal promoter region contains several transcription factor-binding sites including a binding site for the products of the developmental control genes Hox. The enhancer consists of at least 11 transcription factor-binding sites and is responsive to various signal transduction pathways including cAMP, retinoic acid, endothelin-1, and cytokines, all of which are known to alter renin mRNA levels. Furthermore, in vivo models have validated several of these key components found within the proximal promoter region and the enhancer as well as other key sites necessary for renin gene transcription. PMID:22576577

  3. Combinatorial methods for gene recognition

    SciTech Connect

    Pevzner, P.A.

    1997-10-29

    The major result of the project is the development of a new approach to gene recognition called spliced alignment algorithm. They have developed an algorithm and implemented a software tool (for both IBM PC and UNIX platforms) which explores all possible exon assemblies in polynomial time and finds the multi-exon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully performs exons assemblies even in the case of short exons or exons with unusual codon usage; they also report correct assemblies for the genes with more than 10 exons provided a homologous protein is already known. On a test sample of human genes with known mammalian relatives the average overlap between the predicted and the actual genes was 99%, which is remarkably well as compared to other existing methods. At that, the algorithm absolute correctly reconstructed 87% of genes. The rare discrepancies between the predicted and real axon-intron structures were restricted either to extremely short initial or terminal exons or proved to be results of alternative splicing. Moreover, the algorithm performs reasonably well with non-vertebrate and even prokaryote targets. The spliced alignment software PROCRUSTES has been in extensive use by the academic community since its announcement in August, 1996 via the WWW server (www-hto.usc.edu/software/procrustes) and by biotech companies via the in-house UNIX version.

  4. Gene Body Methylation Patterns in Daphnia Are Associated with Gene Family Size

    PubMed Central

    Asselman, Jana; De Coninck, Dieter I. M.; Pfrender, Michael E.; De Schamphelaere, Karel A. C.

    2016-01-01

    The relation between gene body methylation and gene function remains elusive. Yet, our understanding of this relationship can contribute significant knowledge on how and why organisms target specific gene bodies for methylation. Here, we studied gene body methylation patterns in two Daphnia species. We observed both highly methylated genes and genes devoid of methylation in a background of low global methylation levels. A small but highly significant number of genes was highly methylated in both species. Remarkably, functional analyses indicate that variation in methylation within and between Daphnia species is primarily targeted to small gene families whereas large gene families tend to lack variation. The degree of sequence similarity could not explain the observed pattern. Furthermore, a significant negative correlation between gene family size and the degree of methylation suggests that gene body methylation may help regulate gene family expansion and functional diversification of gene families leading to phenotypic variation. PMID:27017526

  5. Activities of Human Gene Nomenclature Committee

    SciTech Connect

    2002-07-16

    The objective of this project, shared between NIH and DOE, has been and remains to enable the medical genetics communities to use common names for genes that are discovered by different gene hunting groups, in different species. This effort provides consistent gene nomenclature and approved gene symbols to the community at large. This contributes to a uniform and consistent understanding of genomes, particularly the human as well as functional genomics based on comparisons between homologous genes in related species (human and mice).

  6. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1995-01-01

    Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.

  7. Gene-Gene and Gene-Environment Interactions in Ulcerative Colitis

    PubMed Central

    Wang, Ming-Hsi; Fiocchi, Claudio; Zhu, Xiaofeng; Ripke, Stephan; Kamboh, M. Ilyas; Rebert, Nancy; Duerr, Richard H.; Achkar, Jean-Paul

    2014-01-01

    Genome-wide association studies (GWAS) have identified at least 133 ulcerative colitis (UC) associated loci. The role of genetic factors in clinical practice is not clearly defined. The relevance of genetic variants to disease pathogenesis is still uncertain because of not characterized gene-gene and gene-environment interactions. We examined the predictive value of combining the 133 UC risk loci with genetic interactions in an ongoing inflammatory bowel disease (IBD) GWAS. The Wellcome Trust Case-Control Consortium (WTCCC) IBD GWAS was used as a replication cohort. We applied logic regression (LR), a novel adaptive regression methodology, to search for high order interactions. Exploratory genotype correlations with UC sub-phenotypes (extent of disease, need of surgery, age of onset, extra-intestinal manifestations and primary sclerosing cholangitis (PSC)) were conducted. The combination of 133 UC loci yielded good UC risk predictability (area under the curve [AUC] of 0.86). A higher cumulative allele score predicted higher UC risk. Through LR, several lines of evidence for genetic interactions were identified and successfully replicated in the WTCCC cohort. The genetic interactions combined with the gene-smoking interaction significantly improved predictability in the model (AUC, from 0.86 to 0.89, P=3.26E-05). Explained UC variance increased from 37% to 42% after adding the interaction terms. A within case analysis found suggested genetic association with PSC. Our study demonstrates that the LR methodology allows the identification and replication of high order genetic interactions in UC GWAS datasets. UC risk can be predicted by a 133 loci and improved by adding gene-gene and gene-environment interactions. PMID:24241240

  8. [Gene therapy for osteoarticular disorders].

    PubMed

    Gouze, Jean-Noël; Evans, Christopher H; Ghivizzani, Steven C; Gouze, Elvire

    2007-03-01

    Osteoarticular disorders are the major cause of disability in Europe and North America. It is estimated that rheumatoid arthritis affects 1 % of the population and that more than two third of people over age 55 develop osteoarthritis. Because there are no satisfactory treatments, gene therapy offers a new therapeutic approach. The delivery of cDNA encoding anti-arthritic proteins to articular cells has shown therapeutic efficacy in numerous animal models in vivo. Through the development and the experimental progresses that have been made for both rheumatoid arthritis and osteoarthritis, this review discusses the different gene therapy strategies available today and the safety issues with which they may be associated. Among the different vectors available today, adeno-associated virus seems the best candidate for a direct in vivo gene delivery approach for the treatment of joint disorders. PMID:17349293

  9. Nuclear Neighborhoods and Gene Expression

    PubMed Central

    Zhao, Rui; Bodnar, Megan S.; Spector, David L.

    2009-01-01

    Summary The eukaryotic nucleus is a highly compartmentalized and dynamic environment. Chromosome territories are arranged non-randomly within the nucleus and numerous studies have indicated that a gene’s position in the nucleus can impact its transcriptional activity. Here, we focus on recent advances in our understanding of the influence of specific nuclear neighborhoods on gene expression or repression. Nuclear neighborhoods associated with transcriptional repression include the inner nuclear membrane/nuclear lamina and peri-nucleolar chromatin, whereas neighborhoods surrounding the nuclear pore complex, PML nuclear bodies, and nuclear speckles seem to be transcriptionally permissive. While nuclear position appears to play an important role in gene expression, it is likely to be only one piece of a flexible puzzle that incorporates numerous parameters. We are still at a very early, yet exciting stage in our journey toward deciphering the mechanism(s) that govern the permissiveness of gene expression/repression within different nuclear neighborhoods. PMID:19339170

  10. Lateral gene transfer in eukaryotes.

    PubMed

    Andersson, J O

    2005-06-01

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular. PMID:15761667

  11. Search for Basonuclin Target Genes

    PubMed Central

    Wang, Junwen; Zhang, Shengliang; Schultz, Richard M.; Tseng, Hung

    2006-01-01

    Basonuclin (Bnc 1) is a transcription factor that has an unusual ability to interact with promoters of both RNA polymerases I and II. The action of basonuclin is mediated through three pairs of evolutionarily conserved zinc fingers, which produce three DNase I footprints on the promoters of rDNA and the basonuclin gene. Using these DNase footprints, we built a computational model for the basonuclin DNA-binding module, which was used to identify in silico potential RNA polymerase II target genes in the human and mouse promoter databases. The target genes of basonuclin show that it regulates the expression of proteins involved in chromatin structure, transcription/DNA-binding, ion-channels, adhesion/cell-cell junction, signal transduction and intracellular transport. Our results suggest that basonuclin, like MYC, may coordinate transcriptional activities among the three RNA polymerases. But basonuclin regulates a distinctive set of pathways, which differ from that regulated by MYC. PMID:16919236

  12. Gene Therapy in Corneal Transplantation

    PubMed Central

    Qazi, Yureeda; Hamrah, Pedram

    2014-01-01

    Corneal transplantation is the most commonly performed organ transplantation. Immune privilege of the cornea is widely recognized, partly because of the relatively favorable outcome of corneal grafts. The first-time recipient of corneal allografts in an avascular, low-risk setting can expect a 90% success rate without systemic immunosuppressive agents and histocompatibility matching. However, immunologic rejection remains the major cause of graft failure, particularly in patients with a high risk for rejection. Corticosteroids remain the first-line therapy for the prevention and treatment of immune rejection. However, current pharmacological measures are limited in their side-effect profiles, repeated application, lack of targeted response, and short duration of action. Experimental ocular gene therapy may thus present new horizons in immunomodulation. From efficient viral vectors to sustainable alternative splicing, we discuss the progress of gene therapy in promoting graft survival and postulate further avenues for gene-mediated prevention of allogeneic graft rejection. PMID:24138037

  13. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  14. Differential Gene Expression in Glaucoma

    PubMed Central

    Jakobs, Tatjana C.

    2014-01-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell–matrix interactions and adhesion, the cell cycle, and the endothelin system. PMID:24985133

  15. Gene Expression Studies in Mosquitoes

    PubMed Central

    Chen, Xlao-Guang; Mathur, Geetika; James, Anthony A.

    2009-01-01

    Research on gene expression in mosquitoes is motivated by both basic and applied interests. Studies of genes involved in hematophagy, reproduction, olfaction, and immune responses reveal an exquisite confluence of biological adaptations that result in these highly-successful life forms. The requirement of female mosquitoes for a bloodmeal for propagation has been exploited by a wide diversity of viral, protozoan and metazoan pathogens as part of their life cycles. Identifying genes involved in host-seeking, blood feeding and digestion, reproduction, insecticide resistance and susceptibility/refractoriness to pathogen development is expected to provide the bases for the development of novel methods to control mosquito-borne diseases. Advances in mosquito transgenesis technologies, the availability of whole genome sequence information, mass sequencing and analyses of transcriptomes and RNAi techniques will assist development of these tools as well as deepen the understanding of the underlying genetic components for biological phenomena characteristic of these insect species. PMID:19161831

  16. Metagenomics and novel gene discovery

    PubMed Central

    Culligan, Eamonn P; Sleator, Roy D; Marchesi, Julian R; Hill, Colin

    2014-01-01

    Metagenomics provides a means of assessing the total genetic pool of all the microbes in a particular environment, in a culture-independent manner. It has revealed unprecedented diversity in microbial community composition, which is further reflected in the encoded functional diversity of the genomes, a large proportion of which consists of novel genes. Herein, we review both sequence-based and functional metagenomic methods to uncover novel genes and outline some of the associated problems of each type of approach, as well as potential solutions. Furthermore, we discuss the potential for metagenomic biotherapeutic discovery, with a particular focus on the human gut microbiome and finally, we outline how the discovery of novel genes may be used to create bioengineered probiotics. PMID:24317337

  17. Gene transfer for erythropoiesis enhancement.

    PubMed

    Naffakh, N; Danos, O

    1996-08-01

    The spectrum of anemias treated with recombinant human erythropoietin is rapidly broadening. Lifelong treatment with very high doses is now under evaluation for beta-thalassemia and sickle cell anemia. These indications make it worthwhile to search for methods that will allow a permanent systemic delivery of the hormone. Here, we review experimental gene-transfer-based procedures for erythropoietin delivery in vivo. In mice, both ex vivo and direct in vivo approaches for gene transfer have resulted in the long-term production of therapeutic levels of the hormone. Gene transfer of erythropoietin could become a viable alternative to the injection of the purified recombinant protein once reliable procedures for controlling transgene expression are available. PMID:8796920

  18. GeneMANIA prediction server 2013 update.

    PubMed

    Zuberi, Khalid; Franz, Max; Rodriguez, Harold; Montojo, Jason; Lopes, Christian Tannus; Bader, Gary D; Morris, Quaid

    2013-07-01

    GeneMANIA (http://www.genemania.org) is a flexible user-friendly web interface for generating hypotheses about gene function, analyzing gene lists and prioritizing genes for functional assays. Given a query gene list, GeneMANIA extends the list with functionally similar genes that it identifies using available genomics and proteomics data. GeneMANIA also reports weights that indicate the predictive value of each selected data set for the query. GeneMANIA can also be used in a function prediction setting: given a query gene, GeneMANIA finds a small set of genes that are most likely to share function with that gene based on their interactions with it. Enriched Gene Ontology categories among this set can sometimes point to the function of the gene. Seven organisms are currently supported (Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Homo sapiens, Rattus norvegicus and Saccharomyces cerevisiae), and hundreds of data sets have been collected from GEO, BioGRID, IRefIndex and I2D, as well as organism-specific functional genomics data sets. Users can customize their search by selecting specific data sets to query and by uploading their own data sets to analyze. PMID:23794635

  19. Gene expression throughout a vertebrate's embryogenesis

    PubMed Central

    2011-01-01

    Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development. PMID:21356103

  20. Does inbreeding affect gene expression in birds?

    PubMed Central

    Hansson, Bengt; Naurin, Sara; Hasselquist, Dennis

    2014-01-01

    Inbreeding increases homozygosity, exposes genome-wide recessive deleterious alleles and often reduces fitness. The physiological and reproductive consequences of inbreeding may be manifested already during gene regulation, but the degree to which inbreeding influences gene expression is unknown in most organisms, including in birds. To evaluate the pattern of inbreeding-affected gene expression over the genome and in relation to sex, we performed a transcriptome-wide gene expression (10 695 genes) study of brain tissue of 10-day-old inbred and outbred, male and female zebra finches. We found significantly lower gene expression in females compared with males at Z-linked genes, confirming that dosage compensation is incomplete in female birds. However, inbreeding did not affect gene expression at autosomal or sex-linked genes, neither in males nor in females. Analyses of single genes again found a clear sex-biased expression at Z-linked genes, whereas only a single gene was significantly affected by inbreeding. The weak effect of inbreeding on gene expression in zebra finches contrasts to the situation, for example, in Drosophila where inbreeding has been found to influence gene expression more generally and at stress-related genes in particular. PMID:25232028

  1. Gene Therapy for Coagulation Disorders.

    PubMed

    Swystun, Laura L; Lillicrap, David

    2016-04-29

    Molecular genetic details of the human coagulation system were among the first successes of the genetic revolution in the 1980s. This information led to new molecular diagnostic strategies for inherited disorders of hemostasis and the development of recombinant clotting factors for the treatment of the common inherited bleeding disorders. A longer term goal of this knowledge has been the establishment of gene transfer to provide continuing access to missing or defective hemostatic proteins. Because of the relative infrequency of inherited coagulation factor disorders and the availability of safe and effective alternative means of management, the application of gene therapy for these conditions has been slow to realize clinical application. Nevertheless, the tools for effective and safe gene transfer are now much improved, and we have started to see examples of clinical gene therapy successes. Leading the way has been the use of adeno-associated virus-based strategies for factor IX gene transfer in hemophilia B. Several small phase 1/2 clinical studies using this approach have shown prolonged expression of therapeutically beneficial levels of factor IX. Nevertheless, before the application of gene therapy for coagulation disorders becomes widespread, several obstacles need to be overcome. Immunologic responses to the vector and transgenic protein need to be mitigated, and production strategies for clinical grade vectors require enhancements. There is little doubt that with the development of more efficient and facile strategies for genome editing and the application of other nucleic acid-based approaches to influence the coagulation system, the future of genetic therapies for hemostasis is bright. PMID:27126652

  2. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.

    PubMed Central

    van der Krol, A R; Mur, L A; Beld, M; Mol, J N; Stuitje, A R

    1990-01-01

    To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits. PMID:2152117

  3. The yeast ubiquitin genes: a family of natural gene fusions.

    PubMed

    Ozkaynak, E; Finley, D; Solomon, M J; Varshavsky, A

    1987-05-01

    Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress. PMID:3038523

  4. Identification of genes and gene clusters involved in mycotoxin synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Research methods to identify and characterize genes involved in mycotoxin biosynthetic pathways have evolved considerably over the years. Before whole genome sequences were available (e.g. pre-genomics), work focused primarily on chemistry, biosynthetic mutant strains and molecular analysis of sing...

  5. Horizontal gene transfer of stress resistance genes through plasmid transport.

    PubMed

    Shoeb, Erum; Badar, Uzma; Akhter, Jameela; Shams, Hina; Sultana, Maria; Ansari, Maqsood A

    2012-03-01

    The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature. PMID:22805823

  6. The KCNE genes in hypertrophic cardiomyopathy: a candidate gene study

    PubMed Central

    2011-01-01

    Background The gene family KCNE1-5, which encode modulating β-subunits of several repolarising K+-ion channels, has been associated with genetic cardiac diseases such as long QT syndrome, atrial fibrillation and Brugada syndrome. The minK peptide, encoded by KCNE1, is attached to the Z-disc of the sarcomere as well as the T-tubules of the sarcolemma. It has been suggested that minK forms part of an "electro-mechanical feed-back" which links cardiomyocyte stretching to changes in ion channel function. We examined whether mutations in KCNE genes were associated with hypertrophic cardiomyopathy (HCM), a genetic disease associated with an improper hypertrophic response. Results The coding regions of KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5 were examined, by direct DNA sequencing, in a cohort of 93 unrelated HCM probands and 188 blood donor controls. Fifteen genetic variants, four previously unknown, were identified in the HCM probands. Eight variants were non-synonymous and one was located in the 3'UTR-region of KCNE4. No disease-causing mutations were found and no significant difference in the frequency of genetic variants was found between HCM probands and controls. Two variants of likely functional significance were found in controls only. Conclusions Mutations in KCNE genes are not a common cause of HCM and polymorphisms in these genes do not seem to be associated with a propensity to develop arrhythmia PMID:21967835

  7. Lipid Nanoparticles for Gene Delivery

    PubMed Central

    Zhao, Yi; Huang, Leaf

    2016-01-01

    Nonviral vectors which offer a safer and versatile alternative to viral vectors have been developed to overcome problems caused by viral carriers. However, their transfection efficacy or level of expression is substantially lower than viral vectors. Among various nonviral gene vectors, lipid nanoparticles are an ideal platform for the incorporation of safety and efficacy into a single delivery system. In this chapter, we highlight current lipidic vectors that have been developed for gene therapy of tumors and other diseases. The pharmacokinetic, toxic behaviors and clinic trials of some successful lipids particles are also presented. PMID:25409602

  8. GENE PROFILING: IMPLICATIONS IN DERMATOLOGY

    PubMed Central

    Blumenberg, Miroslav; Tomic-Canic, Marjana

    2016-01-01

    Summary DNA microarrays are capable of following the level of expression of, virtually, all genes in a human tissue. This has been employed to determine the aberrant gene expression profiles in many skin diseases, including ultraviolet light damage, inflammatory processes and cancers. Because of its accessibility, skin also served as one of the initial targets of basic research using DNA microarrays. Both the epidermis and dermis have been extensively investigated. Development of bed-side uses of DNA arrays, and the concomitant price reduction of the materials and methods of microarray analyses, holds great promise for improved diagnosis, treatment and prevention of dermatologic disorders.

  9. Cytoskeletal genes regulating brain size.

    PubMed

    Bond, Jacquelyn; Woods, C Geoffrey

    2006-02-01

    One of the most notable trends in human evolution is the dramatic increase in brain size that has occurred in the great ape clade, culminating in humans. Of particular interest is the vast expanse of the cerebral cortex, which is believed to have resulted in our ability to perform higher cognitive functions. Recent investigations of congenital microcephaly in humans have resulted in the identification of several genes that non-redundantly and specifically influence mammalian brain size. These genes appear to affect neural progenitor cell number through microtubular organisation at the centrosome. PMID:16337370

  10. Persistence drives gene clustering in bacterial genomes

    PubMed Central

    Fang, Gang; Rocha, Eduardo PC; Danchin, Antoine

    2008-01-01

    Background Gene clustering plays an important role in the organization of the bacterial chromosome and several mechanisms have been proposed to explain its extent. However, the controversies raised about the validity of each of these mechanisms remind us that the cause of this gene organization remains an open question. Models proposed to explain clustering did not take into account the function of the gene products nor the likely presence or absence of a given gene in a genome. However, genomes harbor two very different categories of genes: those genes present in a majority of organisms – persistent genes – and those present in very few organisms – rare genes. Results We show that two classes of genes are significantly clustered in bacterial genomes: the highly persistent and the rare genes. The clustering of rare genes is readily explained by the selfish operon theory. Yet, genes persistently present in bacterial genomes are also clustered and we try to understand why. We propose a model accounting specifically for such clustering, and show that indispensability in a genome with frequent gene deletion and insertion leads to the transient clustering of these genes. The model describes how clusters are created via the gene flux that continuously introduces new genes while deleting others. We then test if known selective processes, such as co-transcription, physical interaction or functional neighborhood, account for the stabilization of these clusters. Conclusion We show that the strong selective pressure acting on the function of persistent genes, in a permanent state of flux of genes in bacterial genomes, maintaining their size fairly constant, that drives persistent genes clustering. A further selective stabilization process might contribute to maintaining the clustering. PMID:18179692

  11. Gene therapy on demand: site specific regulation of gene therapy.

    PubMed

    Jazwa, Agnieszka; Florczyk, Urszula; Jozkowicz, Alicja; Dulak, Jozef

    2013-08-10

    Since 1990 when the first clinical gene therapy trial was conducted, much attention and considerable promise have been given to this form of treatment. Gene therapy has been used with success in patients suffering from severe combined immunodeficiency syndromes (X-SCID and ADA-deficiency), Leber's congenital amaurosis, hemophilia, β-thalassemia and adrenoleukodystrophy. Last year, the first therapeutic vector (Glybera) for treatment of lipoprotein lipase deficiency has been registered in the European Union. Nevertheless, there are still several numerous issues that need to be improved to make this technique more safe, effective and easily accessible for patients. Introduction of the therapeutic gene to the given cells should provide the level of expression which will restore the production of therapeutic protein to normal values or will provide therapeutic efficacy despite not fully physiological expression. However, in numerous diseases the expression of therapeutic genes has to be kept at certain level for some time, and then might be required to be switched off to be activated again when worsening of the symptoms may aggravate the risk of disease relapse. In such cases the promoters which are regulated by local conditions may be more required. In this article the special emphasis is to discuss the strategies of regulation of gene expression by endogenous stimuli. Particularly, the hypoxia- or miRNA-regulated vectors offer the possibilities of tight but, at the same time, condition-dependent and cell-specific expression. Such means have been already tested in certain pathophysiological conditions. This creates the chance for the translational approaches required for development of effective treatments of so far incurable diseases. PMID:23566848

  12. Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism

    PubMed Central

    Ma, D. Q.; Whitehead, P. L.; Menold, M. M.; Martin, E. R.; Ashley-Koch, A. E.; Mei, H.; Ritchie, M. D.; DeLong, G. R.; Abramson, R. K.; Wright, H. H.; Cuccaro, M. L.; Hussman, J. P.; Gilbert, J. R.; Pericak-Vance, M. A.

    2005-01-01

    Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis—with the pedigree disequilibrium test and the family-based association test—and for genotypic and haplotypic association analysis—with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a

  13. Evolution of Gene Duplication in Plants.

    PubMed

    Panchy, Nicholas; Lehti-Shiu, Melissa; Shiu, Shin-Han

    2016-08-01

    Ancient duplication events and a high rate of retention of extant pairs of duplicate genes have contributed to an abundance of duplicate genes in plant genomes. These duplicates have contributed to the evolution of novel functions, such as the production of floral structures, induction of disease resistance, and adaptation to stress. Additionally, recent whole-genome duplications that have occurred in the lineages of several domesticated crop species, including wheat (Triticum aestivum), cotton (Gossypium hirsutum), and soybean (Glycine max), have contributed to important agronomic traits, such as grain quality, fruit shape, and flowering time. Therefore, understanding the mechanisms and impacts of gene duplication will be important to future studies of plants in general and of agronomically important crops in particular. In this review, we survey the current knowledge about gene duplication, including gene duplication mechanisms, the potential fates of duplicate genes, models explaining duplicate gene retention, the properties that distinguish duplicate from singleton genes, and the evolutionary impact of gene duplication. PMID:27288366

  14. Genome Majority Vote Improves Gene Predictions

    PubMed Central

    Wall, Michael E.; Raghavan, Sindhu; Cohn, Judith D.; Dunbar, John

    2011-01-01

    Recent studies have noted extensive inconsistencies in gene start sites among orthologous genes in related microbial genomes. Here we provide the first documented evidence that imposing gene start consistency improves the accuracy of gene start-site prediction. We applied an algorithm using a genome majority vote (GMV) scheme to increase the consistency of gene starts among orthologs. We used a set of validated Escherichia coli genes as a standard to quantify accuracy. Results showed that the GMV algorithm can correct hundreds of gene prediction errors in sets of five or ten genomes while introducing few errors. Using a conservative calculation, we project that GMV would resolve many inconsistencies and errors in publicly available microbial gene maps. Our simple and logical solution provides a notable advance toward accurate gene maps. PMID:22131910

  15. Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli

    PubMed Central

    Eppler, Tanja; Postma, Pieter; Schütz, Alexandra; Völker, Uwe; Boos, Winfried

    2002-01-01

    The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as d-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and d-tagatose-1,6-bisphosphate aldolase. PMID:12003946

  16. Genes and Syndromic Hearing Loss.

    ERIC Educational Resources Information Center

    Keats, Bronya J. B.

    2002-01-01

    This article provides a description of the human genome and patterns of inheritance and discusses genes that are associated with some of the syndromes for which hearing loss is a common finding, including: Waardenburg, Stickler, Jervell and Lange-Neilsen, Usher, Alport, mitochondrial encephalomyopathy, and sensorineural hearing loss. (Contains…

  17. Circadian gene variants in cancer

    PubMed Central

    Kettner, Nicole M.; Katchy, Chinenye A.; Fu, Loning

    2014-01-01

    Humans as diurnal beings are active during the day and rest at night. This daily oscillation of behavior and physiology is driven by an endogenous circadian clock not environmental cues. In modern societies, changes in lifestyle have led to a frequent disruption of the endogenous circadian homeostasis leading to increased risk of various diseases including cancer. The clock is operated by the feedback loops of circadian genes and controls daily physiology by coupling cell proliferation and metabolism, DNA damage repair, and apoptosis in peripheral tissues with physical activity, energy homeostasis, immune and neuroendocrine functions at the organismal level. Recent studies have revealed that defects in circadian genes due to targeted gene ablation in animal models or single nucleotide polymorphism, deletion, deregulation and/or epigenetic silencing in humans are closely associated with increased risk of cancer. In addition, disruption of circadian rhythm can disrupt the molecular clock in peripheral tissues in the absence of circadian gene mutations. Circadian disruption has recently been recognized as an independent cancer risk factor. Further study of the mechanism of clock-controlled tumor suppression will have a significant impact on human health by improving the efficiencies of cancer prevention and treatment. PMID:24901356

  18. Gene-Culture Coevolutionary Games

    ERIC Educational Resources Information Center

    Blute, Marion

    2006-01-01

    Gene-culture interactions have largely been modelled employing population genetic-type models. Moreover, in the most notable application to date, the "interactive" modes have been one way rather than bidirectional. This paper suggests using game theoretic, fully interactive models. Employing the logic utilized in population ecology for coevolution…

  19. Genes, Environment, and Human Behavior.

    ERIC Educational Resources Information Center

    Bloom, Mark V.; Cutter, Mary Ann; Davidson, Ronald; Dougherty, Michael J.; Drexler, Edward; Gelernter, Joel; McCullough, Laurence B.; McInerney, Joseph D.; Murray, Jeffrey C.; Vogler, George P.; Zola, John

    This curriculum module explores genes, environment, and human behavior. This book provides materials to teach about the nature and methods of studying human behavior, raise some of the ethical and public policy dilemmas emerging from the Human Genome Project, and provide professional development for teachers. An extensive Teacher Background…

  20. Gene therapy for bone healing

    PubMed Central

    Evans, Christopher H.

    2015-01-01

    Clinical problems in bone healing include large segmental defects, nonunion and delayed union of fractures, and spinal fusions. Gene-transfer technologies have the potential to aid healing by permitting the local delivery and sustained expression of osteogenic gene products within osseous lesions. Key questions for such an approach include the choice of transgene, vector and gene-transfer strategy. Most experimental data have been obtained using cDNAs encoding osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), BMP-4 and BMP-7, in conjunction with both nonviral and viral vectors using in vivo and ex vivo delivery strategies. Proof of principle has been convincingly demonstrated in small-animal models. Relatively few studies have used large animals, but the results so far are encouraging. Once a reliable method has been developed, it will be necessary to perform detailed pharmacological and toxicological studies, as well as satisfy other demands of the regulatory bodies, before human clinical trials can be initiated. Such studies are very expensive and often protracted. Thus, progress in developing a clinically useful gene therapy for bone healing is determined not only by scientific considerations, but also by financial constraints and the ambient regulatory environment. PMID:20569532