Science.gov

Sample records for acidic protein gene

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

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

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

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

    PubMed Central

    Pederson, Thoru

    1974-01-01

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

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    2013-05-01

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

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

    PubMed Central

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

    2013-01-01

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

  8. Interactions Between Fatty Acid Transport Proteins, Genes That Encode for Them, and Exercise: A Systematic Review.

    PubMed

    Jayewardene, Avindra F; Mavros, Yorgi; Reeves, Anneliese; Hancock, Dale P; Gwinn, Tom; Rooney, Kieron B

    2016-08-01

    Long-chain fatty acid (LCFA) movement into skeletal muscle involves a highly mediated process in which lipid rafts are utilized in the cellular membrane, involving numerous putative plasma membrane-associated LCFA transport proteins. The process of LCFA uptake and oxidation is of particular metabolic significance both at rest and during light to moderate exercise. A comprehensive systematic search of electronic databases was conducted to investigate whether exercise alters protein and/or gene expression of putative LCFA transport proteins. There were 31 studies meeting all eligibility criteria, of these 13 utilized an acute exercise protocol and 18 examined chronic exercise adaptations. Seventeen involved a study design incorporating an exercise stimulus, while the remaining 14 incorporated a combined exercise and diet stimulus. Divergent data relating to acute exercise, as well as prolonged exercise training (≥3 weeks), on protein content (PC) response was identified for proteins CD36, FABPpm and CAV1. Messenger ribonucleic acid (mRNA) data did not always correspond to functional PC, supporting previous suggestions of a disconnect due to potentially limiting factors post gene expression. The large array of study designs, cohorts, and primary dependent variables within the studies included in the present review elucidate the complexity of the interaction between exercise and LCFA transport proteins. Summary of the results in the present review validate the need for further targeted investigation within this topic, and provide an important information base for such research. J. Cell. Physiol. 231: 1671-1687, 2016. © 2015 Wiley Periodicals, Inc. PMID:26638980

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

    SciTech Connect

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

    1990-06-01

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

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed

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

    2015-01-01

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

  12. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

    PubMed

    Moon, Yunwon; Choi, Su Mi; Chang, Soojeong; Park, Bongju; Lee, Seongyeol; Lee, Mi-Ock; Choi, Hueng-Sik; Park, Hyunsung

    2015-01-01

    This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2) and severe hypoxia (0.1% O2). We found that chenodeoxy cholic acid (CDCA) reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR), a CDCA receptor and its target gene, Small heterodimer partner (SHP) are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein. PMID:26098428

  13. The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

    PubMed Central

    Pédrono, Frédérique; Blanchard, Hélène; Kloareg, Maela; D'andréa, Sabine; Daval, Stéphanie; Rioux, Vincent; Legrand, Philippe

    2010-01-01

    In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176–183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Δ5- and Δ6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3. PMID:19752397

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences. PMID:18397498

  16. Intracellular calcium-release and protein kinase C-activation stimulate sonic hedgehog gene expression during gastric acid secretion

    PubMed Central

    El-Zaatari, Mohamad; Zavros, Yana; Tessier, Art; Waghray, Meghna; Lentz, Steve; Gumucio, Deborah; Todisco, Andrea; Merchant, Juanita L.

    2010-01-01

    Introduction Hypochlorhydria during Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2+i-dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)-activation. Method We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1 (sHip-1), a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, EGTA+BAPTA, PKC-overexpressing adenoviruses, and PKC-inhibitors were used to modulate Ca2+i-release, PKC-activity and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H+/K+-β-cholera-toxin overexpressing mice (Ctox). Results Mice that expressed sHip-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression was also repressed in the hyperchlorhydric Ctox model with elevated cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca2+i-release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin- and carbachol-mediated release of Ca2+i induced Shh expression. Ca2+-chelation with BAPTA+EGTA reduced Shh expression. Overexpression of PKC-α, -β and -δ (but not PKC-ε) induced Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Conclusion Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca2+i-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin. PMID:20816837

  17. Genome-Wide Methylation and Gene Expression Changes in Newborn Rats following Maternal Protein Restriction and Reversal by Folic Acid

    PubMed Central

    Stupka, Elia; Clark, Adrian J. L.; Langley-Evans, Simon

    2013-01-01

    A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures. PMID:24391732

  18. Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores.

    PubMed Central

    Connors, M J; Mason, J M; Setlow, P

    1986-01-01

    Three Bacillus subtilis genes (termed sspA, sspB, and sspD) which code for small, acid-soluble spore proteins (SASPs) have been cloned, and their complete nucleotide sequence has been determined. The amino acid sequences of the SASPs coded for by these genes are similar to each other and to those of the SASP-1 of B. subtilis (coded for by the sspC gene) and the SASP-A/C family of B. megaterium. The sspA and sspB genes are expressed only in sporulation, in parallel with each other and with the sspC gene. Two regions upstream of the postulated transcription start sites for the sspA and B genes have significant homology with the analogous regions of the sspC gene and the SASP-A/C gene family. Purification of two of the three major B, subtilis SASPs (alpha and beta) and determination of their amino-terminal sequences indicated that the sspA gene codes for SASP-alpha and that the sspB gene codes for SASP-beta. This was confirmed by the introduction of deletion mutations into the cloned sspA and sspB genes and transfer of these deletions into the B. subtilis chromosome with concomitant loss of the wild-type gene. Images PMID:3009398

  19. Okadaic acid mimics multiple changes in early protein phosphorylation and gene expression induced by tumor necrosis factor or interleukin-1.

    PubMed

    Guy, G R; Cao, X; Chua, S P; Tan, Y H

    1992-01-25

    Okadaic acid, a phosphatase inhibitor from a marine organism, mimics tumor necrosis factor/interleukin-1 (TNF/IL-1) in inducing changes in early cellular protein phosphorylation. A total of approximately 116 proteins exhibit significant and concordant changes in phosphorylation or dephosphorylation within 15 min in human fibroblasts activated by either okadaic acid, TNF, or IL-1. The fidelity of this mimicry by okadaic acid extends to the phosphorylation of the 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, epidermal growth factor receptor, and other cdc2-kinase substrates (c-abl, RB, and p53). The okadaic acid-induced pattern of protein phosphorylation is distinct from that observed in cells treated with phorbol 12-myristate 13-acetate or with ligands like epidermal growth factor, cyclic AMP agonists, bradykinin, or interferons. Like TNF, okadaic acid also induces the transcription of immediate early response genes like c-jun and Egr-1 as well as the interleukin-6 genes. The overall early effects of okadaic acid uniquely parallel those of TNF/IL-1 and not those of other cytokines or ligands. Regulation of protein phosphatase inhibition is discussed as a mechanism for TNF/IL-1 signal transduction. PMID:1370482

  20. Fatty acid-binding protein (fabp) genes of spotted green pufferfish (Tetraodon nigroviridis): comparative genomics and spatial transcriptional regulation.

    PubMed

    Thirumaran, Aruloli; Wright, Jonathan M

    2014-05-01

    The fatty acid-binding protein (fabp) genes belong to the multigene family of intracellular lipid-binding proteins. To date, 12 different FABPs have been identified in vertebrate genomes. Owing to the teleost-specific genome duplication event, many fishes have duplicated copies of the fabp genes. Here, we identified and characterized the fabp genes of spotted green pufferfish (Tetraodon nigroviridis). Seven fabp genes were identified, out of which, two were retained in the pufferfish genome as duplicated copies. Each putative pufferfish Fabp protein shares greatest sequence identity and similarity with their teleost and tetrapod orthologs, and clustered together as a distinct clade in phylogenetic analysis. Conserved gene synteny was evident between the pufferfish fabp genes and the orthologs of human, zebrafish, three-spined stickleback, and medaka FABP/fabp genes, providing evidence that the duplicated copies of pufferfish fabp genes most likely arose as a result of the teleost-specific genome duplication event. The differential tissue-specific distribution of pufferfish fabp transcripts suggests divergent spatial regulation of duplicated pairs of fabp genes. PMID:25153522

  1. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  2. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    PubMed

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  3. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

    PubMed Central

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-01-01

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the “lipolysome.” Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

  4. Liver fatty acid binding protein gene ablation potentiates hepatic cholesterol accumulation in cholesterol-fed female mice.

    PubMed

    Martin, Gregory G; Atshaves, Barbara P; McIntosh, Avery L; Mackie, John T; Kier, Ann B; Schroeder, Friedhelm

    2006-01-01

    Although liver fatty acid binding protein (L-FABP) is postulated to influence cholesterol homeostasis, the physiological significance of this hypothesis remains to be resolved. This issue was addressed by examining the response of young (7 wk) female mice to L-FABP gene ablation and a cholesterol-rich diet. In control-fed mice, L-FABP gene ablation alone induced hepatic cholesterol accumulation (2.6-fold), increased bile acid levels, and increased body weight gain (primarily as fat tissue mass). In cholesterol-fed mice, L-FABP gene ablation further enhanced the hepatic accumulation of cholesterol (especially cholesterol ester, 12-fold) and potentiated the effects of dietary cholesterol on increased body weight gain, again mainly as fat tissue mass. However, in contrast to the effects of L-FABP gene ablation in control-fed mice, biliary levels of bile acids (as well as cholesterol and phospholipids) were reduced. These phenotypic alterations were not associated with differences in food intake. In conclusion, it was shown for the first time that L-FABP altered cholesterol metabolism and the response of female mice to dietary cholesterol. While the biliary and lipid phenotype of female wild-type L-FABP+/+ mice was sensitive to dietary cholesterol, L-FABP gene ablation dramatically enhanced many of the effects of dietary cholesterol to greatly induce hepatic cholesterol (primarily cholesterol ester) and triacylglycerol accumulation as well as to potentiate body weight gain (primarily as fat tissue mass). Taken together, these data support the hypothesis that L-FABP is involved in the physiological regulation of cholesterol metabolism, body weight gain, and obesity. PMID:16123197

  5. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  6. The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

    SciTech Connect

    Klapper, Maja . E-mail: klapper@molnut.uni-kiel.de; Boehme, Mike; Nitz, Inke; Doering, Frank

    2007-04-27

    The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

  7. Functional roles of the pepper RING finger protein gene, CaRING1, in abscisic acid signaling and dehydration tolerance.

    PubMed

    Lim, Chae Woo; Hwang, Byung Kook; Lee, Sung Chul

    2015-09-01

    Plants are constantly exposed to a variety of biotic and abiotic stresses, which include pathogens and conditions of high salinity, low temperature, and drought. Abscisic acid (ABA) is a major plant hormone involved in signal transduction pathways that mediate the defense response of plants to abiotic stress. Previously, we isolated Ring finger protein gene (CaRING1) from pepper (Capsicum annuum), which is associated with resistance to bacterial pathogens, accompanied by hypersensitive cell death. Here, we report a new function of the CaRING1 gene product in the ABA-mediated defense responses of plants to dehydration stress. The expression of the CaRING1 gene was induced in pepper leaves treated with ABA or exposed to dehydration or NaCl. Virus-induced gene silencing of CaRING1 in pepper plants exhibited low degree of ABA-induced stomatal closure and high levels of transpirational water loss in dehydrated leaves. These led to be more vulnerable to dehydration stress in CaRING1-silenced pepper than in the control pepper, accompanied by reduction of ABA-regulated gene expression and low accumulation of ABA and H2O2. In contrast, CaRING1-overexpressing transgenic plants showed enhanced sensitivity to ABA during the seedling growth and establishment. These plants were also more tolerant to dehydration stress than the wild-type plants because of high ABA accumulation, enhanced stomatal closure and increased expression of stress-responsive genes. Together, these results suggest that the CaRING1 acts as positive factor for dehydration tolerance in Arabidopsis by modulating ABA biosynthesis and ABA-mediated stomatal closing and gene expression. PMID:26249046

  8. Liver Fatty Acid Binding Protein Gene-ablation Exacerbates Weight Gain in High-Fat Fed Female Mice

    PubMed Central

    McIntosh, Avery L.; Atshaves, Barbara P.; Landrock, Danilo; Landrock, Kerstin K.; Martin, Gregory G.; Storey, Stephen M.; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Loss of liver fatty acid binding protein (L-FABP) decreases long chain fatty acid uptake and oxidation in primary hepatocytes and in vivo. On this basis, L-FABP gene ablation would potentiate high-fat diet-induced weight gain and weight gain/energy intake. While this was indeed the case when L-FABP null (−/−) mice on the C57BL/6NCr background were pair-fed high fat diet, whether this would also be observed under high-fat diet fed ad libitum was not known. Therefore, this possibility was examined in female L-FABP (−/−) mice on the same background. L-FABP (−/−) mice consumed equal amounts of defined high-fat or isocaloric control diets fed ad libitum. However, on the ad libitum fed high-fat diet the L-FABP (−/−) mice exhibited: 1) Decreased hepatic long chain fatty acid (LCFA) β-oxidation as indicated by lower serum β–hydroxybutyrate level; 2) Decreased hepatic protein levels of key enzymes mitochondrial (rate limiting carnitine palmitoyl acyltransferase A1, CPT1A; HMG-CoA synthase) and peroxisomal (acyl CoA oxidase 1, ACOX1) LCFA β-oxidation; 3) Increased fat tissue mass (FTM) and FTM/energy intake to the greatest extent; and 4) Exacerbated body weight gain, weight gain/energy intake, liver weight, and liver weight/body weight to the greatest extent. Taken together, these findings showed that L-FABP gene-ablation exacerbated diet-induced weight gain and fat tissue mass gain in mice fed high-fat diet ad libitum—consistent with the known biochemistry and cell biology of L-FABP. PMID:23539345

  9. Proteins and Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  10. T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells.

    PubMed

    Boskovic, Goran; Niles, Richard M

    2004-05-01

    Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes. PMID:15093729

  11. Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

  12. Effects of abscisic acid and high osmoticum on storage protein gene expression in microspore embryos of Brassica napus

    SciTech Connect

    Wilen, R.W.; Mandel, R.M.; Pharis, R.P.; Moloney, M.M. ); Holbrook, L.A. )

    1990-11-01

    Storage protein gene expression, characteristic of mid- to late embryogenesis, was investigated in microspore embryos of rapeseed (Brassica napus). These embryos, derived from the immature male gametophyte, accumulate little or no detectable napin or cruciferin mRNA when cultured on hormone-free medium containing 13% sucrose. The addition of abscisic acid (ABA) to the medium results in an increase in detectable transcripts encoding both these polypeptides. Storage protein mRNA is induced at 1 micromolar ABA with maximum stimulation occurring between 5 and 50 micromolar. This hormone induction results in a level of storage protein mRNA that is comparable to that observed in zygotic embryos of an equivalent morphological stage. Effects similar to that of ABA are noted when 12.5% sorbitol is added to the microspore embryo medium (osmotic potential = 25.5 bars). Time course experiments, to study the induction of napin and cruciferin gene expression demonstrated that the ABA effect occurred much more rapidly than the high osmoticum effect, although after 48 hours, the levels of napin or cruciferin mRNA detected were similar in both treatments. This difference in the rates of induction is consistent with the idea that the osmotic effect may be mediated by ABA which is synthesized in response to the reduced water potential. Measurements of ABA (by gas chromatography-mass spectrometry using ({sup 2}H{sub 6})ABA as an internal standard) present in microspore embryos during sorbitol treatment and in embryos treated with 10 micromolar ABA were performed to investigate this possibility. Within 2 hours of culture on high osmoticum the level of ABA increased substantially and significantly above control and reached a maximum concentration within 24 hours. This elevated concentration was maintained for 48 hours after culturing and represents a sixfold increase over control embryos.

  13. The cancer-promoting gene fatty acid-binding protein 5 (FABP5) is epigenetically regulated during human prostate carcinogenesis.

    PubMed

    Kawaguchi, Koichiro; Kinameri, Ayumi; Suzuki, Shunsuke; Senga, Shogo; Ke, Youqiang; Fujii, Hiroshi

    2016-02-15

    FABPs (fatty-acid-binding proteins) are a family of low-molecular-mass intracellular lipid-binding proteins consisting of ten isoforms. FABPs are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting these ligands to the appropriate compartments in the cell. FABP5 is overexpressed in multiple types of tumours. Furthermore, up-regulation of FABP5 is strongly associated with poor survival in triple-negative breast cancer. However, the mechanisms underlying the specific up-regulation of the FABP5 gene in these cancers remain poorly characterized. In the present study, we determined that FABP5 has a typical CpG island around its promoter region. The DNA methylation status of the CpG island in the FABP5 promoter of benign prostate cells (PNT2), prostate cancer cells (PC-3, DU-145, 22Rv1 and LNCaP) and human normal or tumour tissue was assessed by bisulfite sequencing analysis, and then confirmed by COBRA (combined bisulfite restriction analysis) and qAMP (quantitative analysis of DNA methylation using real-time PCR). These results demonstrated that overexpression of FABP5 in prostate cancer cells can be attributed to hypomethylation of the CpG island in its promoter region, along with up-regulation of the direct trans-acting factors Sp1 (specificity protein 1) and c-Myc. Together, these mechanisms result in the transcriptional activation of FABP5 expression during human prostate carcinogenesis. Importantly, silencing of Sp1, c-Myc or FABP5 expression led to a significant decrease in cell proliferation, indicating that up-regulation of FABP5 expression by Sp1 and c-Myc is critical for the proliferation of prostate cancer cells. PMID:26614767

  14. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  15. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles.

    PubMed

    Yan, Jingjing; Du, Yong-Zhong; Chen, Feng-Ying; You, Jian; Yuan, Hong; Hu, Fu-Qiang

    2013-07-01

    A stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection. PMID:23679858

  16. Species specific identification of spore-producing microbes using the gene sequence of small acid-soluble spore coat proteins for amplification based diagnostics

    DOEpatents

    McKinney, Nancy

    2002-01-01

    PCR (polymerase chain reaction) primers for the detection of certain Bacillus species, such as Bacillus anthracis. The primers specifically amplify only DNA found in the target species and can distinguish closely related species. Species-specific PCR primers for Bacillus anthracis, Bacillus globigii and Clostridium perfringens are disclosed. The primers are directed to unique sequences within sasp (small acid soluble protein) genes.

  17. Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer

    PubMed Central

    Avondo, Federica; Roncaglia, Paola; Crescenzio, Nicoletta; Krmac, Helena; Garelli, Emanuela; Armiraglio, Marta; Castagnoli, Carlotta; Campagnoli, Maria Francesca; Ramenghi, Ugo; Gustincich, Stefano; Santoro, Claudio; Dianzani, Irma

    2009-01-01

    Background Diamond-Blackfan anaemia (DBA) is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. Patients have an increased risk of solid tumors. Mutations have been found in several ribosomal protein (RP) genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A. Studies in haematopoietic progenitors from patients show that haplo-insufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes show reduced protein synthesis and fibroblasts display abnormal rRNA processing and impaired proliferation. Results To evaluate the involvement of non-haematopoietic tissues in DBA, we have analysed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are differentially expressed in DBA patient fibroblasts. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. Conclusion This analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in DBA. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis. PMID:19765279

  18. Soy protein diet alters expression of hepatic genes regulating fatty acid and thyroid hormone metabolism in the male rat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined effects of soy protein (SPI) and the isoflavone genistein (GEN) on mRNA expression of key lipid metabolism and thyroid hormone system genes in young adult, male Sprague-Dawley rats. SPI-fed rats had less retroperitoneal fat and less hepato-steatosis than casein (CAS, control protein)-...

  19. Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

    PubMed Central

    Hong, S W; Jon, J H; Kwak, J M; Nam, H G

    1997-01-01

    A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

  20. A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

    NASA Technical Reports Server (NTRS)

    Lu, C.; Fedoroff, N.

    2000-01-01

    Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

  1. Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

    PubMed

    Kitsy, Abderrazak; Carney, Skyla; Vivar, Juan C; Knight, Megan S; Pointer, Mildred A; Gwathmey, Judith K; Ghosh, Sujoy

    2014-01-01

    The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of

  2. Levels of mRNAs which code for small, acid-soluble spore proteins and their LacZ gene fusions in sporulating cells of Bacillus subtilis.

    PubMed Central

    Mason, J M; Fajardo-Cavazos, P; Setlow, P

    1988-01-01

    The levels of mRNAs from genes (sspA, B and E) which code for major small, acid-soluble, spore proteins of Bacillus subtilis have been determined, as well as the levels of mRNAs from ssp-lacZ gene fusions. Increasing the gene dosage of ssp-lacZ fusions resulted in parallel increases in both the ssp-lacZ mRNA level and the rate of b-galactosidase accumulation. Similarly, an 11-fold increase in sspE gene dosage gave a comparable increase in sspE mRNA, but at most a 1.5-fold increase in the amount of sspE gene product accumulated. In contrast, an 11-fold increase in the dosage of the sspA or B genes had no significant effect on the level of total sspA plus sspB mRNA, but did alter the ratios of these mRNAs as well as the amount of their gene products, to reflect the altered ratio of the two genes. These results suggest that intact ssp genes, but not ssp-lacZ gene fusions, are subject to feedback regulation of gene expression, with this regulation of the sspA and B genes effected by modulation of mRNA levels, while the feedback regulation of the sspE gene is at the post-transcriptional level. Images PMID:2456528

  3. Amino acid regulation of gene expression.

    PubMed Central

    Fafournoux, P; Bruhat, A; Jousse, C

    2000-01-01

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

  4. A novel polymorphism in the chicken adipocyte fatty acid-binding protein gene (FABP4) that alters ligand-binding and correlates with fatness.

    PubMed

    Wang, Qigui; Guan, Tianzhu; Li, Hui; Bernlohr, David A

    2009-11-01

    Similar to the mammalian FABP4 gene, the chicken (Gallus gallus) FABP4 gene consists of four exons separated by three introns and encodes a 132 amino acid protein termed the adipocyte fatty acid-binding protein (AFABP). In the current study, a novel G/A polymorphism in exon 3 of the chicken FABP4 gene was identified associated with different chicken breeds that leads to either Ser or Asn at amino acid 89 of the AFABP protein. The Baier chicken averages 0.89+/-0.12% abdominal fat and expresses the G allele (Ser 89 isoform) while the Broiler chicken typically has 3.74+/-0.23% abdominal fat and expresses the A allele (Asn 89 isoforms). cDNAs corresponding to the two AFABP isoforms were cloned and expressed in Escherichia coli as GST fusions, purified by using glutathione sepharose 4B chromatography and evaluated for lipid binding using the fluorescent surrogate ligand 1-anilinonaphthalene 8-sulphonic acid (1,8-ANS). The results showed that AFABP Ser89 exhibited a lower ligand-binding affinity with apparent dissociation constants (Kd) of 7.31+/-3.75 microM, while the AFABP Asn89 isoform bound 1,8-ANS with an apparent dissociation constant of 2.99+/-1.00 microM (P=0.02). These results suggest that the Ser89Asn polymorphism may influence chicken AFABP function and ultimately lipid deposition through changing the ligand-binding activity of AFABP. PMID:19595785

  5. The cpc-2 gene of Neurospora crassa encodes a protein entirely composed of WD-repeat segments that is involved in general amino acid control and female fertility.

    PubMed

    Müller, F; Krüger, D; Sattlegger, E; Hoffmann, B; Ballario, P; Kanaan, M; Barthelmess, I B

    1995-07-28

    Phenotypic and molecular studies of the mutation U142 indicate that the cpc-2+ gene is required to activate general amino acid control under conditions of amino acid limitation in the vegetative growth phase, and for formation of protoperithecia in preparation for the sexual phase of the life cycle of Neurospora crassa. The cpc-2 gene was cloned by complementation of the cpc-2 mutation in a his-2ts bradytrophic background. Genomic and cDNA sequence analysis indicated a 1636 bp long open reading frame interrupted by four introns. The deduced 316 amino acid polypeptide reveals 70% positional identity over its full length with G-protein beta-subunit-related polypeptides found in humans, rat (RACK1), chicken, tobacco and Chlamydomonas. With the exception of RACK1 the function of these proteins is obscure. All are entirely made up of seven WD-repeats. Expression studies of cpc-2 revealed one abundant transcript in the wild type; in the mutant its level is drastically reduced. In mutant cells transformed with the complementing sequence, the transcript level, enzyme regulation and female fertility are restored. In the wild type the cpc-2 transcript is down-regulated under conditions of amino acid limitation. With cpc-2 a new element involved in general amino acid control has been identified, indicating a function for a WD-repeat protein that belongs to a class that is conserved throughout the evolution of eukaryotes. PMID:7651339

  6. Role of GlnR in Acid-Mediated Repression of Genes Encoding Proteins Involved in Glutamine and Glutamate Metabolism in Streptococcus mutans▿ †

    PubMed Central

    Chen , Pei-Min; Chen, Yi-Ywan M.; Yu, Sung-Liang; Sher, Singh; Lai, Chern-Hsiung; Chia, Jean-San

    2010-01-01

    The acid tolerance response (ATR) is one of the major virulence traits of Streptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR in S. mutans was investigated. Using a whole-genome microarray and in silico analyses, we demonstrated that GlnR and the GlnR box (ATGTNAN7TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in the citBZC operon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates in S. mutans ATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H+ and potential ATP synthesis. Such regulation will ensure an optimal acid adaption in S. mutans. PMID:20173059

  7. Identification of Exonic Nucleotide Variants of the Thyroid Hormone Responsive Protein Gene Associated with Carcass Traits and Fatty Acid Composition in Korean Cattle

    PubMed Central

    Oh, Dong-yep; Lee, Yoon-seok; La, Boo-mi; Lee, Jea-young; Park, Yong-soo; Lee, Ji-hong; Ha, Jae-jung; Yi, Jun-koo; Kim, Byung-ki; Yeo, Jung-sou

    2014-01-01

    The thyroid hormone responsive protein (THRSP) gene is a functional gene that can be used to indicate the fatty acid compositions. This study investigates the relationships of exonic single nucleotide polymorphisms (SNPs) in the THRSP gene and fatty acid composition of muscle fat and marbling score in the 612 Korean cattle. The relationships between fatty acid composition and eight SNPs in the THRSP gene (g.78 G>A, g.173 C>T, g.184 C>T, g.190 C>A, g.194 C>T, g.277 C>G, g.283 T>G and g.290 T>G) were investigated, and according to the results, two SNPs (g.78 G>A and g.184 C>T) in exon 1 were associated with fatty acid composition. The GG and CC genotypes of g.78 G>A and g.184 C>T had higher unsaturated fatty acid (UFA) and monounsaturated fatty acid (MUFA) content (p<0.05). In addition, the ht1*ht1 group (Val/Ala haplotype) in a linkage disequilibrium increased MUFAs and marbling scores for carcass traits (p<0.05). As a result, g.78 G>A and g.184 C>T had significantly relationships with UFAs and MUFAs. Two SNPs in the THRSP gene affected fatty acid composition, suggesting that GG and CC genotypes and the ht1*ht1 group (Val/Ala haplotype) can be markers to genetically improve the quality and flavor of beef. PMID:25178286

  8. Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor.

    PubMed

    Ishii, Satoshi; Kihara, Yasuyuki; Shimizu, Takao

    2005-03-11

    T cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor mainly expressed in lymphoid organs and cancer tissues. TDAG8 shares high amino acid sequence homologies with recently reported proton-sensing G-protein-coupled receptors, G2A, OGR1, and GPR4. Here we have identified TDAG8 as a novel proton-sensing receptor. Upon acid stimulation, stably expressed TDAG8 was internalized from the plasma membrane. As a signaling pathway downstream of TDAG8, accumulation of cyclic AMP was observed in response to solutions with a pH value lower than 7.2. Furthermore, RhoA activation and actin rearrangement were elicited by acid-stimulated TDAG8. These results suggest that TDAG8 may play biological roles in immune response and cellular transformation under conditions accompanying tissue acidosis. PMID:15618224

  9. AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

    PubMed

    Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; Müller, Rolf; Brüne, Bernhard; Namgaladze, Dmitry

    2015-01-01

    AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload. PMID:26098914

  10. AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages

    PubMed Central

    Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; Müller, Rolf; Brüne, Bernhard; Namgaladze, Dmitry

    2015-01-01

    AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload. PMID:26098914

  11. A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction.

    PubMed Central

    Nishio, Y; Isshiki, H; Kishimoto, T; Akira, S

    1993-01-01

    The acute-phase reaction is accompanied by an increase in a variety of serum proteins, named acute-phase proteins. The synthesis of these proteins is synergistically controlled by glucocorticoids and inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha. Recently, we have cloned nuclear factor-IL-6 (NF-IL6), a transcription factor that activates the IL-6 gene, and have demonstrated its involvement in the expression of acute-phase-protein genes. We report here an analysis of the molecular mechanisms by which inflammatory cytokines and glucocorticoid act synergistically to activate expression of the rat alpha 1-acid glycoprotein (AGP) gene. We found that NF-IL6 and ligand-activated rat glucocorticoid receptor acted synergistically to transactivate the AGP gene and that maximal transcriptional activation of the AGP gene required expression of both intact NF-IL6 and rat glucocorticoid receptor. Surprisingly, however, transcriptional synergism was still observed even when one of the two factors lacked either its DNA-binding or transcriptional-activation function. We present evidence for a direct protein-protein interaction between these two distinct transcription factors and propose that this may be responsible for the synergistic activation of the rat AGP gene. Images PMID:8441418

  12. Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

    PubMed

    Kirimura, Kohtaro; Kobayashi, Keiichi; Ueda, Yuka; Hattori, Takasumi

    2016-09-01

    The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger. PMID:27088852

  13. STAT5 plays a critical role in regulating the 5'-flanking region of the porcine whey acidic protein gene in transgenic mice.

    PubMed

    Ji, Mi-Ran; Lee, Sang In; Jang, Ye-Jin; Jeon, Mi-Hyang; Kim, Jeom Sun; Kim, Kyung-Woon; Park, Jin-Ki; Yoo, Jae Gyu; Jeon, Ik-Soo; Kwon, Dae-Jin; Park, Choon-Keun; Byun, Sung June

    2015-12-01

    The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical-grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5'-flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene-expression activity. In the present study, we investigated how lactogenic hormones--including prolactin, hydrocortisone, and insulin--contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5'-flanking region. To confirm the expression and hormonal regulation of the 5'-flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk. PMID:26256125

  14. [Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence].

    PubMed

    Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

    2016-01-01

    An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen. PMID:27239860

  15. Effect of the ratios of unsaturated fatty acids on the expressions of genes related to fat and protein in the bovine mammary epithelial cells.

    PubMed

    Sheng, R; Yan, S M; Qi, L Z; Zhao, Y L

    2015-04-01

    The objective of this study was to evaluate the effects of the different ratios of unsaturated fatty acids (UFAs) (oleic acid, linoleic acid, and linolenic acid) on the cell viability and triacylglycerol (TAG) content, as well as the mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Afterward, the cells were randomly allocated to six treatments, five UFA-treated groups, and one control group. For all of the treatments, the the fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L), and the cells were treated with different ratios of oleic, linoleic, and linolenic acids (0.75:4:1, 1.5:10:1, 2:13.3:1, 3:20:1, and 4:26.7:1) for 48 h, which were group 1 to group 5. The control culture solution contained only fatty acid-free BSA without UFAs (0 μM). The results indicated that the cell viability was not affected by adding different ratios of UFAs, but the accumulation of TAG was significantly influenced by supplementing with different ratios of UFAs. Adding different ratios of UFAs suppressed the expression of ACACA and FASN but had the opposite effect on the abundances of FABP3 and CD36 mRNA. The expression levels of PPARG, SPEBF1, CSN1S1, and CSN3 mRNA in the BMECs were affected significantly after adding different ratios of UFAs. Our results suggested that groups 1, 2, and 3 (0.75:4:1, 1.5:10:1, and 2:13.3:1) had stronger auxo-action on fat synthesis in the BMECs, where group 3 (2:13.3:1) was the best, followed by group 4 (3:20:1). However, group 5 (4:26.7:1) was the worst. Genes related to protein synthesis in the BMECs were better promoted in groups 2 and 3, and group 3 had the strongest auxo-action, whereas the present study only partly examined the regulation of protein synthesis at the transcriptional level; more studies on translation level are needed in the future

  16. The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression.

    PubMed

    Brock, Anita K; Willmann, Roland; Kolb, Dagmar; Grefen, Laure; Lajunen, Heini M; Bethke, Gerit; Lee, Justin; Nürnberger, Thorsten; Gust, Andrea A

    2010-07-01

    Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal opening and seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggested to be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of the Protein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. The PP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus. Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhanced ABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in the opposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not being activated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABA treatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resulted in an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seed germination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genes such as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPK phosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression. PMID:20488890

  17. Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain)

    PubMed Central

    Zeng, Xianglan; Ye, Haihui; Yang, Ya’nan; Wang, Guizhong; Huang, Huiyang

    2013-01-01

    Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab. PMID:23569421

  18. Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis.

    PubMed Central

    Shen, Z; Byers, D M

    1996-01-01

    We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes. Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins. Five of the 11 sequence differences between V. harveyi and E. coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II. PMID:8550484

  19. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    SciTech Connect

    Waters, Katrina M.; Jacobs, Jon M.; Gritsenko, Marina A.; Karin, Norman J.

    2011-02-26

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  20. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease

    SciTech Connect

    Kane, S.E.; Troen, B.R.; Gal, S.; Ueda, K.; Pastan, I.; Gottesman, M.M.

    1988-08-01

    Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, the authors transfected cloned genes for mouse or human MEP into mouse MIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes were also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

  1. Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid1

    PubMed Central

    Wang, Ning Ning; Yang, Shang Fa; Charng, Yee-yung

    2001-01-01

    Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower. PMID:11351088

  2. Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP) gene in female Chinese mitten crab (Eriocheir sinensis)

    PubMed Central

    2010-01-01

    Background Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Results Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP) was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January. Conclusions Evidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes. PMID:20846381

  3. DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements.

    PubMed Central

    Foufelle, F; Lepetit, N; Bosc, D; Delzenne, N; Morin, J; Raymondjean, M; Ferré, P

    1995-01-01

    We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7772036

  4. Fatty acid represses insulin receptor gene expression by impairing HMGA1 through protein kinase C{epsilon}

    SciTech Connect

    Dey, Debleena; Bhattacharya, Anirban; Roy, SibSankar; Bhattacharya, Samir . E-mail: smrbhattacharya@gmail.com

    2007-06-01

    It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p < 0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKC{epsilon} was also inhibited. Surprisingly, phosphorylation of cytosolic PKC{epsilon} was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKC{epsilon} translocation inhibitor peptide, {epsilon}V1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKC{epsilon} migration to implement palmitate effect. Experimental evidences indicate that phospho-PKC{epsilon} adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.

  5. Yeast Cyc8p and Tup1p proteins function as coactivators for transcription of Stp1/2p-dependent amino acid transporter genes.

    PubMed

    Tanaka, Naoko; Mukai, Yukio

    The yeast Cyc8p-Tup1p complex is known to serve primarily as a transcriptional corepressor in a variety of biological processes. However, less is known about its function as a coactivator. Herein, we found tryptophan transporter genes, TAT1 and TAT2, that, when overexpressed, suppressed the slow growth of Δcyc8. We observed that the addition of tryptophan to Δcyc8 cultures partially restored cell growth, and the deletion of CYC8 and TUP1 reduced transcriptional levels of TAT1 and TAT2. Tup1p bound to the promoter region of TAT1 and TAT2 genes that were dependent on STP1 and STP2 (encoding DNA-binding activator proteins) for expression. Similarly, transcription of the other Stp1/2p-dependent amino acid transporter (AAT) genes also required CYC8 and TUP1 gene functions. These data indicate that Cyc8p-Tup1p plays a role as a transcriptional coactivator for AAT genes via Stp1/2p activators and that lowering intracellular tryptophan by CYC8 deletion causes slow growth. PMID:26546823

  6. Effects of supplementation with branched-chain amino acids to low-protein diets on expression of genes related to lipid metabolism in skeletal muscle of growing pigs.

    PubMed

    Duan, Yehui; Duan, Yangmiao; Li, Fengna; Li, Yinghui; Guo, Qiuping; Ji, Yujiao; Tan, Bie; Li, Tiejun; Yin, Yulong

    2016-09-01

    Branched-chain amino acids (BCAA), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in energy homeostasis and lipid metabolism in addition to their other functions, such as in protein metabolism. This study investigated the effects of different dietary BCAA ratios on the intramuscular fat (IMF) content and fatty acid composition in different location of skeletal muscles, including the longissimus dorsi (LD), biceps femoris (BF), and psoas major (PM) muscles of growing pigs, and also examined the mRNA expression levels of genes involved in lipid metabolism in these muscle tissues. The experiment was performed on 40 growing pigs (Large White × Landrace) with a similar initial weight (9.85 ± 0.35 kg). The pigs were randomly assigned to one of five diets: diet A was a positive control and contained 20 % crude protein (CP) with a Leu:Ile:Val ratio of 1:0.51:0.63 according to the recommendation of the National Research Council (NRC); for diets B to E, the CP level was reduced to 17 %, and the Leu:Ile:Val ratios were 1:1:1, 1:0.75:0.75, 1:0.51:0.63, and 1:0.25:0.25, respectively. No significant difference was observed in the average feed intake and feed efficiency of the pigs fed the low protein diet (17 % CP) with BCAA treatments relative to the positive control. However, there was a tendency for increased feed efficiency of the 1:0.75:0.75 group compared with the 1:1:1 group (P = 0.09). The BCAA ratio of 1:0.75:0.75 (17 % CP) increased the IMF content of BF muscle (P < 0.01). Moreover, varied dietary BCAA supplementation with a reduced protein level had different effects on the fatty acid composition of the LD, BF, and PM muscles. The BCAA ratio of 1:0.51:0.63-1:0.75:0.75 (17 % CP) significantly lowered the ratio of n-6 to n-3 polyunsaturated fatty acid in these muscles compared with the positive control group (20 % CP). This effect was associated with an increase in mRNA expression levels of acetyl-CoA carboxylase

  7. Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

    PubMed

    Kobayashi, Michie; Yamamoto-Katou, Ayako; Katou, Shinpei; Hirai, Katsuyuki; Meshi, Tetsuo; Ohashi, Yuko; Mitsuhara, Ichiro

    2011-07-01

    The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death. PMID:21310506

  8. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis.

    PubMed

    Jiang, Shanshan; Zhang, Dan; Wang, Li; Pan, Jiaowen; Liu, Yang; Kong, Xiangpei; Zhou, Yan; Li, Dequan

    2013-10-01

    Calcium-dependent protein kinases (CDPKs) play essential roles in calcium-mediated signal transductions in plant response to abiotic stress. Several members have been identified to be regulators for plants response to abscisic acid (ABA) signaling. Here, we isolated a subgroup I CDPK gene, ZmCPK4, from maize. Quantitative real time PCR (qRT-PCR) analysis revealed that the ZmCPK4 transcripts were induced by various stresses and signal molecules. Transient and stable expression of the ZmCPK4-GFP fusion proteins revealed ZmCPK4 localized to the membrane. Moreover, overexpression of ZmCPK4 in the transgenic Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth and stomatal movement. The transgenic plants also enhanced drought stress tolerance. Taken together, the results suggest that ZmCPK4 might be involved in ABA-mediated regulation of stomatal closure in response to drought stress. PMID:23911729

  9. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  10. Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells.

    PubMed Central

    Kuo, A; Cappelluti, S; Cervantes-Cervantes, M; Rodriguez, M; Bush, D S

    1996-01-01

    The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling. PMID:8742711

  11. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    SciTech Connect

    Benson, D.L.; Isackson, P.J.; Hendry, S.H.; Jones, E.G. )

    1991-06-01

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate.

  12. The bovine fatty acid binding protein 4 gene is significantly associated with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses.

    PubMed

    Michal, J J; Zhang, Z W; Gaskins, C T; Jiang, Z

    2006-08-01

    Fatty acid binding protein 4 (FABP4), which is expressed in adipose tissue, interacts with peroxisome proliferator-activated receptors and binds to hormone-sensitive lipase and therefore, plays an important role in lipid metabolism and homeostasis in adipocytes. The objective of this study was to investigate associations of the bovine FABP4 gene with fat deposition. Both cDNA and genomic DNA sequences of the bovine gene were retrieved from the public databases and aligned to determine its genomic organization. Primers targeting two regions of the FABP4 gene were designed: from nucleotides 5433-6106 and from nucleotides 7417-7868 (AAFC01136716). Direct sequencing of polymerase chain reaction (PCR) products on two DNA pools from high- and low-marbling animals revealed two single nucleotide polymorphisms (SNPs): AAFC01136716.1:g.7516G>C and g.7713G>C. The former SNP, detected by PCR-restriction fragment length polymorphism using restriction enzyme MspA1I, was genotyped on 246 F2 animals in a Waygu x Limousin F2 reference population. Statistical analysis showed that the FABP4 genotype significantly affected marbling score (P = 0.0398) and subcutaneous fat depth (P = 0.0246). The FABP4 gene falls into a suggestive/significant quantitative trait loci interval for beef marbling that was previously reported on bovine chromosome 14 in three other populations. PMID:16879357

  13. The Vibrio cholerae Fatty Acid Regulatory Protein, FadR, Represses Transcription of plsB, the Gene Encoding the First Enzyme of Membrane Phospholipid Biosynthesis

    PubMed Central

    Feng, Youjun; Cronan, John E.

    2011-01-01

    SUMMARY Glycerol-3-phosphate (sn-glycerol-3-P, G3P) acyltransferase catalyzes the first committed step in the biosynthesis of membrane phospholipids, the acylation of G3P to form 1-acyl G3P (lysophosphatidic acid). The paradigm G3P acyltransferase is the Escherichia coli plsB gene product which acylates position-1 of G3P using fatty acids in thioester linkage to either acyl carrier protein (ACP) or CoA as acyl-donors. Although the Escherichia coli plsB gene was discovered about 30 years ago, no evidence for transcriptional control of its expression has been reported. However Kazakov and coworkers (Kazakov, A. E. et al. (2009) J Bacteriol, 191, 52–64) reported the presence of a putative FadR-binding site upstream of the candidate plsB genes of V. cholerae and three other Vibrio species suggesting that plsB might be regulated by FadR, a GntR-family transcription factor thus far known only to regulate fatty acid synthesis and degradation. We report that the V. cholerae plsB homologue restored growth of E. coli strain BB26-36 which is a G3P auxotroph due to an altered G3P acyltransferase activity. The plsB promoter was also mapped and the predicted FadR-binding palindrome was found to span positions -19 to -35, upstream of the transcription start site. Gel shift assays confirmed that both V. cholerae FadR and E. coli FadR bound the V. cholerae plsB promoter region and binding was reversed upon addition of long chain fatty acyl-CoA thioesters. The expression level of the V. cholerae plsB gene was elevated 2–3 fold in an E. coli fadR null mutant strain indicating that FadR acts as a repressor of V. cholerae plsB expression. In both E. coli and V. cholerae the β-galactosidase activity of transcriptional fusions of the V. cholerae plsB promoter to lacZ increased 2–3 fold upon supplementation of growth media with oleic acid. Therefore, V. cholerae coordinates fatty acid metabolism with 1-acyl G3P synthesis. PMID:21771112

  14. Effects of reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in weaned piglets*

    PubMed Central

    Wu, Li; He, Liu-qin; Cui, Zhi-jie; Liu, Gang; Yao, Kang; Wu, Fei; Li, Jun; Li, Tie-jun

    2015-01-01

    The effects of crude protein (CP) levels in the diet on the mRNA expression of amino acid (AA) transporters were studied in a 45-d trial. Eighteen piglets with an initial body weight (BW) of 9.57 kg were assigned to three groups (14%, 17%, and 20% CP in the diet) in a completely randomized design (six replicates per treatment). Diets were supplemented with crystalline AA to achieve equal standardized ileal digestible contents of Lys, Met plus Cys, Thr, and Trp, and were provided ad libitum. After 45 d, all piglets were slaughtered to collect small intestine samples. Compared with the values in the 14% CP group, the expressions of ASCT2, 4F2hc, and ATB0 mRNA in the jejunum were increased by 23.00%, 12.00%, 6.00% and 48.00%, 47.00%, 56.00% in the 17% and 20% CP groups, respectively. These results indicate that a 14% CP diet supplemented with crystalline AA may not transport enough AA into the body and maintain growth performance of piglets. However, a reduction of dietary 17% CP may reduce the excretion of nitrogen into the environment while supporting the development of piglets. Therefore, the 17% CP level is more suitable than 14% CP level. PMID:26055911

  15. Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: different gene expression of nuclear receptors and ECM proteins.

    PubMed

    Fernández, Ignacio; Tiago, Daniel M; Laizé, Vincent; Leonor Cancela, M; Gisbert, Enric

    2014-03-01

    Retinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 μM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36-59% in VSa13 and 17-46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50-62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11-57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner. PMID:24291400

  16. The structure of the gene ATRC1 coding for a cationic amino acid transport system in man: Molecular studies in lysinuric protein intolerance

    SciTech Connect

    Incerti, B.; Sebastio, G.; Parenti, G.

    1994-09-01

    The human cDNA (ATRC1) homologue of a murine gene encoding for a transporter specific for cationic amino acid (CAA) has been isolated. ATRC1 stimulates the uptake of CAA and shows the kinetic properties of system y+ when expressed in frog oocytes. To characterize the organization of the ATRC1 gene, a {lambda} phages genomic DNA library has been screened using an ATRC1 full length cDNA clone as a probe. Nine positive phages have been subcloned in plasmids and sequenced using cDNA specific primers to identify intron-exon junctions. The ATRC1 gene consists of 13 exons with an alternative first exon. Analysis of the intron/exon boundaries showed canonical sequences at the splice junction sites. ATRC1 expression pattern has been analyzed by RT-PCR. ATRC1 is expressed in adult fibroblasts and enterocytes, in fetal kidney, brain and heart, and in lymphoblastoid cell lines. The knowledge of structure and organization of ATRC1 can help in studying inborn errors of CAA transport. The best characterized among these diseases is Lysinuric Protein Intolerance (LPI) a multisystem disorder with impaired formation of urea and hyperammonemia after protein ingestion. Linkage analysis performed on 10 LPI patients from 9 Italian families using two intragenic RFLPs revealed 3 informative families and no recombinations. Using the CA-repeat microsatellite D12S120 (2 cM far from ATRC-1 locus) we found 7 informative families and 3 recombinational events. The sequence of the entire coding region of an LPI patient failed to show mutations. The data so far obtained do not seem to support the hypothesis that ATRC1 is the LPI gene.

  17. Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism.

    PubMed

    Shandilya, Umesh K; Sharma, Ankita; Sodhi, Monika; Kapila, Neha; Kishore, Amit; Mohanty, Ashok; Kataria, Ranjit; Malakar, Dhruva; Mukesh, Manishi

    2016-02-01

    Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three-dimensional (3D) environment as important signals are lost when cells are cultured in two-dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1-casein (CSN1S1), alpha S2-casein (CSN1S2), beta-casein (CSN2), kappa-casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol-3-phosphate acyltransferase (GPAM), fatty acid-binding protein 3 (FABP3), and stearoyl-CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT-PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome-like structures were formed by day 5, whereas polarized acinus-like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10-day-old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism-related genes. Therefore, matrix-based 3D culture of MEC that recapitulate the structural and functional context of normal tissues

  18. Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

    PubMed

    Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

    2010-05-01

    A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava. PMID:20336312

  19. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

    PubMed Central

    Martínez, M A; Dopazo, J; Hernández, J; Mateu, M G; Sobrino, F; Domingo, E; Knowles, N J

    1992-01-01

    The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time. PMID:1316467

  20. Identification of a functional polymorphism at the Adipose Fatty Acid Binding protein gene (FABP4) and demonstration of its association with cardiovascular disease: A path to follow

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty acid binding proteins (FABPs) are proteins that reversibly bind fatty acids and other lipids. So far, 9 tissue-specific cytoplasmic FABPs have been identified. Adipose tissue FABP (FABP4) has been suggested to be a bridge between inflammation and other pathways related to the metabolic syndrom...

  1. Molecular Identification and Functional Characterization of the Fatty Acid- and Retinoid-Binding Protein Gene Rs-far-1 in the Burrowing Nematode Radopholus similis (Tylenchida: Pratylenchidae)

    PubMed Central

    Zhang, Chao; Xie, Hui; Cheng, Xi; Wang, Dong-Wei; Li, Yu; Xu, Chun-Ling; Huang, Xin

    2015-01-01

    Fatty acid- and retinoid-binding protein (FAR) is a nematode-specific protein expressed in the nematode hypodermis. It is involved in nematode development, reproduction, and infection and can disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the far gene from Radopholus similis (Rs-far-1), which is 828 bp long and includes a 558 bp ORF encoding 186 amino acids. A protein homology analysis revealed that Rs-FAR-1 is 75% similar to Mj-FAR-1 from Meloidogyne javanica. A neighbor-joining phylogenetic tree was inferred and showed that Rs-FAR-1 is most similar to Pv-FAR-1 from Pratylenchus vulnus. A fluorescence-based ligand-binding analysis confirmed that Rs-FAR-1 can combine with fatty acids and retinol. qPCR was used to assess Rs-far-1 expression levels at different developmental stages in different R. similis populations, and its expression was 2.5 times greater in the highly pathogenic Rs-C population than in the less pathogenic Rs-P population. The highest expression was found in females, followed by eggs, juveniles and males. When R. similis was treated with Rs-far-1 dsRNA for 36 h, the reproduction and pathogenicity decreased significantly. In situ hybridization revealed Rs-far-1 transcripts in the R. similis hypodermis. Additionally, R. similis treated with Rs-far-1 dsRNA or water were inoculated into Arabidopsis thaliana. Allene oxide synthase (AOS) expression in A. thaliana was upregulated during early infection in both treatments and then returned to the expression levels of the control plant. Compared with the control plant, AOS expression significantly decreased in A. thaliana inoculated with water-treated R. similis but significantly increased in A. thaliana inoculated with Rs-far-1 dsRNA-treated R. similis. This finding indicates that Rs-far-1 regulates AOS expression in A. thaliana. Rs-FAR-1 plays a critical role in R. similis development, reproduction, and infection and can disturb the plant defense reaction

  2. M-protein gene-type distribution and hyaluronic acid capsule in group A Streptococcus clinical isolates in Chile: association of emm gene markers with csrR alleles.

    PubMed

    Wozniak, A; Rojas, P; Rodríguez, C; Undabarrena, A; Garate, C; Riedel, I; Román, J C; Kalergis, A M; García, P

    2012-07-01

    Streptococcus pyogenes causes a variety of infections because of virulence factors such as capsular hyaluronic acid and M protein. The aim of this study was to determine emm types and capsule phenotype in 110 isolates of S. pyogenes from patients with invasive (sterile sites) and non-invasive (mainly pharyngitis) infections in Chile, and the relationship between both virulence factors. The most abundant types found were emm12, emm1, emm4 and emm28 and their distribution was similar to that seen in Latin America and developed countries, but very different from that in Asia and Pacific Island countries. Ten of 16 emm types identified in pharyngeal isolates were found in sterile-site isolates, and three of nine emm types of sterile-site isolates occurred in pharyngeal isolates; three emm subtypes were novel. The amount of hyaluronic acid was significantly higher in sterile-site isolates but did not differ substantially among emm types. Only three isolates were markedly capsulate and two of them had mutations in the csrR gene that codes for a repressor of capsule synthesis genes. We found a non-random association between emm types and csrR gene alleles suggesting that horizontal gene transfer is not freely occurring in the population. PMID:21906413

  3. EFFECTS OF METHOPRENE DERIVATIVES ON THE EXPRESSION OF RETINOIC ACID SENSITIVE GENES AND PROTEINS IN CULTURED CELLS

    EPA Science Inventory

    The insect juvenile hormone analog methoprene has been suggested as a possible cause of malformations in frogs and other amphibians. Methoprene has structural similarities to the ubiquitous development regulator, retinoic acid, and thus, may bind to retinoid receptors and consequ...

  4. Cloning and nucleotide sequences of livB and livC, the structural genes encoding binding proteins of the high-affinity branched-chain amino acid transport in Salmonella typhimurium.

    PubMed

    Ohnishi, K; Nakazima, A; Matsubara, K; Kiritani, K

    1990-02-01

    The liv gene cluster responsible for encoding the high-affinity branched-chain amino acid transport proteins in Salmonella typhimurium was mapped in the 7.6-kilobase HindIII-SacI segment of plasmid pMN12 by utilizing the gene dosage effect. By subcloning and biochemical analysis, the livB and livC structural genes encoding the leucine-, isoleucine-, valine-, threonine-binding protein (LIVT-BP) and the leucine-specific binding protein (L-BP), respectively, were localized within the 3,617-base HindIII-BstEII segment. Upon determining the nucleotide sequence of the 3,617 bases, we found that the coding sequence of the livB gene (1,095 base pairs) starts at the position 355 and specifies the precursor LIVT-BP of 365 amino acid residues, and the livC gene (1,107 base pairs) starts at the position 2,452 and encodes the precursor L-BP of 369 amino acid residues. The two genes, separated by a 1-kilobase intergenic region, each possess potential promoters and rho-independent transcriptional terminators. The mature LIVT-BP and L-BP are produced by removing the putative 21 and 23 signal peptides from the respective precursors. In comparison with the analogous two binding proteins from Escherichia coli K-12, strong homologies are observed. PMID:2193932

  5. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. PMID:25749303

  6. A STRESS-RESPONSIVE NAC1-Regulated Protein Phosphatase Gene Rice Protein Phosphatase18 Modulates Drought and Oxidative Stress Tolerance through Abscisic Acid-Independent Reactive Oxygen Species Scavenging in Rice1[W][OPEN

    PubMed Central

    You, Jun; Zong, Wei; Hu, Honghong; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2014-01-01

    Plants respond to abiotic stresses through a complexity of signaling pathways, and the dephosphorylation mediated by protein phosphatase (PP) is an important event in this process. We identified a rice (Oryza sativa) PP2C gene, OsPP18, as a STRESS-RESPONSIVE NAC1 (SNAC1)-regulated downstream gene. The ospp18 mutant was more sensitive than wild-type plants to drought stress at both the seedling and panicle development stages. Rice plants with OsPP18 suppressed through artificial microRNA were also hypersensitive to drought stress. Microarray analysis of the mutant revealed that genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant exhibited reduced activities of ROS scavenging enzymes and increased sensitivity to oxidative stresses. Overexpression of OsPP18 in rice led to enhanced osmotic and oxidative stress tolerance. The expression of OsPP18 was induced by drought stress but not induced by abscisic acid (ABA). Although OsPP18 is a typical PP2C with enzymatic activity, it did not interact with SNF1-RELATED PROTEIN KINASE2 protein kinases, which function in ABA signaling. Meanwhile, the expression of ABA-responsive genes was not affected in the ospp18 mutant, and the ABA sensitivities of the ospp18 mutant and OsPP18-overexpressing plants were also not altered. Together, these findings suggest that OsPP18 is a unique PP2C gene that is regulated by SNAC1 and confers drought and oxidative stress tolerance by regulating ROS homeostasis through ABA-independent pathways. PMID:25318938

  7. Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis).

    PubMed

    Dubey, Praveen Kumar; Goyal, Shubham; Mishra, Shailendra Kumar; Arora, Reena; Mukesh, Manishi; Niranjan, Saket Kumar; Kathiravan, Periasamy; Kataria, Ranjit Singh

    2016-04-01

    The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes. PMID:26894500

  8. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis.

    PubMed

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries. PMID:27379111

  9. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis

    PubMed Central

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E.; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries. PMID:27379111

  10. Dietary ascorbic acid modulates the expression profile of stress protein genes in hepatopancreas of adult Pacific abalone Haliotis discus hannai Ino.

    PubMed

    Wu, Chenglong; Wang, Jia; Xu, Wei; Zhang, Wenbing; Mai, Kangsen

    2014-12-01

    This study was conducted to investigate the effects of dietary ascorbic acid (AA) on transcriptional expression patterns of antioxidant proteins, heat shock proteins (HSP) and nuclear factor kappa B (NF-κB) in the hepatopancreas of Pacific abalone Haliotis discus hannai Ino (initial average length: 84.36 ± 0.24 mm) using real-time quantitative PCR assays. L-ascorbyl-2-molyphosphate (LAMP) was added to the basal diet to formulate four experimental diets containing 0.0, 70.3, 829.8 and 4967.5 mg AA equivalent kg(-1) diets, respectively. Each diet was fed to triplicate groups of adult abalone in acrylic tanks (200 L) in a flow-through seawater system. Each tank was stocked with 15 abalone. Animals were fed once daily (17:00) to apparent satiation for 24 weeks. The results showed that the dietary AA (70.3 mg kg(-1)) could significantly up-regulate the expression levels of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), feritin (FT) and heat shock protein 26 (HSP26) in the hepatopancreas of abalone in this treatment compared to the controls. However, the expression levels of Mn-SOD, glutathione peroxidase (GPX), thioredoxin peroxidase (TPx), selenium-binding protein (SEBP), HSP70 and HSP90 were significantly down-regulated. Compared with those in the group with 70.3 mg kg(-1) dietary AA, the expression levels of CAT, GST and HSP26 were decreased in abalone fed with very high dietary AA (4967.5 mg kg(-1)). In addition, significant up-regulations of expression levels of Mn-SOD, GPX, TPx, SEBP, FT, HSP70, HSP90 and NF-κB were observed in abalone fed with apparently excessive dietary AA (829.8 and 4967.5 mg kg(-1)) as compared to those fed 70.3 mg kg(-1) dietary AA. These findings showed that dietary AA influenced the expression levels of antioxidant proteins, heat shock proteins and NF-κB in the hepatopancreas of abalone at transcriptional level. Levels of dietary AA that appeared adequate (70.3 mg kg(-1)) reduced the oxidative stress

  11. Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors α- and γ-mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus

    PubMed Central

    Wolfrum, Christian; Borrmann, Carola M.; Börchers, Torsten; Spener, Friedrich

    2001-01-01

    Peroxisome proliferator-activated receptor α (PPARα) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARα colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARα. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARα and PPARγ but not with PPARβ and retinoid X receptor-α by protein–protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARα and PPARγ transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARα and PPARγ agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes. PMID:11226238

  12. Inhibition of gene expression of carnitine palmitoyltransferase I and heart fatty acid binding protein in cyclophosphamide and ifosfamide-induced acute cardiotoxic rat models.

    PubMed

    Sayed-Ahmed, Mohamed M; Aldelemy, Meshan L; Al-Shabanah, Othman A; Hafez, Mohamed M; Al-Hosaini, Khaled A; Al-Harbi, Naif O; Al-Sharary, Shakir D; Al-Harbi, Mohamed M

    2014-09-01

    This study investigated whether cyclophosphamide (CP) and ifosfamide (IFO) therapy alters the expression of the key genes engaged in long-chain fatty acid (LCFA) oxidation outside rat heart mitochondria, and if so, whether these alterations should be viewed as a mechanism during CP- and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of the six treatment groups: Rats in group 1 (control) and group 2 (L-carnitine) were injected intraperitoneal (i.p.) with normal saline and L-carnitine (200 mg/kg/day), respectively, for 10 successive days. Animals in group 3 (CP group) were injected i.p. with normal saline for 5 days before and 5 days after a single dose of CP (200 mg/kg, i.p.). Rats in group 4 (IFO group) received normal saline for 5 successive days followed by IFO (50 mg/kg/day, i.p.) for 5 successive days. Rats in group 5 (CP-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5 days before and 5 days after a single dose of CP as group 3. Rats in group 6 (IFO-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5 days before and 5 days concomitant with IFO as group 4. Immediately, after the last dose of the treatment protocol, blood samples were withdrawn and animals were killed for biochemical, histopathological and gene expression studies. Treatment with CP and IFO significantly decreased expression of heart fatty acid binding protein (H-FABP) and carnitine palmitoyltransferase I (CPT I) genes in cardiac tissues. Moreover, CP but not IFO significantly increased acetyl-CoA carboxylase2 mRNA expression. Conversely, IFO but not CP significantly decreased mRNA expression of malonyl-CoA decarboxylase. Both CP and IFO significantly increased serum lactate dehydrogenase, creatine kinase isoenzyme MB and malonyl-CoA content and histopathological lesions in cardiac tissues. Interestingly, carnitine supplementation completely reversed all the biochemical, histopathological and

  13. Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.

    PubMed

    Jing, C; Beesley, C; Foster, C S; Chen, H; Rudland, P S; West, D C; Fujii, H; Smith, P H; Ke, Y

    2001-06-01

    Human cutaneous fatty acid-binding protein (C-FABP) gene is capable of inducing the metastatic phenotype when overexpressed in nonmetastatic rat Rama 37 cells. However, the mechanism of how it induces metastasis is not clear. Northern and slot blot analyses revealed that expression of the endogenous vascular endothelial growth factor (VEGF) gene was increased by 3.8-5.2-fold in the C-FABP-transfected cells (pSV-CFABP-R37) and in their metastatic sublines (e.g., Met-1) when compared with that in the nonmetastatic control transfectant pSV-R37 cells generated by transfection of only plasmid DNA. Higher levels of VEGF immunoreactive protein were also secreted from the malignant C-FABP-expressing cells. Reverse transcription-PCR detected two VEGF transcript isoforms, VEGF(164) and VEGF(188), in both the nonmetastatic control transfectant pSV-R37 cells and the malignant metastatic Met-1 cells. Chick chorioallantoic membrane assays showed that the conditioned medium of the control pSV-R37 cells possessed only very weak angiogenic activity, whereas conditioned media from the metastatic C-FABP transfectants and their sublines were strongly angiogenic and could be inhibited by antibodies to VEGF. Transfection of VEGF(164) cDNA in an expression vector into nonmetastatic Rama 37 cells produced a cell clone (R37-VEGF-2) that expressed high levels of VEGF. Inoculation of R37-VEGF-2 cells into syngeneic Wistar Furth rats produced metastases in a significant number (Fisher's exact test, P < 0.01) of animals (18 of 31 animals), whereas the control, vector alone-transfected R37-PSV cells produced no metastases (0 of 30 animals). Immunocytochemical methods demonstrated a strong positive staining for VEGF and an increased microvessel density in the primary tumors produced from PSV-VEGF-2 cells in comparison with tumors produced from control transfectants. Immunocytochemical staining for factor VIII detected a 3.5-fold increase in microvessel density of the primary tumors produced by

  14. Ectopic expression of Arabidopsis genes encoding salicylic acid- and jasmonic acid-related proteins confers partial resistance to soybean cyst nematode (Heterodera glycines) in transgenic soybean roots

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background. Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) an...

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

    PubMed

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

    2013-04-01

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

  16. Computer programs for the characterization of protein coding genes.

    PubMed

    Pierno, G; Barni, N; Candurro, M; Cipollaro, M; Franzè, A; Juliano, L; Macchiato, M F; Mastrocinque, G; Moscatelli, C; Scarlato, V

    1984-01-11

    Computer programs, implemented on an Univac II00/80 computer system, for the identification and characterization of protein coding genes and for the analysis of nucleic acid sequences, are described. PMID:6546420

  17. Computer programs for the characterization of protein coding genes.

    PubMed Central

    Pierno, G; Barni, N; Candurro, M; Cipollaro, M; Franzè, A; Juliano, L; Macchiato, M F; Mastrocinque, G; Moscatelli, C; Scarlato, V

    1984-01-01

    Computer programs, implemented on an Univac II00/80 computer system, for the identification and characterization of protein coding genes and for the analysis of nucleic acid sequences, are described. PMID:6546420

  18. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken.

    PubMed

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-10-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  19. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I

    PubMed Central

    Park, Seung Bum; Seronello, Scott; Mayer, Wasima; Ojcius, David M.

    2016-01-01

    Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity. PMID:27404108

  20. Correlation between Heart-type Fatty Acid-binding Protein Gene Polymorphism and mRNA Expression with Intramuscular Fat in Baicheng-oil Chicken

    PubMed Central

    Wang, Yong; He, Jianzhong; Yang, Wenxuan; Muhantay, Gemenggul; Chen, Ying; Xing, Jinming; Liu, Jianzhu

    2015-01-01

    This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time. PMID:26323394

  1. Gene expression of fatty acid transport and binding proteins in the blood-brain barrier and the cerebral cortex of the rat: differences across development and with different DHA brain status.

    PubMed

    Pélerin, Hélène; Jouin, Mélanie; Lallemand, Marie-Sylvie; Alessandri, Jean-Marc; Cunnane, Stephen C; Langelier, Bénédicte; Guesnet, Philippe

    2014-11-01

    Specific mechanisms for maintaining docosahexaenoic acid (DHA) concentration in brain cells but also transporting DHA from the blood across the blood-brain barrier (BBB) are not agreed upon. Our main objective was therefore to evaluate the level of gene expression of fatty acid transport and fatty acid binding proteins in the cerebral cortex and at the BBB level during the perinatal period of active brain DHA accretion, at weaning, and until the adult age. We measured by real time RT-PCR the mRNA expression of different isoforms of fatty acid transport proteins (FATPs), long-chain acyl-CoA synthetases (ACSLs), fatty acid binding proteins (FABPs) and the fatty acid transporter (FAT)/CD36 in cerebral cortex and isolated microvessels at embryonic day 18 (E18) and postnatal days 14, 21 and 60 (P14, P21 and P60, respectively) in rats receiving different n-3 PUFA dietary supplies (control, totally deficient or DHA-supplemented). In control rats, all the genes were expressed at the BBB level (P14 to P60), the mRNA levels of FABP5 and ACSL3 having the highest values. Age-dependent differences included a systematic decrease in the mRNA expressions between P14-P21 and P60 (2 to 3-fold), with FABP7 mRNA abundance being the most affected (10-fold). In the cerebral cortex, mRNA levels varied differently since FATP4, ACSL3 and ACSL6 and the three FABPs genes were highly expressed. There were no significant differences in the expression of the 10 genes studied in n-3 deficient or DHA-supplemented rats despite significant differences in their brain DHA content, suggesting that brain DHA uptake from the blood does not necessarily require specific transporters within cerebral endothelial cells and could, under these experimental conditions, be a simple passive diffusion process. PMID:25123062

  2. Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

    PubMed Central

    Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

    2009-01-01

    Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis

  3. Human Protein and Amino Acid Requirements.

    PubMed

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. PMID:26796095

  4. Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

    PubMed

    Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

    2013-01-01

    Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. PMID:23797655

  5. Detecting patterns of protein distribution and gene expression in silico

    PubMed Central

    Geraghty, Michael T.; Bassett, Doug; Morrell, James C.; Gatto, Gregory J.; Bai, Jianwu; Geisbrecht, Brian V.; Hieter, Phil; Gould, Stephen J.

    1999-01-01

    Most biological information is contained within gene and genome sequences. However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities. We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches. CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae. Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes. CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes. More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes. Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins. Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine, a heretofore unrecognized role for peroxisomes. These results expand our understanding of peroxisome content and function, demonstrate the utility of CoSMoS for context-sensitive motif scanning, and point to the benefits of improved in silico genome analysis. PMID:10077615

  6. Genes and proteins of Escherichia coli K-12.

    PubMed

    Riley, M

    1998-01-01

    GenProtEC is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins, representing groups of paralogous genes, with PAM values, percent identity of amino acids, length of alignment and percent aligned. GenProtEC can be accessed at the URL http://www.mbl.edu/html/ecoli.html PMID:9399799

  7. Axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes

    NASA Astrophysics Data System (ADS)

    Menet, V.; Prieto, M.; Privat, A.; Giménez Y Ribotta, M.

    2003-07-01

    The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional disabilities. The inability of neurons to regenerate their axon is appreciably due to an inhospitable environment made of an astrocytic scar. We generated mice knock-out for glial fibrillary acidic protein and vimentin, the major proteins of the astrocyte cytoskeleton, which are upregulated in reactive astrocytes. These animals, after a hemisection of the spinal cord, presented reduced astroglial reactivity associated with increased plastic sprouting of supraspinal axons, including the reconstruction of circuits leading to functional restoration. Therefore, improved anatomical and functional recovery in the absence of both proteins highlights the pivotal role of reactive astrocytes in axonal regenerative failure in adult CNS and could lead to new therapies of spinal cord lesions.

  8. Evolution and organization of the human protein C gene

    SciTech Connect

    Plutzky, J.; Hoskins, J.A.; Long, G.L.; Crabtree, G.R.

    1986-02-01

    The authors have isolated overlapping phage genomic clones covering an area of 21 kilobases that encodes the human protein C gene. The gene is at least 11.2 kilobases long and is made up of nine exons and eight introns. Two regions homologous to epidermal growth factor and transforming growth factor are encoded by amino acids 46-91 and 92-136 and are precisely delimited by introns, as is a similar sequence in the genes for coagulation factor IX and tissue plasminogen activator. When homologous amino acids of factor IX and protein C are aligned, the positions of all eight introns correspond precisely, suggesting that these genes are the product of a relatively recent gene duplication. Nevertheless, the two genes are sufficiently distantly related that no nucleic acid homology remains in the intronic regions and that the size of the introns varies dramatically between the two genes. The similarity of the genes for factor IX and protein C suggests that they may be the most closely related members of the serine protease gene family involved in coagulation and fibrinolysis.

  9. Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

    PubMed

    De Marchis, Francesca; Valeri, Maria Cristina; Pompa, Andrea; Bouveret, Emmanuelle; Alagna, Fiammetta; Grisan, Simone; Stanzione, Vitale; Mariotti, Roberto; Cultrera, Nicolò; Baldoni, Luciana; Bellucci, Michele

    2016-02-01

    Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3. PMID:26560313

  10. Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

    PubMed Central

    Sadiq, Fouzia; Crompton, Leslie A; Scaife, Jes R; Lomax, Michael A

    2008-01-01

    Background Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose (to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between

  11. Protein biosynthesis with conformationally restricted amino acids

    SciTech Connect

    Mendel, D. Lawrence Berkeley Lab., CA ); Ellman, J.; Schultz, P.G. )

    1993-05-19

    The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. The ability to site-specifically introduce constrained amino acids into large polypeptide chains would provide a similar opportunity to probe the flexibility, conformation, folding and stability of proteins. To this end, we have examined the competence of the Escherichia coli protein biosynthetic machinery to incorporate a number of these unnatural amino acids into the 164 residue protein T4 lysozyme (T4L). Results clearly demonstrate that the protein biosynthetic machinery can accommodate a wide variety of conformationally constrained amino acids. The expansion of structural motifs that can be biosynthetically incorporated into proteins to include a large number of conformationally constrained amino acids significantly increases the power of mutagenesis methods as probes of protein structure and function and provides additional insights into the steric requirements of the translational machinery. 13 refs., 2 figs.

  12. Divinyl ether synthase gene and protein, and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  13. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  14. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  15. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  16. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  17. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  18. Polyunsaturated fatty acids and gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans).

    PubMed

    Suriyanarayanan, Balasubramanian; Lakshmi, Praveena Pothuraju; Santhosh, Ramachandran Sarojini; Dhevendaran, Kandasamy; Priya, Balakrishnan; Krishna, Shivaani

    2016-06-01

    Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms. PMID:26198082

  20. Nucleotide sequence of Bacillus phage Nf terminal protein gene.

    PubMed Central

    Leavitt, M C; Ito, J

    1987-01-01

    The nucleotide sequence of Bacillus phage Nf gene E has been determined. Gene E codes for phage terminal protein which is the primer necessary for the initiation of DNA replication. The deduced amino acid sequence of Nf terminal protein is approximately 66% homologous with the terminal proteins of Bacillus phages PZA and luminal diameter 29, and shows similar hydropathy and secondary structure predictions. A serine which has been identified as the residue which covalently links the protein to the 5' end of the genome in luminal diameter 29, is conserved in all three phages. The hydropathic and secondary structural environment of this serine is similar in these phage terminal proteins and also similar to the linking serine of adenovirus terminal protein. PMID:3601672

  1. Non-protein amino acids and neurodegeneration: the enemy within.

    PubMed

    Rodgers, Kenneth J

    2014-03-01

    Animals, in common with plants and microorganisms, synthesise proteins from a pool of 20 protein amino acids (plus selenocysteine and pyrolysine) (Hendrickson et al., 2004). This represents a small proportion (~2%) of the total number of amino acids known to exist in nature (Bell, 2003). Many 'non-protein' amino acids are synthesised by plants, and in some cases constitute part of their chemical armoury against pathogens, predators or other species competing for the same resources (Fowden et al., 1967). Microorganisms can also use selectively toxic amino acids to gain advantage over competing organisms (Nunn et al., 2010). Since non-protein amino acids (and imino acids) are present in legumes, fruits, seeds and nuts, they are ubiquitous in the diets of human populations around the world. Toxicity to humans is unlikely to have been the selective force for their evolution, but they have the clear potential to adversely affect human health. In this review we explore the links between exposure to non-protein amino acids and neurodegenerative disorders in humans. Environmental factors play a major role in these complex disorders which are predominantly sporadic (Coppede et al., 2006). The discovery of new genes associated with neurodegenerative diseases, many of which code for aggregation-prone proteins, continues at a spectacular pace but little progress is being made in identifying the environmental factors that impact on these disorders. We make the case that insidious entry of non-protein amino acids into the human food chain and their incorporation into protein might be contributing significantly to neurodegenerative damage. PMID:24374297

  2. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    PubMed Central

    Thomsen, Rune; Daugaard, Tina F.; Holm, Ida E.; Nielsen, Anders Lade

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease. PMID:23991052

  3. From Gene Mutation to Protein Characterization

    ERIC Educational Resources Information Center

    Moffet, David A.

    2009-01-01

    A seven-week "gene to protein" laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the…

  4. Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

    PubMed Central

    Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J.; Peng, Deliang

    2016-01-01

    Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in

  5. Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

    PubMed

    Qiao, Fen; Luo, Lilian; Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J; Peng, Deliang

    2016-01-01

    Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in

  6. Protective effect of chronic caffeine intake on gene expression of brain derived neurotrophic factor signaling and the immunoreactivity of glial fibrillary acidic protein and Ki-67 in Alzheimer’s disease

    PubMed Central

    Ghoneim, Fatma M; Khalaf, Hanaa A; Elsamanoudy, Ayman Z; Abo El-khair, Salwa M; Helaly, Ahmed MN; Mahmoud, El-Hassanin M; Elshafey, Saad H

    2015-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disorder with progressive degeneration of the hippocampal and cortical neurons. This study was designed to demonstrate the protective effect of caffeine on gene expression of brain derived neurotrophic factor (BDNF) and its receptor neural receptor protein-tyrosine kinase-β (TrkB) as well as glial fibrillary acidic protein (GFAP) and Ki-67 immunoreactivity in Aluminum chloride (AlCl3) induced animal model of AD. Fifty adult rats included in this study were classified into 5 group (10 rats each); negative and positive control groups (I&II), AD model group (III), group treated with caffeine from the start of AD induction (IV) and group treated with caffeine two weeks before AD induction (V). Hippocampal tissue BDNF and its receptor (TrkB) gene expression by real time RT-PCR in addition to immunohistochemical study of GFAP and Ki67 immunoreactivity were performed for all rats in the study. The results of this study revealed that caffeine has protective effect through improving the histological and immunohistochemical findings induced by AlCl3 as well as BDNF and its receptor gene expression. It could be concluded from the current study, that chronic caffeine consumption in a dose of 1.5 mg/kg body weight daily has a potentially good protective effect against AD. PMID:26339337

  7. The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

    PubMed Central

    Evers, R; Cornelissen, A W

    1990-01-01

    We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit. Images PMID:2169604

  8. The Ala54Thr Polymorphism of the Fatty Acid Binding Protein 2 Gene Modulates HDL Cholesterol in Mexican-Americans with Type 2 Diabetes

    PubMed Central

    Salto, Lorena M.; Bu, Liming; Beeson, W. Lawrence; Firek, Anthony; Cordero-MacIntyre, Zaida; De Leon, Marino

    2015-01-01

    The alanine to threonine amino acid substitution at codon 54 (Ala54Thr) of the intestinal fatty acid binding protein (FABP2) has been associated with elevated levels of insulin and blood glucose as well as with dyslipidemia. The aim of this study was to characterize the effect of this FABP2 polymorphism in Mexican-Americans with type 2 diabetes (T2D) in the context of a three-month intervention to determine if the polymorphism differentially modulates selected clinical outcomes. For this study, we genotyped 43 participant samples and performed post-hoc outcome analysis of the profile changes in fasting blood glucose, HbA1c, insulin, lipid panel and body composition, stratified by the Ala54Thr polymorphism. Our results show that the Thr54 allele carriers (those who were heterozygous or homozygous for the threonine-encoding allele) had lower HDL cholesterol and higher triglyceride levels at baseline compared to the Ala54 homozygotes (those who were homozygous for the alanine-encoding allele). Both groups made clinically important improvements in lipid profiles and glycemic control as a response to the intervention. Whereas the Ala54 homozygotes decreased HDL cholesterol in the context of an overall total cholesterol decrease, Thr54 allele carriers increased HDL cholesterol as part of an overall total cholesterol decrease. We conclude that the Ala54Thr polymorphism of FABP2 modulates HDL cholesterol in Mexican-Americans with T2D and that Thr54 allele carriers may be responsive in interventions that include dietary changes. PMID:26703680

  9. Amino acid repeats and the structure and evolution of proteins.

    PubMed

    Albà, M M; Tompa, P; Veitia, R A

    2007-01-01

    Many proteins have repeats or runs of single amino acids. The pathogenicity of some repeat expansions has fueled proteomic, genomic and structural explorations of homopolymeric runs not only in human but in a wide variety of other organisms. Other types of amino acid repetitive structures exhibit more complex patterns than homopeptides. Irrespective of their precise organization, repetitive sequences are defined as low complexity or simple sequences, as one or a few residues are particularly abundant. Prokaryotes show a relatively low frequency of simple sequences compared to eukaryotes. In the latter the percentage of proteins containing homopolymeric runs varies greatly from one group to another. For instance, within vertebrates, amino acid repeat frequency is much higher in mammals than in amphibians, birds or fishes. For some repeats, this is correlated with the GC-richness of the regions containing the corresponding genes. Homopeptides tend to occur in disordered regions of transcription factors or developmental proteins. They can trigger the formation of protein aggregates, particularly in 'disease' proteins. Simple sequences seem to evolve more rapidly than the rest of the protein/gene and may have a functional impact. Therefore, they are good candidates to promote rapid evolutionary changes. All these diverse facets of homopolymeric runs are explored in this review. PMID:18753788

  10. Porin protein of Neisseria gonorrhoeae: cloning and gene structure.

    PubMed Central

    Gotschlich, E C; Seiff, M E; Blake, M S; Koomey, M

    1987-01-01

    The outer membrane porin molecule of Neisseria gonorrhoeae is known as protein I (PI). Among different strains of gonococci there is variability of PI, and two main classes, PIA and PIB, have been recognized. A lambda gt11 bank of gonococcal DNA was screened using monoclonal antibodies directed to a PIB-type porin molecule of N. gonorrhoeae, and three immunoreactive clones were isolated. DNA sequence analysis indicated that each contained only portions of the PI structural gene, but that together they contained the complete gene, and its structure was determined. The DNA sequence predicts a protein of 348 amino acids with a typical 19 amino acid signal peptide. The PI protein resembles Escherichia coli porins in size, lack of long hydrophobic sequences, and absence of cysteine residues. Sequence homologies between PI and the E. coli porins were found, particularly in the 100 N-terminal and the 110 C-terminal amino acids. In addition to the coding sequence of PI, the complementary strand contains a large open reading frame. At the 3' end of the PI gene, immediately following an inverted repeat (probably the transcription terminator), the clone contains an unusual sequence consisting of 31 perfect repeats of the heptamer CTGTTTT. Hybridization analysis suggests that there is a single structural gene for PI and that it is homologous to the gene found in a PIA-bearing strain of gonococcus. Images PMID:2825179

  11. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  12. Correlation of gene and protein structures in the FXYD family proteins.

    PubMed

    Franzin, Carla M; Yu, Jinghua; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M

    2005-12-01

    The FXYD family proteins are auxiliary subunits of the Na,K-ATPase, expressed primarily in tissues that specialize in fluid or solute transport, or that are electrically excitable. These proteins range in size from about 60 to 160 amino acid residues, and share a core homology of 35 amino acid residues in and around a single transmembrane segment. Despite their relatively small sizes, they are all encoded by genes with six to nine small exons. We show that the helical secondary structures of three FXYD family members, FXYD1, FXYD3, and FXYD4, determined in micelles by NMR spectroscopy, reflect the structures of their corresponding genes. The coincidence of helical regions, and connecting segments, with the positions of intron-exon junctions in the genes, support the hypothesis that the FXYD proteins may have been assembled from discrete structural modules through exon shuffling. PMID:16288923

  13. Probing protein stability with unnatural amino acids

    SciTech Connect

    Mendel, D.; Ellman, J.A.; Zhiyuh Chang; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G. )

    1992-06-26

    Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (1) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (2) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (3) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

  14. (Genetic engineering with a gene encoding a soybean storage protein)

    SciTech Connect

    Beachy, R.N.

    1985-12-18

    We have isolated and characterized a gene which encodes the alpha prime subunit of beta conglycinin. This gene was fully sequenced by DNA sequence analysis and a report of that work was prepared and submitted for publication in early November 1985. This represented the culmination of several years of research effort by several scientists. A preprint of that work is attached to this report and has been offered by Dr. J.J. Doyle, Dr. Mary A. Schuler and Dr. Jerry Slighton, as well as myself. This paper is a comparison of the alpha prime subunit gene with a similar gene from phaseolus vulgaris, the common garden bean. In this paper we compare the sequences that are 5' of the gene, and which would represent the transcriptional promoter, as well as the sequences within the structural region of the gene. The sequence paper also compares the amino acid sequence of these two genes with that of other genes from Phaseolus, peas and from soybeans. On the basis of this comparison, we predict evolutionary trends within the multigene families which encode these proteins in the various plants, as well as to look at the protein itself to try to predict regions of the protein that might have functional significance. All of this work was done on a prior DOE-BER grant and has simply been reported here for the first time.

  15. Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes*

    PubMed Central

    Choi, Won-Il; Yoon, Jae-Hyeon; Kim, Min-Young; Koh, Dong-In; Licht, Jonathan D.; Kim, Kunhong; Hur, Man-Wook

    2014-01-01

    Promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) is an oncogene transcriptional repressor that is generated by a chromosomal translocation between the PLZF and RARα genes in acute promyelocytic leukemia (APL-type) patients. The molecular interaction between PLZF-RARα and the histone deacetylase corepressor was proposed to be important in leukemogenesis. We found that PLZF-RARα can repress transcription of the p21WAF/CDKN1A gene, which encodes the negative cell cycle regulator p21 by binding to its proximal promoter Sp1-binding GC-boxes 3, 4, 5/6, a retinoic acid response element (RARE), and distal p53-responsive elements (p53REs). PLZF-RARα also acts as a competitive transcriptional repressor of p53, RARα, and Sp1. PLZF-RARα interacts with co-repressors such as mSin3A, NCoR, and SMRT, thereby deacetylating histones Ac-H3 and Ac-H4 at the CDKN1A promoter. PLZF-RARα also interacts with the MBD3-NuRD complex, leading to epigenetic silencing of CDKN1A through DNA methylation. Furthermore, PLZF-RARα represses TP53 and increases p53 protein degradation by ubiquitination, further repressing p21 expression. Resultantly, PLZF-RARα promotes cell proliferation and significantly increases the number of cells in S-phase. PMID:24821728

  16. Cycloheximide resistance in yeast: the gene and its protein.

    PubMed Central

    Käufer, N F; Fried, H M; Schwindinger, W F; Jasin, M; Warner, J R

    1983-01-01

    Mutations in the yeast gene CYH2 can lead to resistance to cycloheximide, an inhibitor of eukaryotic protein synthesis. The gene product of CYH2 is ribosomal protein L29, a component of the 60S ribosomal subunit. We have cloned the wild-type and resistance alleles of CYH2 and determined their nucleotide sequence. Transcription of CYH2 appears to initiate and terminate at multiple sites, as judged by S1 nuclease analysis. The gene is transcribed into an RNA molecule of about 1082 nucleotides, containing an intervening sequence of 510 nucleotides. The splice junction of the intron resides within a codon near the 5' end of the gene. In confirmation of peptide analysis by Stocklein et al. (1) we find that resistance to cycloheximide is due to a transversion mutation resulting in the replacement of a glutamine by glutamic acid in position 37 of L29. Images PMID:6304624

  17. Abscisic acid represses the transcription of chloroplast genes*

    PubMed Central

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

    2013-01-01

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

  18. A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

    PubMed

    Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

    2015-10-01

    During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. PMID:25926684

  19. Making the Chromosome-Gene-Protein Connection.

    ERIC Educational Resources Information Center

    Mulvihill, Charlotte

    1996-01-01

    Presents an exercise that demonstrates the chromosome-gene-protein connection using sickle-cell anemia, a genetic disease with a well-characterized molecular basis. Involves connecting changes in DNA to protein outcomes and tying them into the next generation by meiosis and gamete formation with genetic crosses. Motivates students to integrate…

  20. Human protein kinase CK2 genes.

    PubMed

    Wirkner, U; Voss, H; Lichter, P; Pyerin, W

    1994-01-01

    We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome. PMID:7735323

  1. Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

    PubMed Central

    Schultz, L D; Friesen, J D

    1983-01-01

    The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes. PMID:6305925

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses

    PubMed Central

    Guan, Jiahn-Chou; Yeh, Ching-Hui; Lin, Ya-Ping; Ke, Yi-Ting; Chen, Ming-Tse; You, Jia-Wen; Liu, Yi-Hsin; Lu, Chung-An; Wu, Shaw-Jye; Lin, Chu-Yung

    2010-01-01

    In rice, the class I small heat shock protein (sHSP-CI) genes were found to be selectively induced by L-azetidine-2-carboxylic acid (AZC) on chromosome 3 but not chromosome 1. Here it is shown that a novel cis-responsive element contributed to the differential regulation. By serial deletion and computational analysis, a 9 bp putative AZC-responsive element (AZRE), GTCCTGGAC, located between nucleotides –186 and –178 relative to the transcription initiation site of Oshsp17.3 was revealed. Deletion of this putative AZRE from the promoter abolished its ability to be induced by AZC. Moreover, electrophoretic mobility shift assay (EMSA) revealed that the AZRE interacted specifically with nuclear proteins from AZC-treated rice seedlings. Two AZRE–protein complexes were detected by EMSA, one of which could be competed out by a canonical heat shock element (HSE). Deletion of the AZRE also affected the HS response. Furthermore, transient co-expression of the heat shock factor OsHsfA4b with the AZRE in the promoter of Oshsp17.3 was effective. The requirement for the putative AZRE for AZC and HS responses in transgenic Arabidopsis was also shown. Thus, AZRE represents an alternative form of heat HSE, and its interaction with canonical HSEs through heat shock factors may be required to respond to HS and AZC. PMID:20643810

  4. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  5. Nucleic acid-protein interactions: Wedding for love or circumstances?

    PubMed

    Lavelle, Christophe; Buckle, Malcolm

    2009-08-01

    The sixth Figeac meeting on nucleic acid-protein interactions was held in Figeac, France, from September 26th to October 1st, 2008. It was organized by the working group "nucleic acid-protein interactions and gene expression" from the French Society for Biochemistry and Molecular Biology. This report briefly summarizes the presentations by 40 speakers during the four plenary sessions, which were organised as follows: (1) nucleic acids: targets and tools, (2) RNA superstar, (3) nuclear structure and dynamics, and (4) new concepts - new approaches. A total of 22 plenary lectures, 18 oral communications and 40 posters were presented over the 5 days, providing a highly stimulating environment for scientific exchange between the approximately 80 participants (biochemists, physicists, bio-informaticians and molecular and cellular biologists). PMID:19422875

  6. Protein and amino Acid supplementation in athletes.

    PubMed

    Armsey, Thomas D; Grime, Todd E

    2002-08-01

    Amino acid supplementation is practiced by numerous individuals with the hope of increasing muscle mass and function by increasing available proteins. Theoretically, this makes a great deal of sense; the scientific facts, however, fail to conclusively prove that ingesting more than the recommended dietary allowance of protein has any effect on otherwise healthy adults. Athletes may be the exception to this rule. This review examines the most current literature pertaining to amino acid supplementation, and reports on the potential benefits and risks of this common practice. PMID:12831703

  7. New mutation in Delta-9-Stearoyl-Acyl Carrier Protein desaturase gene associated with enhanced stearic acid levels in soybean seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean [Glycine max (L.) Merr] oil from conventional cultivars typically contains ~3% stearic acid of the total seed oil. Increased stearic acid concentration in the seed oil of soybeans is desirable from both food and industrial use stand-points. To date a small number of mutants have been develop...

  8. Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability

    PubMed Central

    Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

    2015-01-01

    Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability. PMID:26056817

  9. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  10. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  11. DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.

    PubMed Central

    Dong, Q; Ebright, R H

    1992-01-01

    The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2. PMID:1322886

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  14. Dihydrolipoic acid reduces cytochrome b561 proteins.

    PubMed

    Bérczi, Alajos; Zimányi, László; Asard, Han

    2013-03-01

    Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins. PMID:22526465

  15. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html. PMID:8594596

  16. Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

    DOEpatents

    Craft, David L.; Madduri, Krishna M.; Loper, John C.

    2003-01-01

    A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

  17. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  18. Common Variants of the Liver Fatty Acid Binding Protein Gene Influence the Risk of Type 2 Diabetes and Insulin Resistance in Spanish Population

    PubMed Central

    Mansego, Maria Luisa; Martínez, Fernando; Martínez-Larrad, Maria Teresa; Zabena, Carina; Rojo, Gemma; Morcillo, Sonsoles; Soriguer, Federico; Martín-Escudero, Juan Carlos; Serrano-Ríos, Manuel; Redon, Josep; Chaves, Felipe Javier

    2012-01-01

    Summary The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. Methods 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes). DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1-4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. Results One polymorphism (rs2197076) and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. Conclusions The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians. PMID:22396741

  19. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  20. The Exchangeability of Amino Acids in Proteins

    PubMed Central

    Yampolsky, Lev Y.; Stoltzfus, Arlin

    2005-01-01

    The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of “experimental exchangeability” values EXij derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance. PMID:15944362

  1. Structure of the infected cell protein 0 gene of canine herpesvirus.

    PubMed

    Miyoshi, M; Takiguchi, M; Yasuda, J; Hashimoto, A; Takada, A; Okazaki, K; Kida, H

    2000-01-01

    The canine herpesvirus infected cell protein 0 (CICP0) gene was sequenced. The CICP0 gene was transcribed as a 1.4 kb mRNA from the end of the unique long region nearby the internal repeat during early phase of productive infection of the virus. An open reading frame of the gene encodes a polypeptide of 333 amino acids. The RING finger domain and acidic transcriptional activation domain were found at the N-terminus and within the middle region in the deduced amino acid sequence, respectively, suggesting that the CICP0, like the ICP0 of herpes simplex virus 1, is a transactivating protein. PMID:11003479

  2. A single-nucleotide polymorphism in the 3′-UTR region of the adipocyte fatty acid binding protein 4 gene is associated with prognosis of triple-negative breast cancer

    PubMed Central

    Wang, Wenmiao; Yuan, Peng; Yu, Dianke; Du, Feng; Zhu, Anjie; Li, Qing; Zhang, Pin; Lin, Dongxin; Xu, Binghe

    2016-01-01

    Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and high heterogeneity. The aim of this study was to screen patients for single-nucleotide polymorphisms (SNPs) associated with the prognosis of TNBC. Database-derived SNPs (NextBio, Ensembl, NCBI and MirSNP) located in the 3′-untranslated regions (3′-UTRs) of genes that are differentially expressed in breast cancer were selected. The possible associations between 111 SNPs and progression risk among 323 TNBC patients were investigated using a two-step case-control study with a discovery cohort (n=162) and a validation cohort (n=161). We identified the rs1054135 SNP in the adipocyte fatty acid binding protein 4 (FABP4) gene as a predictor of TNBC recurrence. The G allele of rs1054135 was associated with a reduced risk of disease progression as well as a prolonged disease-free survival time (DFS), with a hazard ratio (HR) for recurrence in the combined sample of 0.269 [95%CI: 0.098−0.735;P=0.001]. Notably, for individuals having the rs1054135 SNP with the AA/AG genotype, the magnitude of increased tumour recurrence risk for overweight patients (BMI≥25kg/m2) was significantly elevated (HR2.53; 95%CI: 1.06–6.03). Immunohistochemical staining of adipocytes adjacent to TNBC tissues showed that the expression level of FABP4 was statistically significantly lower in patients with the rs1054135-GG genotype and those in the disease-free group (P=0.0004 and P=0.0091, respectively). These results suggested that the expression of a lipid metabolism-related gene and an important SNP in the 3′-UTR of FABP4 are associated with TNBC prognosis, which may aid in the screening of high-risk patients with TNBC recurrence and the development of novel chemotherapeutic agents. PMID:26959740

  3. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  4. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  5. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene.

    PubMed

    Cerretti, D P; Dean, D; Davis, G R; Bedwell, D M; Nomura, M

    1983-05-11

    The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome. One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O). We now report the entire 5.9 kb nucleotide sequence of the spc operon. DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon. It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon. One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export. In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon. PMID:6222285

  6. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins

    NASA Astrophysics Data System (ADS)

    Tang, Nicholas C.; Chilkoti, Ashutosh

    2016-04-01

    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  7. Protein and Amino Acid Requirements during Pregnancy.

    PubMed

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

  8. Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

    NASA Astrophysics Data System (ADS)

    Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

    2014-04-01

    At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)

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

    SciTech Connect

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

    2000-02-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism.

    PubMed

    Rigamonti, Elena; Parolini, Cinzia; Marchesi, Marta; Diani, Erika; Brambilla, Stefano; Sirtori, Cesare R; Chiesa, Giulia

    2010-05-01

    Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Nath's hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (p<0.05). Pea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (p<0.05). Hepatic mRNA concentration of genes involved in fatty acids synthesis, such as fatty acid synthase and stearoyl-CoA desaturase, was lower in pea protein-fed rats than in rats fed casein (p<0.05). In conclusion, the present study demonstrates a marked cholesterol and triglyceride-lowering activity of pea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes. PMID:20077421

  12. Jussara (Euterpe edulis Mart.) Supplementation during Pregnancy and Lactation Modulates the Gene and Protein Expression of Inflammation Biomarkers Induced by trans-Fatty Acids in the Colon of Offspring

    PubMed Central

    Almeida Morais, Carina; Oyama, Lila Missae; de Oliveira, Juliana Lopez; Carvalho Garcia, Márcia; de Rosso, Veridiana Vera; Sousa Mendes Amigo, Laís; do Nascimento, Claudia Maria Oller; Pisani, Luciana Pellegrini

    2014-01-01

    Maternal intake of trans-fatty acids (TFAs) in the perinatal period triggers a proinflammatory state in offspring. Anthocyanins contained in fruit are promising modulators of inflammation. This study investigated the effect of Jussara supplementation in the maternal diet on the proinflammatory state of the colon in offspring exposed to perinatal TFAs. On the first day of pregnancy rats were divided into four groups: control diet (C), control diet with 0.5% Jussara supplementation (CJ), diet enriched with hydrogenated vegetable fat, rich in TFAs (T), or T diet supplemented with 0.5% Jussara (TJ) during pregnancy and lactation. We showed that Jussara supplementation in maternal diet (CJ and TJ groups) reduced carcass lipid/protein ratios, serum lipids, glucose, IL-6, TNF-α, gene expression of IL-6R, TNF-αR (P < 0.05), TLR-4 (P < 0.01), and increase Lactobacillus spp. (P < 0.05) in the colon of offspring compared to the T group. The IL-10 (P = 0.035) and IL-10/TNF-α ratio (P < 0.01) was higher in the CJ group than in the T group. The 0.5% Jussara supplementation reverses the adverse effects of perinatal TFAs, improving lipid profiles, glucose levels, body composition, and gut microbiota and reducing low-grade inflammation in the colon of 21-day-old offspring, and could contribute to reducing chronic disease development. PMID:25276060

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

    PubMed

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

    2015-03-01

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

  14. Stress genes and proteins in the archaea.

    PubMed

    Macario, A J; Lange, M; Ahring, B K; Conway de Macario, E

    1999-12-01

    The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of approximately 60 k

  15. Stress Genes and Proteins in the Archaea

    PubMed Central

    Macario, Alberto J. L.; Lange, Marianne; Ahring, Birgitte K.; De Macario, Everly Conway

    1999-01-01

    The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of ∼60 kDa. These

  16. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  17. Cloning, sequencing, gene organization, and localization of the human ribosomal protein RPL23A gene

    SciTech Connect

    Fan, Wufang; Christensen, M.; Eichler, E.

    1997-12-01

    The intron-containing gene for human ribosomal protein RPL23A has been cloned, sequenced, and localized. The gene is approximately 4.0 kb in length and contains five exons and four introns. All splice sites exactly match the AG/GT consensus rule. The transcript is about 0.6 kb and is detected in all tissues examined. In adult tissues, the RPL23A transcript is dramatically more abundant in pancreas, skeletal muscle, and heart, while much less abundant in kidney, brain, placenta, lung, and liver. A full-length cDNA clone of 576 nt was identified, and the nucleotide sequence was found to match the exon sequence precisely. The open reading frame encodes a polypeptide of 156 amino acids, which is absolutely conserved with the rat RPL23A protein. In the 5{prime} flanking region of the gene, a canonical TATA sequence and a defined CAAT box were found for the first time in a mammalian ribosomal protein gene. The intron-containing RPL23A gene was mapped to cytogenetic band 17q11 by fluorescence in situ hybridization. 33 refs., 4 figs.

  18. Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC).

    PubMed

    Hubner, Nina C; Mann, Matthias

    2011-04-01

    Large-scale proteomic screens are increasingly employed for placing genes into specific pathways. Therefore generic methods providing a physiological context for protein-protein interaction studies are of great interest. In recent years many protein-protein interactions have been determined by affinity purification followed by mass spectrometry (AP-MS). Among many different AP-MS approaches, the recently developed Quantitative BAC InteraCtomics (QUBIC) approach is particularly attractive as it uses tagged, full-length baits that are expressed under endogenous control. For QUBIC large cell line collections expressing tagged proteins from BAC transgenes or gene trap loci have been developed and are freely available. Here we describe detailed workflows on how to obtain specific protein binding partners with high confidence under physiological conditions. The methods are based on fast, streamlined and generic purification procedures followed by single run liquid chromatography-mass spectrometric analysis. Quantification is achieved either by the stable isotope labeling of amino acids in cell culture (SILAC) method or by a 'label-free' procedure. In either case data analysis is performed by using the freely available MaxQuant environment. The QUBIC approach enables biologists with access to high resolution mass spectrometry to perform small and large-scale protein interactome mappings. PMID:21184827

  19. Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene.

    PubMed

    Li, Z Y; Song, H Q; Wang, C R; Zhu, X Q

    2016-01-01

    Toxoplasma gondii is a successful opportunistic protozoan distributed worldwide, which can infect all vertebrates, leading to serious infection, blindness, and abortion. Micronemal (MIC) proteins are critically important for T. gondii infection, as they participate in various stages of the Toxoplasma life cycle, including invasion and attachment to host cells. MIC8 secretion relies on the concentration of intracellular calcium, and can mediate the invasion of T. gondii by interacting with soluble MIC3. To investigate genetic diversity of the MIC8 gene, 16 T. gondii strains from different hosts and geographical locations, and two reference isolates (ToxoDB: TGME49_245490 and TGVEG_245490) were examined in this study. The results showed that all the examined MIC8 genes are 2055 bp, with an A+T content ranging from 50.2 to 50.6%. Conversely, lower levels of variation were detected within their nucleotide and amino acid sequences. Phylogenetic analyses indicated that three classical genotypes of T. gondii and the ToxoDB#9 genotype did not group exclusively via Bayesian inference, maximum parsimony, neighbor joining, and/or maximum likelihood assays based on the nucleotide and amino acid sequences of the MIC8 gene. In summary, the T. gondii MIC8 gene is not a suitable marker for population genetic studies of this parasite. PMID:27173337

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

    PubMed Central

    Williford, Anna; Demuth, Jeffery P.

    2012-01-01

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

  1. Expression and Functional Characterization of two Pathogenesis-Related Protein 10 Genes from Zea mays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogenesis-related protein 10 (PR10) is one of seventeen PR protein families and plays important roles in plant response to biotic and abiotic stresses. A novel PR10 gene (ZmPR10.1), which shares 89.8% and 85.7% identity to the previous ZmPR10 at the nucleotide and amino acid sequence level, respe...

  2. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

    PubMed

    Salwiczek, Mario; Nyakatura, Elisabeth K; Gerling, Ulla I M; Ye, Shijie; Koksch, Beate

    2012-03-21

    Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references). PMID:22130572

  3. Oleic acid enhances G protein coupled receptor 43 expression in bovine intramuscular adipocytes but not in subcutaneous adipocytes.

    PubMed

    Chung, K Y; Smith, S B; Choi, S H; Johnson, B J

    2016-05-01

    We hypothesized that fatty acids would differentially affect G protein coupled receptor (GPR) 43 mRNA expression and GPR43 protein concentrations in bovine intramuscular (IM) and subcutaneous (SC) adipocytes. The GPR43 protein was detected in bovine liver, pancreas, and semimembranosus (MUS) muscle in samples taken at slaughter. Similarly, GPR43 protein levels were similar in IM adipose tissue and SM muscle but was barely detectable in SC adipose tissue. Primary cultures of IM and SC stromal vascular cells were isolated from bovine adipose tissues. Oleic acid (100 μ) stimulated PPARγ gene expression and decreased stearoyl-CoA desaturase (SCD) gene expression but had no effect on GPR43 gene expression, which was readily detectable in both IM and SC adipocytes. Differentiation cocktail (Diff; 10 μ insulin, 4 μ dexamethasone, and 10 μ ciglitizone) stimulated CCAAT/enhancer-binding protein β (C/EBPβ) and PPARγ gene expression in SC but not IM adipocytes, but Diff increased SCD gene expression in both cell types. Linoleic acid (10 µ) increased PPARγ gene expression relative to Diff cocktail in SC adipocytes, whereas linoleic acid and α-linolenic decreased SCD gene expression relative to control adipocytes and adipocytes incubated with Diff ( < 0.05). Increasing concentrations of oleic acid (1, 10, 100, and 500 μM) increased GPR43 protein and mRNA expression in IM but not SC adipocytes. These data indicated that oleic acid alters mRNA and protein concentrations of GPR43 in bovine IM adipocytes. PMID:27285685

  4. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  5. Foamy Virus Protein-Nucleic Acid Interactions during Particle Morphogenesis.

    PubMed

    Hamann, Martin V; Lindemann, Dirk

    2016-01-01

    Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches. PMID:27589786

  6. Characterization of three novel fatty acid- and retinoid-binding protein genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the cereal cyst nematodes Heterodera avenae and H. filipjevi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinoid-binding (FAR) proteins are nematode-spe...

  7. Regulation of gene transcription by Polycomb proteins

    PubMed Central

    Aranda, Sergi; Mas, Gloria; Di Croce, Luciano

    2015-01-01

    The Polycomb group (PcG) of proteins defines a subset of factors that physically associate and function to maintain the positional identity of cells from the embryo to adult stages. PcG has long been considered a paradigmatic model for epigenetic maintenance of gene transcription programs. Despite intensive research efforts to unveil the molecular mechanisms of action of PcG proteins, several fundamental questions remain unresolved: How many different PcG complexes exist in mammalian cells? How are PcG complexes targeted to specific loci? How does PcG regulate transcription? In this review, we discuss the diversity of PcG complexes in mammalian cells, examine newly identified modes of recruitment to chromatin, and highlight the latest insights into the molecular mechanisms underlying the function of PcGs in transcription regulation and three-dimensional chromatin conformation. PMID:26665172

  8. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  9. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  10. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  11. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  12. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  13. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  14. Identification of new human cadherin genes using a combination of protein motif search and gene finding methods.

    PubMed

    Hoeng, Julia C; Höng, Julia C; Ivanov, Nikolai V; Hodor, Paul; Xia, Menghang; Wei, Nan; Blevins, Richard; Gerhold, David; Borodovsky, Mark; Liu, Yuan

    2004-03-19

    We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains. PMID:15003449

  15. General Theory for Integrated Analysis of Growth, Gene, and Protein Expression in Biofilms

    PubMed Central

    Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S.

    2013-01-01

    A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques. PMID:24376726

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

    PubMed

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

    2015-08-01

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

  17. Molecular mechanisms of ribosomal protein gene coregulation.

    PubMed

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B Franklin

    2015-09-15

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. PMID:26385964

  18. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    NASA Technical Reports Server (NTRS)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  19. The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

    PubMed

    Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

    1992-02-01

    The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

  20. The structure of the human sterol carrier protein X/sterol carrier protein 2 gene (SCP2)

    SciTech Connect

    Ohba, Takashi; Rennert, H.; Pfeifer, S.M.

    1994-11-15

    Sterol carrier protein X (SCPx) is a 58-kDa protein that is localized to peroxisomes. The amino acid sequence of the protein suggests that SCPx may function as a thiolase. The gene encoding SCPx also codes for a 15.3-kDa protein called sterol carrier protein 2 (SCP{sub 2}). Here the authors report the structure of this gene (SCP2), which spans approximately 80 kb and consists of 16 exons and 15 introns. Multiple transcription start sites were identified. The 5{prime} flanking region has characteristics of other peroxisomal protein promoters, which include the absence of a TATA box and G+C-enriched region containing several reverse GC boxes. 24 refs., 3 figs., 1 tab.

  1. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    PubMed

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  2. Relatedness of acyl carrier proteins shown by amino acid compositions.

    PubMed

    Walker, T A; Ernst-Fonberg, M L

    1982-01-01

    1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552. PMID:7128903

  3. Retinoic acid-binding protein, rhombomeres and the neural crest.

    PubMed

    Maden, M; Hunt, P; Eriksson, U; Kuroiwa, A; Krumlauf, R; Summerbell, D

    1991-01-01

    We have investigated by immunocytochemistry the spatial and temporal distribution of cellular retinoic acid-binding protein (CRABP) in the developing nervous system of the chick embryo in order to answer two specific questions: do neural crest cells contain CRABP and where and when do CRABP-positive neuroblasts first arise in the neural tube? With regard to the neural crest, we have compared CRABP staining with HNK-1 staining (a marker of migrating neural crest) and found that they do indeed co-localise, but cephalic and trunk crest behave slightly differently. In the cephalic region in tissues such as the frontonasal mass and branchial arches, HNK-1 immunoreactivity is intense at early stages, but it disappears as CRABP immunoreactivity appears. Thus the two staining patterns do not overlap, but are complementary. In the trunk, HNK-1 and CRABP stain the same cell populations at the same time, such as those migrating through the anterior halves of the somites. In the neural tube, CRABP-positive neuroblasts first appear in the rhombencephalon just after the neural folds close and then a particular pattern of immunoreactivity appears within the rhombomeres of the hindbrain. Labelled cells are present in the future spinal cord, the posterior rhombencephalon up to rhombomere 6 and in rhombomere 4 thus producing a single stripe pattern. This pattern is dynamic and gradually changes as anterior rhombomeres begin to label. The similarity of this initial pattern to the arrangement of certain homeobox genes in the mouse stimulated us to examine the expression of the chicken Hox-2.9 gene. We show that at stage 15 the pattern of expression of this gene is closely related to that of CRABP. The relationship between retinoic acid, CRABP and homeobox genes is discussed. PMID:1707786

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

    PubMed Central

    2012-01-01

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

  5. Beta-galactosidase and selective neutrality. [amino acid composition of proteins

    NASA Technical Reports Server (NTRS)

    Holmquist, R.

    1979-01-01

    Three hypotheses to explain the amino acid composition of proteins are inconsistent (about 10 to the minus 9th) with the experimental data for beta-galactosidase from Escherichia coli. The exceptional length of this protein, 1021 residues, permits rigorous tests of these hypotheses without complication from statistical artifacts. Either this protein is not at compositional equilibrium, which is unlikely from knowledge about other proteins, or the evolution of this protein and its coding gene have not been selectively neutral. However, the composition of approximately 60% of the molecule is consistent with either a selectively neutral or nonneutral evolutionary process.

  6. Optimizing Scoring Function of Protein-Nucleic Acid Interactions with Both Affinity and Specificity

    PubMed Central

    Yan, Zhiqiang; Wang, Jin

    2013-01-01

    Protein-nucleic acid (protein-DNA and protein-RNA) recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions) for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions. PMID:24098651

  7. Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes.

    PubMed

    Sawaki, Yoshiharu; Kobayashi, Yuriko; Kihara-Doi, Tomonori; Nishikubo, Nobuyuki; Kawazu, Tetsu; Kobayashi, Masatomo; Kobayashi, Yasufumi; Iuchi, Satoshi; Koyama, Hiroyuki; Sato, Shigeru

    2014-06-01

    Tolerance to soil acidity is an important trait for eucalyptus clones that are introduced to commercial forestry plantations in pacific Asian countries, where acidic soil is dominant in many locations. A conserved transcription factor regulating aluminum (Al) and proton (H⁺) tolerance in land-plant species, STOP1 (SENSITIVE TOPROTON RHIZOTOXICITY 1)-like protein, was isolated by polymerase chain reaction-based cloning, and then suppressed by RNA interference in hairy roots produced by Agrobacterium rhizogenes-mediated transformation. Eucalyptus STOP1-like protein complemented proton tolerance in an Arabidopsis thaliana stop1-mutant, and localized to the nucleus in a transient assay of a green fluorescent protein fusion protein expressed in tobacco leaves by Agrobacterium tumefaciens-mediated transformation. Genes encoding a citrate transporting MULTIDRUGS AND TOXIC COMPOUND EXTRUSION protein and an orthologue of ALUMINUM SENSITIVE 3 were suppressed in transgenic hairy roots in which the STOP1 orthologue was knocked down. In summary, we identified a series of genes for Al-tolerance in eucalyptus, including a gene for STOP1-like protein and the Al-tolerance genes it regulates. These genes may be useful for molecular breeding and genomic selection of elite clones to introduce into acid soil regions. PMID:24767110

  8. Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation

    PubMed Central

    Gao, Wei-wei; Xiao, Rong-quan; Peng, Bing-ling; Xu, Huan-teng; Shen, Hai-feng; Huang, Ming-feng; Shi, Tao-tao; Yi, Jia; Zhang, Wen-juan; Wu, Xiao-nan; Gao, Xiang; Lin, Xiang-zhi; Dorrestein, Pieter C.; Rosenfeld, Michael G.; Liu, Wen

    2015-01-01

    Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control. PMID:26080448

  9. RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

    SciTech Connect

    Reynolds, P.; Weber, S.; Prakash, L.

    1985-01-01

    The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures.

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

    PubMed

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

    2007-08-01

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

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

    PubMed Central

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

    2007-01-01

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

  12. Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

    PubMed Central

    Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

    2012-01-01

    Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

  13. Prediction of protein-protein interactions with clustered amino acids and weighted sparse representation.

    PubMed

    Huang, Qiaoying; You, Zhuhong; Zhang, Xiaofeng; Zhou, Yong

    2015-01-01

    With the completion of the Human Genome Project, bioscience has entered into the era of the genome and proteome. Therefore, protein-protein interactions (PPIs) research is becoming more and more important. Life activities and the protein-protein interactions are inseparable, such as DNA synthesis, gene transcription activation, protein translation, etc. Though many methods based on biological experiments and machine learning have been proposed, they all spent a long time to learn and obtained an imprecise accuracy. How to efficiently and accurately predict PPIs is still a big challenge. To take up such a challenge, we developed a new predictor by incorporating the reduced amino acid alphabet (RAAA) information into the general form of pseudo-amino acid composition (PseAAC) and with the weighted sparse representation-based classification (WSRC). The remarkable advantages of introducing the reduced amino acid alphabet is being able to avoid the notorious dimensionality disaster or overfitting problem in statistical prediction. Additionally, experiments have proven that our method achieved good performance in both a low- and high-dimensional feature space. Among all of the experiments performed on the PPIs data of Saccharomyces cerevisiae, the best one achieved 90.91% accuracy, 94.17% sensitivity, 87.22% precision and a 83.43% Matthews correlation coefficient (MCC) value. In order to evaluate the prediction ability of our method, extensive experiments are performed to compare with the state-of-the-art technique, support vector machine (SVM). The achieved results show that the proposed approach is very promising for predicting PPIs, and it can be a helpful supplement for PPIs prediction. PMID:25984606

  14. Analysis of protein-protein interactions in MCF-7 and MDA-MB-231 cell lines using phthalic acid chemical probes.

    PubMed

    Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

    2014-01-01

    Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein-protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

  15. Strained cycloalkynes as new protein sulfenic acid traps.

    PubMed

    Poole, Thomas H; Reisz, Julie A; Zhao, Weiling; Poole, Leslie B; Furdui, Cristina M; King, S Bruce

    2014-04-30

    Protein sulfenic acids are formed by the reaction of biologically relevant reactive oxygen species with protein thiols. Sulfenic acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein sulfenic acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing sulfenic acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein sulfenic acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivatives as concerted traps of sulfenic acids. These strained cycloalkynes react efficiently with sulfenic acids in proteins and small molecules yielding stable alkenyl sulfoxide products at rates more than 100× greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiological sulfur chemistry. Similar to the 1,3-dicarbonyl reagents, the BCN compounds distinguish the sulfenic acid oxoform from the thiol, disulfide, sulfinic acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown sulfenic acid sites and their parent proteins. PMID:24724926

  16. Atomic force microscopy and anodic porous allumina of nucleic acid programmable protein arrays.

    PubMed

    Nicolini, Claudio; Correia, Tercio Bezerra; Stura, Enrico; Larosa, Claudio; Spera, Rosanna; Pechkova, Eugenia

    2013-08-01

    The methodological aspects are here presented for the NAPPA (Nucleic Acid Programmable Protein Arrays) characterization by atomic force microscopy and anodic porous alumina. Anodic Porous Alumina represents also an advanced on chip laboratory for gene expression contained in an engineered plasmid vector. The results obtained with CdK2, CDKN1A, p53 and Jun test genes expressed on NAPPA and the future developments are discussed in terms of our pertinent and recent Patents and of their possibility to overcome some limitations of present fluorescence detection in probing protein-protein interaction in both basic sciences and clinical studies. PMID:23848275

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. What's that gene (or protein)? Online resources for exploring functions of genes, transcripts, and proteins

    PubMed Central

    Hutchins, James R. A.

    2014-01-01

    The genomic era has enabled research projects that use approaches including genome-scale screens, microarray analysis, next-generation sequencing, and mass spectrometry–based proteomics to discover genes and proteins involved in biological processes. Such methods generate data sets of gene, transcript, or protein hits that researchers wish to explore to understand their properties and functions and thus their possible roles in biological systems of interest. Recent years have seen a profusion of Internet-based resources to aid this process. This review takes the viewpoint of the curious biologist wishing to explore the properties of protein-coding genes and their products, identified using genome-based technologies. Ten key questions are asked about each hit, addressing functions, phenotypes, expression, evolutionary conservation, disease association, protein structure, interactors, posttranslational modifications, and inhibitors. Answers are provided by presenting the latest publicly available resources, together with methods for hit-specific and data set–wide information retrieval, suited to any genome-based analytical technique and experimental species. The utility of these resources is demonstrated for 20 factors regulating cell proliferation. Results obtained using some of these are discussed in more depth using the p53 tumor suppressor as an example. This flexible and universally applicable approach for characterizing experimental hits helps researchers to maximize the potential of their projects for biological discovery. PMID:24723265

  19. Induction of several acute-phase protein genes by heavy metals: A new class of metal-responsive genes

    SciTech Connect

    Yiangou, Minas; Ge, Xin; Carter, K.C.; Papaconstantinou, J. Shriners Burns Institute, Galveston, TX )

    1991-04-16

    Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals whereas very little is known about the effect of heavy metals on acute-phase-reactant genes. The authors have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants {alpha}{sub 1}-acid glycoprotein, C-reactive protein, {alpha}{sub 1}-antitrypsin and {alpha}{sub 1}-antichymotrypsin. {alpha}{sub 1}-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd > Pb > Cu > Ni > Zn > Mg. None of the metals affected the mRNA levels of albumin, {alpha}{sub 1}-antitrypsin, and {alpha}{sub 1}-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced {alpha}{sub 1}-acid glycoprotein and C-reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the {alpha}{sub 1}-acid glycoprotein and C-reactive protein promoters. The studies indicate that the induction of {alpha}{sub 1}-acid glycoprotein and C-reactive protein by heavy metals may be regulated by these metal-responsive elements at the level of transcription.

  20. Highly efficient extraction of cellular nucleic acid associated proteins in vitro with magnetic oxidized carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Wei, Xiaoluan; Cheng, Kai; Liu, Fangjie; Wu, Ren'an; Zou, Hanfa

    2012-12-01

    Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1,000,000 cells, and 803 groups of NAaP were analyzed with only about 10,000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation. PMID:23121485

  1. Ultraviolet and 5'fluorodeoxyuridine induced random mutagenesis in Chlorella vulgaris and its impact on fatty acid profile: a new insight on lipid-metabolizing genes and structural characterization of related proteins.

    PubMed

    Anthony, Josephine; Rangamaran, Vijaya Raghavan; Gopal, Dharani; Shivasankarasubbiah, Kumar T; Thilagam, Mary Leema J; Peter Dhassiah, Magesh; Padinjattayil, Divya Shridhar M; Valsalan, VinithKumar N; Manambrakat, Vijayakumaran; Dakshinamurthy, Sivakumar; Thirunavukkarasu, Sivaraman; Ramalingam, Kirubagaran

    2015-02-01

    The present study was aimed at randomly mutating the microalga, Chlorella vulgaris, in order to alter its cellular behaviour towards increased lipid production for efficient biodiesel production from algal biomass. Individual mutants from ultraviolet light (UV-1 (30 s exposure), UV-2 (60 s exposure) and UV-3 (90 s exposure)) and 5'fluorodeoxyuridine (5'FDU-1 (0.25 mM) and 5'FDU-2 (0.50 mM)) exposed cells were identified to explore an alternative method for lipid enhancement. A marginally significant decrease in biomass in the UV mutants; marked increase in the lipid content in UV-2 and 5'FDU-1 mutants; significant increase in saturated fatty acids level, especially in UV-2 mutant; insignificant increase in lipid production when these mutants were subjected to an additional stress of nitrogen starvation and predominantly enhanced level of unsaturated fatty acids in all the strains except UV-2 were noted. Chloroplast ultrastructural alterations and defective biosynthesis of chloroplast specific lipid constituents were observed in the mutants. Modelling of three-dimensional structures of acetyl coA carboxylase (ACCase), omega-6, plastid delta-12 and microsomal delta-12 fatty acid desaturases for the first time and ligand-interaction studies greatly substantiated our findings. A replacement of leucine by a serine residue in the acetyl coA carboxylase gene of UV-2 mutant suggests the reason behind lipid enhancement in UV-2 mutant. Higher activity of ACCase in UV-2 and 5'FDU-1 strongly proves the functional consequences of gene mutation to lipid production. In conclusion, algal mutants exhibited significant impact on biodiesel production through structural alterations in the lipid-metabolizing genes, thereby enhancing lipid production and saturated fatty acid levels. PMID:25189135

  2. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  3. Molecular evolution of the fusion protein gene in human respiratory syncytial virus subgroup A.

    PubMed

    Kimura, Hirokazu; Nagasawa, Koo; Tsukagoshi, Hiroyuki; Matsushima, Yuki; Fujita, Kiyotaka; Yoshida, Lay Myint; Tanaka, Ryota; Ishii, Haruyuki; Shimojo, Naoki; Kuroda, Makoto; Ryo, Akihide

    2016-09-01

    We studied the molecular evolution of the fusion protein (F) gene in the human respiratory syncytial virus subgroup A (HRSV-A). We performed time-scaled phylogenetic analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. We also conducted genetic distance (p-distance), positive/negative selection, and Bayesian skyline plot analyses. Furthermore, we mapped the amino acid substitutions of the protein. The MCMC-constructed tree indicated that the HRSV F gene diverged from the bovine RSV (BRSV) gene approximately 550years ago and had a relatively low substitution rate (7.59×10(-4) substitutions/site/year). Moreover, a common ancestor of HRSV-A and -B diverged approximately 280years ago, which has since formed four distinct clusters. The present HRSV-A strains were assigned six genotypes based on F gene sequences and attachment glycoprotein gene sequences. The present strains exhibited high F gene sequence similarity values and low genetic divergence. No positive selection sites were identified; however, 50 negative selection sites were identified. F protein amino acid substitutions at 17 sites were distributed in the F protein. The effective population size of the gene has remained relatively constant, but the population size of the prevalent genotype (GA2) has increased in the last 10years. These results suggest that the HRSV-AF gene has evolved independently and formed some genotypes. PMID:27291709

  4. Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry.

    PubMed

    Trček, Janja; Barja, François

    2015-03-01

    Acetic acid bacteria have attracted much attention over the past few years, due mainly to their metabolic traits that are of interest to the biotechnology industry. In addition, it turns out that their ecological habitats are almost unlimited since they have been found as symbionts in different insects and also as emerging opportunistic human pathogens. Very surprising is the finding that they colonize niches considered anaerobic, disproving the generalized statement that they are strict aerobes. Since they have taken on different biological roles in our environment, more and more people are charged with the task of identifying them. However, this turns out to be not always easy, especially if we are using phenotypic approaches for identification. A substantial step forward in making the identification of acetic acid bacteria easier was made possible using molecular biological methods, which have been extensively tested since 2000. However, some molecular methods require expensive machines and experienced staff, and moreover the level of their discrimination varies. All these factors must be considered when selecting the most appropriate approach for identifying acetic acid bacteria. With this objective in mind, this review article discusses the benefits and drawbacks of molecular biological methods for identification of acetic acid bacteria, with a focus on the 16S-23S rRNA gene ITS regions and the recently described alternative method for identification of acetic acid bacteria, MALDI-TOF MS. PMID:25589227

  5. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. PMID:26873273

  6. Isolation of the mouse (MFH-1) and human (FKHL14) mesenchyme fork head-1 genes reveals conservation of their gene and protein structures

    SciTech Connect

    Miura, Naoyuki; Iida, Kiyoshi; Yang, Xiao-Li

    1997-05-01

    The very recently found evolutionarily conserved DNA-binding domain of 100 amino acids, termed the fork head domain, emerged from a sequence comparison of the rat hepatocyte transcription factor HNF-3{alpha} and the homeotic gene fork head of Drosophila. We previously isolated a new member of this family, the mesenchyme fork head-1 (MFH-1) gene, which is expressed in developing mesenchyme. Here we describe the isolation of the mouse (MFH-1) and human (FKHL14) chromosomal MFH-1 genes and the determination of the gene and protein structures of MFH-1. We found that the MFH-1 gene has no introns and that the identity of the amino acid sequences of mouse and human MFH-1 proteins is 94%. We also investigated the transcriptional activity of the mouse and human MFH-1 proteins and found that both proteins act as positive transactivators. 31 refs., 3 figs.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Cloning and sequencing of a gene coding for an actin binding protein of Saccharomyces exiguus.

    PubMed

    Lange, U; Steiner, S; Grolig, F; Wagner, G; Philippsen, P

    1994-03-01

    The actin binding protein Abp1p of the yeast Saccharomyces cervisiae is thought to be involved in the spatial organisation of cell surface growth. It contains a potential actin binding domain and an SH-3 region, a common motif of many signal transduction proteins [1]. We have cloned and sequenced an ABP1 homologous gene of Saccharomyces exiguus, a yeast which is only distantly related to S. cerevisiae. The protein encoded by this gene is slightly larger than the respective S. cerevisiae protein (617 versus 592 amino acids). The two genes are 67.4% identical and the deduced amino acid sequences share an overall identity of 59.8%. The most conserved regions are the 148 N-terminal amino acids containing the potential actin binding site and the 58 C-terminal amino acids including the SH3 domain. In addition, both proteins contain a repeated motif of unknown function which is rich in glutamic acids with the sequence EEEEEEEAPAPSLPSR in the S. exiguus Abp1p. PMID:8110838

  9. d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

    PubMed Central

    Leiman, Sara A.; May, Janine M.; Lebar, Matthew D.; Kahne, Daniel; Kolter, Roberto

    2013-01-01

    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941

  10. D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis.

    PubMed

    Leiman, Sara A; May, Janine M; Lebar, Matthew D; Kahne, Daniel; Kolter, Roberto; Losick, Richard

    2013-12-01

    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941

  11. Hydrophobicity, expressivity and aromaticity are the major trends of amino-acid usage in 999 Escherichia coli chromosome-encoded genes.

    PubMed Central

    Lobry, J R; Gautier, C

    1994-01-01

    Multivariate analysis of the amino-acid compositions of 999 chromosome-encoded proteins from Escherichia coli showed that three main factors influence the variability of amino-acid composition. The first factor was correlated with the global hydrophobicity of proteins, and it discriminated integral membrane proteins from the others. The second factor was correlated with gene expressivity, showing a bias in highly expressed genes towards amino-acids having abundant major tRNAs. Just as highly expressed genes have reduced codon diversity in protein coding sequences, so do they have a reduced diversity of amino-acid choice. This showed that translational constraints are important enough to affect the global amino-acid composition of proteins. The third factor was correlated with the aromaticity of proteins, showing that aromatic amino-acid content is highly variable. PMID:8065933

  12. Murine cerebellar neurons express a novel gene encoding a protein related to cell cycle control and cell fate determination proteins.

    PubMed

    Taoka, M; Isobe, T; Okuyama, T; Watanabe, M; Kondo, H; Yamakawa, Y; Ozawa, F; Hishinuma, F; Kubota, M; Minegishi, A

    1994-04-01

    We cloned cDNAs of a novel protein (designated V-1) that has been identified from among the developmentally regulated proteins in the rat cerebellum. Protein sequencing analysis (Taoka, M., Yamakuni, T., Song, S.-Y., Yamakawa, Y., Seta, K., Okuyama, T., and Isobe, T. (1992) Eur. J. Biochem. 207, 615-620) and cDNA sequence analysis revealed that the V-1 protein consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, which was originally found in the products of the cell cycle control genes of yeasts and the cell fate determination genes in Drosophila and Caenorhabditis elegans. In situ hybridization histochemistry revealed that the expression of the V-1 gene is transiently increased in postmigratory granule cells during postnatal rat cerebellar development and thereafter is markedly suppressed, whereas Purkinje cells constitutively express V-1 mRNA. In contrast, cerebellar granule cells of the staggerer mutant mouse continue to express the V-1 gene even when the granule cells of the normal mouse have ceased to express the V-1 gene, suggesting that the expression of the V-1 gene in granule cells is regulated through the interaction with Purkinje cells. On the basis of these results, we postulate that the V-1 protein has a potential role in the differentiation of granule cells. PMID:8144589

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

    PubMed

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

    2008-09-15

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

  14. Induction of the hyaluronic acid-binding protein, tumor necrosis factor-stimulated gene-6, in cervical smooth muscle cells by tumor necrosis factor-alpha and prostaglandin E(2).

    PubMed

    Fujimoto, Toshio; Savani, Rashmin C; Watari, Michiko; Day, Anthony J; Strauss, Jerome F

    2002-04-01

    Immediately before parturition the cervix undergoes striking changes in structure (ripening) that facilitate dilatation and effacement. Cervical ripening shares many features in common with inflammation-associated tissue remodeling, making it a valuable process to explore with respect to the biochemical events in extracellular matrix restructuring. Cervical ripening can be pharmacologically induced with prostaglandin E(2) (PGE(2)). Among the biochemical changes in the cervix at parturition is a marked increase in the hyaluronic acid (HA) content. HA and HA-binding proteins have been implicated in tissue hydration, release of collagenase, and leukocyte migration, but their roles in cervical ripening have not been explored. In the present study we examined the ability of PGE(2) to induce expression of the HA-binding protein, tumor necrosis factor-stimulated gene (TSG)-6, in human cervical smooth muscle cells (hCSMCs) and compared the PGE(2) response to that of tumor necrosis factor-alpha (TNF-alpha), an established inducer of TSG-6. TNF-alpha stimulated TSG-6 mRNA accumulation in a dose- and time-dependent manner, with the maximal response observed at 10 ng/ml after 6 hours of incubation. PGE(2) stimulated TSG-6 mRNA expression, but the magnitude of response was substantially less than that produced by TNF-alpha, and it was maximal only after 24 hours of incubation. Quantitative real-time polymerase chain reaction was performed to assess the induction of TSG-6 mRNA and nascent transcripts at 24 hours of treatment. Induction of TSG-6 mRNA and nascent transcripts in response to 10 micromol/L of PGE(2) was 5.7-fold and 6.3-fold greater than control values, respectively, whereas TNF-alpha (10 ng/ml) induced TSG-6 mRNA and nascent transcripts by 80-fold and 134-fold, respectively. TNF-alpha and PGE(2) stimulated secretion of TSG-6 into the culture medium as detected by Western blotting. The effects of PGE(2) on secretion of TSG-6 were delayed compared to TNF-alpha. A 1

  15. Photoaffinity labeling of retinoic acid-binding proteins.

    PubMed Central

    Bernstein, P S; Choi, S Y; Ho, Y C; Rando, R R

    1995-01-01

    Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7846032

  16. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

    PubMed Central

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-01-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932

  17. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

    NASA Astrophysics Data System (ADS)

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-02-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

  18. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis

    PubMed Central

    Piya, Sarbottam; Shrestha, Sandesh K.; Binder, Brad; Stewart, C. Neal; Hewezi, Tarek

    2014-01-01

    The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs. PMID:25566309

  19. Possible eggshell protein gene from Schistosoma mansoni.

    PubMed

    Johnson, K S; Taylor, D W; Cordingley, J S

    1987-01-01

    We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products. PMID:3100949

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

    PubMed

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

    2015-01-01

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

  1. Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth

    PubMed Central

    Kim, Jung-Wook; Yamakoshi, Yasuo; Iwata, Takanori; Hu, Yuan Yuan; Zhang, Hengmin; Hu, Jan C.-C.; Simmer, James P.

    2015-01-01

    Dentin matrix protein 1 (DMP1) is an acidic non-collagenous protein that is necessary for the proper biomineralization of bone, cartilage, cementum, dentin, and enamel. Dentin matrix protein 1 is highly phosphorylated and potentially glycosylated, but there is no experimental data identifying which specific amino acids are modified. For the purpose of facilitating the characterization of DMP1 from pig, which has the advantage of large developing teeth for obtaining protein in quantity and extensive structural information concerning other tooth matrix proteins, we characterized the porcine DMP1 cDNA and gene structure, raised anti-peptide immunoglobulins that are specific for porcine DMP1, and detected DMP1 protein in porcine tooth extracts and histological sections. Porcine DMP1 has 510 amino acids, including a 16-amino acid signal peptide. The deduced molecular weight of the secreted, unmodified protein is 53.5 kDa. The protein has 93 serines and 12 threonines in the appropriate context for phosphorylation, and four asparagines in a context suitable for glycosylation. Dentin matrix protein 1 protein bands with apparent molecular weights between 30 and 45 kDa were observed in partially purified dentin extracts. In developing teeth, immunohistochemistry localized DMP1 in odontoblasts and the dentinal tubules of mineralized dentin and in ameloblasts, but not in the enamel matrix. PMID:16460339

  2. Barley malt increases hindgut and portal butyric acid, modulates gene expression of gut tight junction proteins and Toll-like receptors in rats fed high-fat diets, but high advanced glycation end-products partially attenuate the effects.

    PubMed

    Zhong, Yadong; Teixeira, Cristina; Marungruang, Nittaya; Sae-Lim, Watina; Tareke, Eden; Andersson, Roger; Fåk, Frida; Nyman, Margareta

    2015-09-01

    Barley malt, a product of controlled germination, has been shown to produce high levels of butyric acid in the cecum and portal serum of rats and may therefore have anti-inflammatory effects. The aim of the study was to investigate how four barley malts, caramelized and colored malts, 50-malt and 350-malt, differing in functional characteristics concerning beta-glucan content and color, affect short-chain fatty acids (SCFA), barrier function and inflammation in the hindgut of rats fed high-fat diets. Male Wistar rats were given malt-supplemented high-fat diets for four weeks. Low and high-fat diets containing microcrystalline cellulose were incorporated as controls. All diets contained 70 g kg(-1) dietary fiber. The malt-fed groups were found to have had induced higher amounts of butyric and propionic acids in the hindgut and portal serum compared with controls, while cecal succinic acid only increased to a small extent. Fat increased the mRNA expression of tight junction proteins and Toll-like receptors (TLR) in the small intestine and distal colon of the rats, as well as the concentration of some amino acids in the portal plasma, but malt seemed to counteract these adverse effects to some extent. However, the high content of advanced glycation end-products (AGE) in caramelized malt tended to prohibit the positive effects on occludin in the small intestine and plasma amino acids seen with the other malt products. In conclusion, malting seems to be an interesting process for producing foods with positive health effects, but part of these effects may be destroyed if the malt contains a high content of AGE. PMID:26227569

  3. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  6. Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

    PubMed

    Sokol, F; Tan, K B; McFalls, M L; Madore, P

    1974-07-01

    Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. PMID:4365328

  7. Dissociation between gene and protein expression of metabolic enzymes in a rodent model of heart failure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies in advanced heart failure show down-regulation of fatty acid oxidation genes, possibly due to decreased expression of the nuclear transcription factors peroxisome proliferator activated receptor alpha (PPARalpha) and retinoid X receptor alpha (RXRalpha). We assessed mRNA and protein expressi...

  8. Identification of the mcpA and mcpM Genes, Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis, and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

    PubMed Central

    Hida, Akiko; Oku, Shota; Kawasaki, Takeru; Nakashimada, Yutaka; Tajima, Takahisa

    2015-01-01

    Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand. PMID:26276117

  9. Group I intron located in PR protein homologue gene in Youngia japonica.

    PubMed

    Nishida, H; Ogura, A; Yokota, A; Yamaguchi, I; Sugiyama, J

    2000-03-01

    A Youngia japonica strain had a group I intron that was suggested to have been transferred from Protomyces inouyei, a pathogenic fungus of Y. japonica. It was located in the miraculin homologue coding gene by reverse complementation. The deduced amino acid sequence of this miraculin homologue of Y. japonica was similar to the amino acid sequences of tobacco and tomato pathogenesis-related proteins. PMID:10803963

  10. Protein and amino acid metabolism and requirements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells of the body. Enzymes, membrane carriers, blood transport molecules, intracellular matrix, and even hair and fingernails are proteins, as are many hormones. Proteins also constitute a major portion of all membranes, and the cons...

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

    PubMed

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

    2016-01-01

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

  12. Interaction of milk whey protein with common phenolic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

    2014-01-01

    Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

  13. Relating protein adduction to gene expression changes: a systems approach

    PubMed Central

    Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

    2013-01-01

    Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data. PMID:21594272

  14. Gene cloning and characterization of the protein encoded by the Neospora caninum bradyzoite-specific antigen gene BAG1.

    PubMed

    Kobayashi, T; Narabu, S; Yanai, Y; Hatano, Y; Ito, A; Imai, S; Ike, K

    2013-06-01

    Neospora caninum is an Apicomplexan parasite that causes repeated abortion and stillbirth in cattle. The aim of this study was to clone the gene encoding the N. caninum orthologue (NcBAG1) of the Toxoplasma gondii bradyzoite-specific protein TgBAG1 and characterize its expression pattern in the parasite. Isolation of the full-length 684-bp gene revealed that it shared 78.3% sequence similarity with TgBAG1. NcBAG1 encodes a predicted protein of 227 amino acids with 80.3% similarity to TgBAG1. A putative signal peptide sequence and an invariant GVL motif characteristic of small heat-shock proteins were identified in the predicted N. caninum amino acid sequence. We expressed the NcBAG1 gene as a recombinant glutathione S-transferase fusion protein (rNcBAG1) in Escherichia coli and used the purified 60 kDa protein to obtain a monoclonal antibody (Mab). rNcBAG1 reacted to Mabs specific for NcBAG1 and TgBAG1. No reaction between the NcBAG1 Mab and N. caninum tachyzoites was observed. Although the predicted molecular mass of NcBAG1 is 25 kDa, Western blot analysis of parasite lysates using the NcBAG1 Mab revealed a cross-reactive protein of approximately 30 kDa. Additionally, immunofluorescence assays using the tachyzoite-specific Mab for NcSAG1 and the bradyzoite-specific Mab for TgBAG1 or NcSAG4 revealed NcBAG1-specific expression in bradyzoites in cultures exposed to sodium nitroprusside, a reagent that increases the frequency of bradyzoites. Interestingly, the NcBAG1 protein was identified in the cytoplasm of the bradyzoite-stage parasites. This preliminary analysis of the NcBAG1 gene will assist investigations into the role of this protein in N. caninum . PMID:23245337

  15. Protein secretion controlled by a synthetic gene in Escherichia coli.

    PubMed

    Blanchin-Roland, S; Masson, J M

    1989-03-01

    The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain. PMID:2652141

  16. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. PMID:25640894

  17. Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety.

    PubMed

    Lee, Jong-Yeol; Beom, Hye-Rang; Altenbach, Susan B; Lim, Sun-Hyung; Kim, Yeong-Tae; Kang, Chon-Sik; Yoon, Ung-Han; Gupta, Ravi; Kim, Sun-Tae; Ahn, Sang-Nag; Kim, Young-Mi

    2016-05-01

    Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour. PMID:26882917

  18. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    PubMed

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin. PMID:27348124

  19. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells

    PubMed Central

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin. PMID:27348124

  20. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-dependent transcriptional program controls activation of the early growth response 1 (EGR1) gene during amino acid limitation.

    PubMed

    Shan, Jixiu; Balasubramanian, Mukundh N; Donelan, William; Fu, Lingchen; Hayner, Jaclyn; Lopez, Maria-Cecilia; Baker, Henry V; Kilberg, Michael S

    2014-08-29

    Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. PMID:25028509

  1. A Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK)-dependent Transcriptional Program Controls Activation of the Early Growth Response 1 (EGR1) Gene during Amino Acid Limitation*

    PubMed Central

    Shan, Jixiu; Balasubramanian, Mukundh N.; Donelan, William; Fu, Lingchen; Hayner, Jaclyn; Lopez, Maria-Cecilia; Baker, Henry V.; Kilberg, Michael S.

    2014-01-01

    Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. PMID:25028509

  2. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein.

    PubMed Central

    Celenza, J L; Marshall-Carlson, L; Carlson, M

    1988-01-01

    The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression. Images PMID:3281163

  3. Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49.

    PubMed Central

    Haanes, E J; Cleary, P P

    1989-01-01

    The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes. Images PMID:2687231

  4. Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats

    PubMed Central

    Song, Shangxin; Hooiveld, Guido J.; Li, Mengjie; Zhao, Fan; Zhang, Wei; Xu, Xinglian; Muller, Michael; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets. PMID:26857845

  5. Protein Sialylation Regulates a Gene Expression Signature that Promotes Breast Cancer Cell Pathogenicity

    PubMed Central

    2016-01-01

    Many mechanisms have been proposed for how heightened aerobic glycolytic metabolism fuels cancer pathogenicity, but there are still many unexplored pathways. Here, we have performed metabolomic profiling to map glucose incorporation into metabolic pathways upon transformation of mammary epithelial cells by 11 commonly mutated human oncogenes. We show that transformation of mammary epithelial cells by oncogenic stimuli commonly shunts glucose-derived carbons into synthesis of sialic acid, a hexosamine pathway metabolite that is converted to CMP-sialic acid by cytidine monophosphate N-acetylneuraminic acid synthase (CMAS) as a precursor to glycoprotein and glycolipid sialylation. We show that CMAS knockdown leads to elevations in intracellular sialic acid levels, a depletion of cellular sialylation, and alterations in the expression of many cancer-relevant genes to impair breast cancer pathogenicity. Our study reveals the heretofore unrecognized role of sialic acid metabolism and protein sialylation in regulating the expression of genes that maintain breast cancer pathogenicity. PMID:27380425

  6. Protein Sialylation Regulates a Gene Expression Signature that Promotes Breast Cancer Cell Pathogenicity.

    PubMed

    Kohnz, Rebecca A; Roberts, Lindsay S; DeTomaso, David; Bideyan, Lara; Yan, Peter; Bandyopadhyay, Sourav; Goga, Andrei; Yosef, Nir; Nomura, Daniel K

    2016-08-19

    Many mechanisms have been proposed for how heightened aerobic glycolytic metabolism fuels cancer pathogenicity, but there are still many unexplored pathways. Here, we have performed metabolomic profiling to map glucose incorporation into metabolic pathways upon transformation of mammary epithelial cells by 11 commonly mutated human oncogenes. We show that transformation of mammary epithelial cells by oncogenic stimuli commonly shunts glucose-derived carbons into synthesis of sialic acid, a hexosamine pathway metabolite that is converted to CMP-sialic acid by cytidine monophosphate N-acetylneuraminic acid synthase (CMAS) as a precursor to glycoprotein and glycolipid sialylation. We show that CMAS knockdown leads to elevations in intracellular sialic acid levels, a depletion of cellular sialylation, and alterations in the expression of many cancer-relevant genes to impair breast cancer pathogenicity. Our study reveals the heretofore unrecognized role of sialic acid metabolism and protein sialylation in regulating the expression of genes that maintain breast cancer pathogenicity. PMID:27380425

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

    PubMed Central

    2009-01-01

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

  8. The amino acid sequence of protein SCMK-B2A from the high-sulphur fraction of wool keratin

    PubMed Central

    Elleman, T. C.

    1972-01-01

    1. The amino acid sequence of protein SCMK-B2A, a reduced and S-carboxymethylated protein from the high-sulphur fraction of wool, has been determined. 2. This protein of 171 amino acid residues displays both a high degree of internal homology and extensive external homology with other members of the SCMK-B2 group of proteins. 3. Evidence is presented which suggests that the SCMK-B2 group of proteins are produced by separate non-allelic genes. ImagesPLATE 1 PMID:4679226

  9. Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

    PubMed Central

    Chang, G J; Cropp, B C; Kinney, R M; Trent, D W; Gubler, D J

    1995-01-01

    The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas. PMID:7637022

  10. Fatty acid transport protein 1 can compensate for fatty acid transport protein 4 in the developing mouse epidermis.

    PubMed

    Lin, Meei-Hua; Miner, Jeffrey H

    2015-02-01

    Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very-long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective barrier; they die neonatally because of dehydration and restricted movements. Mutations in SLC27A4, the gene encoding FATP4, cause ichthyosis prematurity syndrome (IPS), characterized by premature birth, respiratory distress, and edematous skin with severe ichthyotic scaling. Symptoms of surviving patients become mild, although atopic manifestations are common. We previously showed that suprabasal keratinocyte expression of a Fatp4 transgene in Fatp4 mutant skin rescues the lethality and ameliorates the skin phenotype. Here we tested the hypothesis that FATP1, the closest FATP4 homolog, can compensate for the lack of FATP4 in our mouse model of IPS, as it might do postnatally in IPS patients. Transgenic expression of FATP1 in suprabasal keratinocytes rescued the phenotype of Fatp4 mutants, and FATP1 sorted to the same intracellular organelles as endogenous FATP4. Thus, FATP1 and FATP4 likely have overlapping substrate specificities, enzymatic activities, and biological functions. These results suggest that increasing expression of FATP1 in suprabasal keratinocytes could normalize the skin of IPS patients and perhaps prevent the atopic manifestations. PMID:25184958

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

    PubMed

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

    2014-10-01

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

  12. Genomic structure of the human D-site binding protein (DBP) gene

    SciTech Connect

    Shutler, G.; Glassco, T.; Kang, Xiaolin

    1996-06-15

    The human gene for the D-Site Binding Protein (DBP) has been sequenced and characterized. This gene is a member of the b/ZIP family of transcription factors and is one of three genes forming the PAR sub-family. DBP has been implicated in the diurnal regulation of a variety of liver-specific genes. Examination of the genomic structure of DBP reveals that the gene is divided into four exons and is contained within a relatively compact region of approximately 6 kb. These exons appear to correspond to functional divisions the DBP protein. Exon 1 contains a long 5{prime} UTR, and conservation between the rat and the human genes of the presence of small open reading frames within this region suggests that is may play a role in translational control. Exon 2 contains a limited region of similarity to the other PAR domain genes, which may be part of a potential activation domain. Exon 3 contains the PAR domain and differs by only 1 of 71 amino acids between rat and human. Exon 4, containing both the basic and the leucine zipper domains, is likewise highly conserved. The overall degree of homology between the rat and the human cDNA sequences is 82% for the nucleic acid sequence and 92% for the protein sequence. comparison of the rat and human proximal promoters reveals extensive sequence conservation, with two previously characterized DNA binding sites being conserved at the functional and sequence levels. 31 refs., 4 figs.

  13. Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

    PubMed Central

    Schnier, J; Thamm, S; Lurz, R; Hussain, A; Faist, G; Dobrinski, B

    1988-01-01

    A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter. Images PMID:3368316

  14. Gene duplication and the evolution of moonlighting proteins.

    PubMed

    Espinosa-Cantú, Adriana; Ascencio, Diana; Barona-Gómez, Francisco; DeLuna, Alexander

    2015-01-01

    Gene duplication is a recurring phenomenon in genome evolution and a major driving force in the gain of biological functions. Here, we examine the role of gene duplication in the origin and maintenance of moonlighting proteins, with special focus on functional redundancy and innovation, molecular tradeoffs, and genetic robustness. An overview of specific examples-mainly from yeast-suggests a widespread conservation of moonlighting behavior in duplicate genes after long evolutionary times. Dosage amplification and incomplete subfunctionalization appear to be prevalent in the maintenance of multifunctionality. We discuss the role of gene-expression divergence and paralog responsiveness in moonlighting proteins with overlapping biochemical properties. Future studies analyzing multifunctional genes in a more systematic and comprehensive manner will not only enable a better understanding of how this emerging class of protein behavior originates and is maintained, but also provide new insights on the mechanisms of evolution by gene duplication. PMID:26217376

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

    PubMed

    Chen, Yingying; Stabryla, Lisa; Wei, Na

    2016-01-01

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

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

    PubMed

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

    2013-09-01

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

  17. Molecular characterization of a human gene for S28 ribosomal binding protein

    SciTech Connect

    Wong, P.; Borst, D.E.; Chader, G.J.

    1994-09-01

    The mechanism of ribosome action and the ribosomal binding proteins which cooperatively interact in the working of this structure are not completely understood. Theoretically, mutations in genes that encode these proteins may compromise the efficiency of protein synthesis and therefore lead to a functional disorder. In the course of our search for human genes which show homology to the C. elegans CED-4 death gene, we have serendipitously identified one of the human S28 ribosomal binding protein genes as a random fragment fused to the end of one of our putative CED-4 positive homologue clones. The cloned S28 fragment consists of 381 nucleotides with a putative open reading frame of 113 amino acids. Sequence comparisons to GenBank revealed significant homologies to ribosomal binding protein genes in other species (including the rat S28 ribosomal binding protein gene) indicating that the S28 gene sequence is highly conserved. This finding is confirmed by zooblot analysis. Significant homologies also exist to two human expressed tagged sites (HUMRIBPROB; L05091 and HSAFIF072; Z21908). Analysis of the putative S28 peptide sequence allows insights into possible functional regions of the protein. The identification of 8 distinct bands upon Southern analysis of the S28 fragments suggests that there are multiple copies of the S28 gene in the human genome. Mapping of the S28 fragment on somatic cell hybrid panels identified distinct S28 gene loci on chromosomes 1, 2, 7, 10, 11, 12, 17 expression in adult tissues (pancreas, kidney, muscle, liver, lung, placenta, brain, heart, and retina) as well as in fetal tissues (kidney, liver, lung, brain, and heart).

  18. A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

    PubMed

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2013-08-01

    Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy. PMID:23851421

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

    PubMed Central

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

    1989-01-01

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

  20. Impact of dietary protein on lipid metabolism-related gene expression in porcine adipose tissue

    PubMed Central

    2010-01-01

    Background High dietary protein can reduce fat deposition in animal subcutaneous adipose tissue, but little is known about the mechanism. Methods Sixty Wujin pigs of about 15 kg weight were fed either high protein (HP: 18%) or low protein (LP: 14%) diets, and slaughtered at body weights of 30, 60 or 100 kg. Bloods were collected to measure serum parameters. Subcutaneous adipose tissues were sampled for determination of adipocyte size, protein content, lipid metabolism-related gene expression, and enzyme activities. Results HP significantly reduced adipocyte size, fat meat percentage and backfat thickness, but significantly increased daily gain, lean meat percentage and loin eye area at 60 and 100 kg. Serum free fatty acid and triglyceride concentrations in the HP group were significantly higher than in the LP group. Serum glucose and insulin concentrations were not significantly affected by dietary protein at any body weight. HP significantly reduced gene expression of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element binding protein 1c (SREBP-1c) at 60 kg and 100 kg; however, the mRNA level and enzyme activity of FAS were increased at 30 kg. HP promoted gene and protein expression and enzyme activities of lipoprotein lipase (LPL), carmitine palmtoyltransferase-1B (CPT-1B), peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte-fatty acid binding proteins (A-FABP) at 60 kg, but reduced their expression at 100 kg. Gene expression and enzyme activity of hormone sensitive lipase (HSL) was reduced markedly at 60 kg but increased at 100 kg by the high dietary protein. Levels of mRNA, enzyme activities and protein expression of ACC, FAS, SREBP-1c and PPARγ in both LP and HP groups increased with increasing body weight. However, gene and protein expression levels/enzyme activities of LPL, CPT-1B, A-FABP and HSL in both groups were higher at 60 kg than at 30 and 100 kg. Conclusion Fat deposition in Wujin pigs fed high

  1. Heat capacities of amino acids, peptides and proteins.

    PubMed

    Makhatadze, G I

    1998-04-20

    The heat capacity is one of the fundamental parameters describing thermodynamic properties of a system. It has wide applications in a number of areas such as polymer chemistry, protein folding and DNA stability. To aid the scientific community in the analysis of such data, I have compiled a database on the experimentally measured heat capacities of amino acids, polyamino acids, peptides, and proteins in solid state and in aqueous solutions. PMID:9648205

  2. Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

    PubMed Central

    Jiang, D W; Englund, P T

    2001-01-01

    As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

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

    PubMed

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

    2015-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  6. DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus.

    PubMed Central

    Ficht, T A; Bearden, S W; Sowa, B A; Adams, L G

    1989-01-01

    The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When

  7. Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.

    PubMed Central

    Chevalier, C; Saillard, C; Bové, J M

    1990-01-01

    The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction. Images PMID:2139649

  8. Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds.

    PubMed

    Sawano, Yoriko; Miyakawa, Takuya; Yamazaki, Hiroshi; Tanokura, Masaru; Hatano, Ken-ichi

    2007-03-01

    A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins. PMID:17338634

  9. GeneSV - an Approach to Help Characterize Possible Variations in Genomic and Protein Sequences.

    PubMed

    Zemla, Adam; Kostova, Tanya; Gorchakov, Rodion; Volkova, Evgeniya; Beasley, David W C; Cardosa, Jane; Weaver, Scott C; Vasilakis, Nikos; Naraghi-Arani, Pejman

    2014-01-01

    A computational approach for identification and assessment of genomic sequence variability (GeneSV) is described. For a given nucleotide sequence, GeneSV collects information about the permissible nucleotide variability (changes that potentially preserve function) observed in corresponding regions in genomic sequences, and combines it with conservation/variability results from protein sequence and structure-based analyses of evaluated protein coding regions. GeneSV was used to predict effects (functional vs. non-functional) of 37 amino acid substitutions on the NS5 polymerase (RdRp) of dengue virus type 2 (DENV-2), 36 of which are not observed in any publicly available DENV-2 sequence. 32 novel mutants with single amino acid substitutions in the RdRp were generated using a DENV-2 reverse genetics system. In 81% (26 of 32) of predictions tested, GeneSV correctly predicted viability of introduced mutations. In 4 of 5 (80%) mutants with double amino acid substitutions proximal in structure to one another GeneSV was also correct in its predictions. Predictive capabilities of the developed system were illustrated on dengue RNA virus, but described in the manuscript a general approach to characterize real or theoretically possible variations in genomic and protein sequences can be applied to any organism. PMID:24453480

  10. GeneSV – an Approach to Help Characterize Possible Variations in Genomic and Protein Sequences

    PubMed Central

    Zemla, Adam; Kostova, Tanya; Gorchakov, Rodion; Volkova, Evgeniya; Beasley, David W. C.; Cardosa, Jane; Weaver, Scott C.; Vasilakis, Nikos; Naraghi-Arani, Pejman

    2014-01-01

    A computational approach for identification and assessment of genomic sequence variability (GeneSV) is described. For a given nucleotide sequence, GeneSV collects information about the permissible nucleotide variability (changes that potentially preserve function) observed in corresponding regions in genomic sequences, and combines it with conservation/variability results from protein sequence and structure-based analyses of evaluated protein coding regions. GeneSV was used to predict effects (functional vs. non-functional) of 37 amino acid substitutions on the NS5 polymerase (RdRp) of dengue virus type 2 (DENV-2), 36 of which are not observed in any publicly available DENV-2 sequence. 32 novel mutants with single amino acid substitutions in the RdRp were generated using a DENV-2 reverse genetics system. In 81% (26 of 32) of predictions tested, GeneSV correctly predicted viability of introduced mutations. In 4 of 5 (80%) mutants with double amino acid substitutions proximal in structure to one another GeneSV was also correct in its predictions. Predictive capabilities of the developed system were illustrated on dengue RNA virus, but described in the manuscript a general approach to characterize real or theoretically possible variations in genomic and protein sequences can be applied to any organism. PMID:24453480

  11. HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA.
    Sponsor: JM Rogers.
    Haloacetic Acids (HAAs) formed during the disinfection process are present in drin...

  12. Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

    PubMed Central

    Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

    2015-01-01

    This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

  13. Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis.

    PubMed

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Suber, Bryce; Kumaraswami, Muthiah; Olsen, Randall J; Flores, Anthony; Musser, James M; Brennan, Richard G; Shelburne, Samuel A

    2011-10-01

    Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis. PMID:22028655

  14. Distinct Single Amino Acid Replacements in the Control of Virulence Regulator Protein Differentially Impact Streptococcal Pathogenesis

    PubMed Central

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Suber, Bryce; Kumaraswami, Muthiah; Olsen, Randall J.; Flores, Anthony; Musser, James M.; Brennan, Richard G.; Shelburne, Samuel A.

    2011-01-01

    Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis. PMID:22028655

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

    PubMed Central

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

    2015-01-01

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

  16. Genetic transformation of genes for protein II in Neisseria gonorrhoeae.

    PubMed Central

    Schwalbe, R S; Cannon, J G

    1986-01-01

    The protein II (PII) outer membrane proteins of Neisseria gonorrhoeae are a family of heat-modifiable proteins that are subject to phase variation, in which the synthesis of different PII species is turned on and off at a high frequency. Transformation of PII genes from a donor gonococcal strain into a recipient strain was detected with monoclonal antibodies specific for the PII proteins of the donor. Individual PII protein-expressing transformants generally bound only one donor-specific PII monoclonal antibody. Recovery of transformants expressing a donor-specific PII protein depended on the PII protein expression state of the donor: the transformed population bound only monoclonal antibodies specific for PII proteins that were expressed in the donor. Colony variants with an altered frequency of switching of PII protein expression were isolated, but the altered switch phenotype did not cotransform with the PII structural gene. These results provide genetic evidence that PII proteins are the products of different genes and that expressed and unexpressed forms of the PII gene are different from each other. Images PMID:3087951

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2015-03-01

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

  19. A Soluble, Folded Protein without Charged Amino Acid Residues.

    PubMed

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall; Johansson, Kristoffer Enøe; Villa, Mara; Willemoës, Martin; Lindorff-Larsen, Kresten; Teilum, Kaare; Winther, Jakob R

    2016-07-19

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced. PMID:27307139

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

    PubMed

    Dehghan Nayeri, Fatemeh; Yarizade, Kazem

    2014-08-01

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

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

    PubMed

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

    2016-07-10

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

  2. Structure and Function of the E. coli Protein YmgB: a Protein Critical for Biofilm Formation and Acid-resistance

    PubMed Central

    Lee, Jintae; Page, Rebecca; García-Contreras, Rodolfo; Palermino, Jeanne-Marie; Zhang, Xue-Song; Doshi, Ojus; Wood, Thomas K.; Peti, Wolfgang

    2007-01-01

    The Escherichia coli gene cluster ymgABC was identified in transcriptome studies to play a role in biofilm development and stability. In this study we show that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB plays a major role in acid-resistance in E. coli. Our data also shows that these phenotypes are potentially mediated through interactions with the important cell signal indole. In addition, gel shift assays suggest that YmgB may be a non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we show that YmgB binds, either directly or indirectly via a second protein, genes important for biofilm formation. To advance our understanding of the function of YmgB, we used X-ray crystallography to solve the structure of the protein to 1.8 Å resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite its low sequence identity of only 5%. This supports our DNA microarray data that YmgB is a gene regulatory protein. Therefore, this protein, which clearly has a critical role in acid-resistance in E. coli, has been renamed as AriR for regulator of acid-resistance influenced by indole. PMID:17765265

  3. Gene Sequence Variability of the Three Surface Proteins of Human Respiratory Syncytial Virus (HRSV) in Texas

    PubMed Central

    Tapia, Lorena I.; Shaw, Chad A.; Aideyan, Letisha O.; Jewell, Alan M.; Dawson, Brian C.; Haq, Taha R.; Piedra, Pedro A.

    2014-01-01

    Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004–2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community. PMID:24625544

  4. [Recombinant proteins containing amino acid sequences of two ectatomin chains].

    PubMed

    Esipov, R S; Gurevich, A I; Kaiushin, A L; Korosteleva, M D; Miroshnikov, A I; Shevchenko, L V; Pluzhnikov, K A; Grishin, E V

    1997-12-01

    Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained. PMID:9499370

  5. GeneSense: a new approach for human gene annotation integrated with protein-protein interaction networks

    PubMed Central

    Chen, Zhongzhong; Zhang, Tianhong; Lin, Jun; Yan, Zidan; Wang, Yongren; Zheng, Weiqiang; Weng, Kevin C.

    2014-01-01

    Virtually all cellular functions involve protein-protein interactions (PPIs). As an increasing number of PPIs are identified and vast amount of information accumulated, researchers are finding different ways to interrogate the data and understand the interactions in context. However, it is widely recognized that a significant portion of the data is scattered, redundant, not considered high quality, and not readily accessible to researchers in a systematic fashion. In addition, it is challenging to identify the optimal protein targets in the current PPI networks. The GeneSense server was developed to integrate gene annotation and PPI networks in an expandable architecture that incorporates selected databases with the aim to assemble, analyze, evaluate and disseminate protein-protein association information in a comprehensive and user-friendly manner. Three network models including nodenet, leafnet and loopnet are used to identify the optimal protein targets in the complex networks. GeneSense is freely available at www.biomedsense.org/genesense.php. PMID:24667292

  6. Methyl-accepting chemotaxis protein III and transducer gene trg.

    PubMed Central

    Hazelbauer, G L; Engström, P; Harayama, S

    1981-01-01

    A comparison of the two-dimensional gel patterns of methyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiple methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed. Images PMID:7007323

  7. Selection for genes encoding secreted proteins and receptors.

    PubMed Central

    Klein, R D; Gu, Q; Goddard, A; Rosenthal, A

    1996-01-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states. Images Fig. 1 PMID:8692953

  8. Graphical Features of Functional Genes in Human Protein Interaction Network.

    PubMed

    Wang, Pei; Chen, Yao; Lü, Jinhu; Wang, Qingyun; Yu, Xinghuo

    2016-06-01

    With the completion of the human genome project, it is feasible to investigate large-scale human protein interaction network (HPIN) with complex networks theory. Proteins are encoded by genes. Essential, viable, disease, conserved, housekeeping (HK) and tissue-enriched (TE) genes are functional genes, which are organized and functioned via interaction networks. Based on up-to-date data from various databases or literature, two large-scale HPINs and six subnetworks are constructed. We illustrate that the HPINs and most of the subnetworks are sparse, small-world, scale-free, disassortative and with hierarchical modularity. Among the six subnetworks, essential, disease and HK subnetworks are more densely connected than the others. Statistical analysis on the topological structures of the HPIN reveals that the lethal, the conserved, the HK and the TE genes are with hallmark graphical features. Receiver operating characteristic (ROC) curves indicate that the essential genes can be distinguished from the viable ones with accuracy as high as almost 70%. Closeness, semi-local and eigenvector centralities can distinguish the HK genes from the TE ones with accuracy around 82%. Furthermore, the Venn diagram, cluster dendgrams and classifications of disease genes reveal that some classes of disease genes are with hallmark graphical features, especially for cancer genes, HK disease genes and TE disease genes. The findings facilitate the identification of some functional genes via topological structures. The investigations shed some light on the characteristics of the compete interactome, which have potential implications in networked medicine and biological network control. PMID:26841412

  9. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  10. Predicting protein disorder by analyzing amino acid sequence

    PubMed Central

    Yang, Jack Y; Yang, Mary Qu

    2008-01-01

    Background Many protein regions and some entire proteins have no definite tertiary structure, presenting instead as dynamic, disorder ensembles under different physiochemical circumstances. These proteins and regions are known as Intrinsically Unstructured Proteins (IUP). IUP have been associated with a wide range of protein functions, along with roles in diseases characterized by protein misfolding and aggregation. Results Identifying IUP is important task in structural and functional genomics. We exact useful features from sequences and develop machine learning algorithms for the above task. We compare our IUP predictor with PONDRs (mainly neural-network-based predictors), disEMBL (also based on neural networks) and Globplot (based on disorder propensity). Conclusion We find that augmenting features derived from physiochemical properties of amino acids (such as hydrophobicity, complexity etc.) and using ensemble method proved beneficial. The IUP predictor is a viable alternative software tool for identifying IUP protein regions and proteins. PMID:18831799

  11. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  12. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    PubMed Central

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts. PMID:10639127

  13. Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum.

    PubMed

    Agarwal, A K; Parrish, S N; Blumberg, D D

    1999-10-01

    Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5' non-translated leader of the ribosomal protein (r-protein) mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5' non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase reporter gene. While beta-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development beta-galactosidase enzyme activity is largely lost from the prespore cells although strong beta-galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner. PMID:10550541

  14. Elongation factor Ts of Chlamydia trachomatis: structure of the gene and properties of the protein.

    PubMed

    Zhang, Y; Tao, J; Zhou, M; Meng, Q; Zhang, L; Shen, L; Klein, R; Miller, D L

    1997-08-01

    A putative structural gene cluster containing four open reading frames (ORFs) located downstream of the omp1 gene of Chlamydia trachomatis mouse pneumonitis (MoPn) was cloned and sequenced. A GenBank survey indicated that the identified cluster is similar to the rpsB-tsf-pyrH(smbA)-frr region of Escherichia coli. The second ORF was 846 bp encoding a 282-amino-acid polypeptide with a calculated M(r) 30,824. Alignment of this deduced protein sequence and E. coli elongation factor Ts (EF-Ts, product of tsf) demonstrated 34% identity and an additional 14% similarity. The putative chlamydial tsf gene was expressed in E. coli as a nonfusion protein and as a 6x His-tagged fusion protein. By SDS-PAGE analysis, the molecular weights of the nonfusion recombinant protein and a protein of chlamydial elementary bodies (EBs), which was recognized by monoclonal antibodies derived from the nonfusion recombinant protein, are 34 kDa. The purified recombinant 6x His-tagged fusion protein increased the rate of GDP exchange with both Chlamydia and E. coli elongation factor Tu (EF-Tu). These data show that the second gene of the identified cluster is tsf. Unlike EF-Ts from any other species, its activity was comparable to that of E. coli EF-Ts in exchange reaction with E. coli EF-Tu. PMID:9244380

  15. The Mycoplasma hyorhinis p37 Protein Rapidly Induces Genes in Fibroblasts Associated with Inflammation and Cancer

    PubMed Central

    Gomersall, Amber Cathie; Li, Song Feng; Parish, Roger W.

    2015-01-01

    The p37 protein at the surface of Mycoplasma hyorhinis cells forms part of a high-affinity transport system and has been found associated with animal and human cancers. Here we show in NIH3T3 fibroblasts, p37 rapidly induces the expression of genes implicated in inflammation and cancer progression. This gene activation was principally via the Tlr4 receptor. Activity was lost from p37 when the C-terminal 20 amino acids were removed or the four amino acids specific for the hydrogen bonding of thiamine pyrophosphate had been replaced by valine. Blocking the IL6 receptor or inhibiting STAT3 signalling resulted in increased p37-induced gene expression. Since cancer associated fibroblasts support growth, invasion and metastasis via their ability to regulate tumour-related inflammation, the rapid induction in fibroblasts of pro-inflammatory genes by p37 might be expected to influence cancer development. PMID:26512722

  16. Primary structure of dihydrofolate reductase and mitochondrial ribosomal protein L36 genes from the basidiomycete Coprinus cinereus.

    PubMed

    Aimi, Tadanori; Fukuhara, Shoji; Ishiguro, Maki; Kitamoto, Yutaka; Morinaga, Tsutomu

    2004-08-01

    We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome. PMID:15620217

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

    PubMed Central

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

    2014-01-01

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

  18. Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2014-01-01

    The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

  19. The KP4 killer protein gene family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  20. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  1. Evolutionary relationship of nuclear genes encoding mitochondrial proteins across grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome studies were done across taxa to provide a basic understanding of genome evolution regarding nuclear genes encoding for mitochondrial proteins and their conservation in grass species. Two different mitochondria-related gene sets, one from rice and another from Arabidopsis, were us...

  2. [Amino acid composition of rice grain proteins].

    PubMed

    Peruanskiĭ, Iu V; Savich, I M

    1976-01-01

    The composition of the major reserve proteins of rice grain--globulins, prolamines and glutelins--was examined in four rice varieties (Dubovsky 129, Kuban 3, Alakul, Ushtobinsky). Globulins proved to be most heterogeneous whereas glutelins appeared to be least heterogeneous. In regards to the ratio of components globulins showed high variability and glutelins displayed high stability. PMID:1005365

  3. Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

    PubMed Central

    Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

    2014-01-01

    Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

  4. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  5. Oleic Acid Stimulates Complete Oxidation of Fatty Acids through Protein Kinase A-dependent Activation of SIRT1-PGC1α Complex*

    PubMed Central

    Lim, Ji-Hong; Gerhart-Hines, Zachary; Dominy, John E.; Lee, Yoonjin; Kim, Sungjin; Tabata, Mitsuhisa; Xiang, Yang K.; Puigserver, Pere

    2013-01-01

    Fatty acids are essential components of the dynamic lipid metabolism in cells. Fatty acids can also signal to intracellular pathways to trigger a broad range of cellular responses. Oleic acid is an abundant monounsaturated omega-9 fatty acid that impinges on different biological processes, but the mechanisms of action are not completely understood. Here, we report that oleic acid stimulates the cAMP/protein kinase A pathway and activates the SIRT1-PGC1α transcriptional complex to modulate rates of fatty acid oxidation. In skeletal muscle cells, oleic acid treatment increased intracellular levels of cyclic adenosine monophosphate (cAMP) that turned on protein kinase A activity. This resulted in SIRT1 phosphorylation at Ser-434 and elevation of its catalytic deacetylase activity. A direct SIRT1 substrate is the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), which became deacetylated and hyperactive after oleic acid treatment. Importantly, oleic acid, but not other long chain fatty acids such as palmitate, increased the expression of genes linked to fatty acid oxidation pathway in a SIRT1-PGC1α-dependent mechanism. As a result, oleic acid potently accelerated rates of complete fatty acid oxidation in skeletal muscle cells. These results illustrate how a single long chain fatty acid specifically controls lipid oxidation through a signaling/transcriptional pathway. Pharmacological manipulation of this lipid signaling pathway might provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation. PMID:23329830

  6. The Structural Characterization of Tumor Fusion Genes and Proteins.

    PubMed

    Wang, Dandan; Li, Daixi; Qin, Guangrong; Zhang, Wen; Ouyang, Jian; Zhang, Menghuan; Xie, Lu

    2015-01-01

    Chromosomal translocation, which generates fusion proteins in blood tumor or solid tumor, is considered as one of the major causes leading to cancer. Recent studies suggested that the disordered fragments in a fusion protein might contribute to its carcinogenicity. Here, we investigated the sequence feature near the breakpoints in the fusion partner genes, the structure features of breakpoints in fusion proteins, and the posttranslational modification preference in the fusion proteins. Results show that the breakpoints in the fusion partner genes have both sequence preference and structural preference. At the sequence level, nucleotide combination AG is preferred before the breakpoint and GG is preferred at the breakpoint. At the structural level, the breakpoints in the fusion proteins prefer to be located in the disordered regions. Further analysis suggests the phosphorylation sites at serine, threonine, and the methylation sites at arginine are enriched in disordered regions of the fusion proteins. Using EML4-ALK as an example, we further explained how the fusion protein leads to the protein disorder and contributes to its carcinogenicity. The sequence and structural features of the fusion proteins may help the scientific community to predict novel breakpoints in fusion genes and better understand the structure and function of fusion proteins. PMID:26347798

  7. The Structural Characterization of Tumor Fusion Genes and Proteins

    PubMed Central

    Wang, Dandan; Li, Daixi; Qin, Guangrong; Zhang, Wen; Ouyang, Jian; Zhang, Menghuan; Xie, Lu

    2015-01-01

    Chromosomal translocation, which generates fusion proteins in blood tumor or solid tumor, is considered as one of the major causes leading to cancer. Recent studies suggested that the disordered fragments in a fusion protein might contribute to its carcinogenicity. Here, we investigated the sequence feature near the breakpoints in the fusion partner genes, the structure features of breakpoints in fusion proteins, and the posttranslational modification preference in the fusion proteins. Results show that the breakpoints in the fusion partner genes have both sequence preference and structural preference. At the sequence level, nucleotide combination AG is preferred before the breakpoint and GG is preferred at the breakpoint. At the structural level, the breakpoints in the fusion proteins prefer to be located in the disordered regions. Further analysis suggests the phosphorylation sites at serine, threonine, and the methylation sites at arginine are enriched in disordered regions of the fusion proteins. Using EML4-ALK as an example, we further explained how the fusion protein leads to the protein disorder and contributes to its carcinogenicity. The sequence and structural features of the fusion proteins may help the scientific community to predict novel breakpoints in fusion genes and better understand the structure and function of fusion proteins. PMID:26347798

  8. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

    PubMed Central

    Gruber, David F.; Gaffney, Jean P.; Mehr, Shaadi; DeSalle, Rob; Sparks, John S.; Platisa, Jelena; Pieribone, Vincent A.

    2015-01-01

    We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. PMID:26561348

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

    PubMed

    Cui, Yuxia; Freedman, Jonathan H

    2009-09-11

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

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

    PubMed Central

    Cui, Yuxia; Freedman, Jonathan H.

    2009-01-01

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

  11. Mutations of 3c and spike protein genes correlate with the occurrence of feline infectious peritonitis.

    PubMed

    Bank-Wolf, Barbara Regina; Stallkamp, Iris; Wiese, Svenja; Moritz, Andreas; Tekes, Gergely; Thiel, Heinz-Jürgen

    2014-10-10

    The genes encoding accessory proteins 3a, 3b, 3c, 7a and 7b, the S2 domain of the spike (S) protein gene and the membrane (M) protein gene of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) samples were amplified, cloned and sequenced. For this faeces and/or ascites samples from 19 cats suffering from feline infectious peritonitis (FIP) as well as from 20 FECV-infected healthy cats were used. Sequence comparisons revealed that 3c genes of animals with FIP were heavily affected by nucleotide deletions and point mutations compared to animals infected with FECV; these alterations resulted either in early termination or destruction of the translation initiation codon. Two ascites-derived samples of cats with FIP which displayed no alterations of ORF3c harboured mutations in the S2 domain of the S protein gene which resulted in amino acid exchanges or deletions. Moreover, changes in 3c were often accompanied by mutations in S2. In contrast, in samples obtained from faeces of healthy cats, the ORF3c was never affected by such mutations. Similarly ORF3c from faecal samples of the cats with FIP was mostly intact and showed only in a few cases the same mutations found in the respective ascites samples. The genes encoding 3a, 3b, 7a and 7b displayed no mutations linked to the feline coronavirus (FCoV) biotype. The M protein gene was found to be conserved between FECV and FIPV samples. Our findings suggest that mutations of 3c and spike protein genes correlate with the occurrence of FIP. PMID:25150756

  12. The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control.

    PubMed Central

    Kinoshita, N; Goebl, M; Yanagida, M

    1991-01-01

    The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis. Images PMID:1944266

  13. FLU, an amino acid substitution model for influenza proteins

    PubMed Central

    2010-01-01

    Background The amino acid substitution model is the core component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Although several general amino acid substitution models have been estimated from large and diverse protein databases, they remain inappropriate for analyzing specific species, e.g., viruses. Emerging epidemics of influenza viruses raise the need for comprehensive studies of these dangerous viruses. We propose an influenza-specific amino acid substitution model to enhance the understanding of the evolution of influenza viruses. Results A maximum likelihood approach was applied to estimate an amino acid substitution model (FLU) from ~113, 000 influenza protein sequences, consisting of ~20 million residues. FLU outperforms 14 widely used models in constructing maximum likelihood phylogenetic trees for the majority of influenza protein alignments. On average, FLU gains ~42 log likelihood points with an alignment of 300 sites. Moreover, topologies of trees constructed using FLU and other models are frequently different. FLU does indeed have an impact on likelihood improvement as well as tree topologies. It was implemented in PhyML and can be downloaded from ftp://ftp.sanger.ac.uk/pub/1000genomes/lsq/FLU or included in PhyML 3.0 server at http://www.atgc-montpellier.fr/phyml/. Conclusions FLU should be useful for any influenza protein analysis system which requires an accurate description of amino acid substitutions. PMID:20384985

  14. Acid Cleavable Surface enhanced Raman Tagging for Protein Detection

    PubMed Central

    Zhang, Dongmao; Vangala, Karthikeshwar; Li, Shaoyong; Yanney, Michael; Xia, Hao; Zou, Sige; Sygula, Andrzej

    2010-01-01

    Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection. PMID:21109888

  15. Calreticulin: one protein, one gene, many functions.

    PubMed Central

    Michalak, M; Corbett, E F; Mesaeli, N; Nakamura, K; Opas, M

    1999-01-01

    The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology. PMID:10567207

  16. Mammalian ets-1 and ets-2 genes encode highly conserved proteins

    SciTech Connect

    Watson, D.K.; McWilliams, M.J.; Lapis, P.; Lautenberger, J.A.; Schweinfest, C.W.; Papas, T.S. )

    1988-11-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, the authors have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is >95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published finding indicates that ets is a family of genes whose members share distinct domains.

  17. Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

    PubMed Central

    Watson, D K; McWilliams, M J; Lapis, P; Lautenberger, J A; Schweinfest, C W; Papas, T S

    1988-01-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains. PMID:2847145

  18. Structure and Function of the Escherichia coli Protein YmgB: A Protein Critical for Biofilm Formation and Acid-resistance

    SciTech Connect

    Lee,J.; Page, R.; Garcia-Contreras, R.; Palermino, J.; Zhang, X.; Doshi, O.; Wood, T.; Peti, W.

    2007-01-01

    The Escherichia coli gene cluster ymgABC was identified in transcriptome studies to have a role in biofilm development and stability. In this study, we showed that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB has a major role in acid-resistance in E. coli. Our data show that these phenotypes are potentially mediated through interactions with the important cell signal indole. In addition, gel mobility-shift assays suggest that YmgB may be a non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we showed that YmgB binds, either directly or indirectly, via a probable ligand, genes important for biofilm formation. To advance our understanding of the function of YmgB, we used X-ray crystallography to solve the structure of the protein to 1.8 A resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite having only 5% sequence identity. This supports our DNA microarray data showing that YmgB is a gene regulatory protein. Therefore, this protein, which clearly has a critical role in acid-resistance in E. coli, has been renamed as AriR for regulator of acid resistance influenced by indole.

  19. Gene evolution and functions of extracellular matrix proteins in teeth.

    PubMed

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-03-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the "core matrisome" in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  20. A Homologue of the 3-Oxoacyl-(Acyl Carrier Protein) Synthase III Gene Located in the Glycosylation Island of Pseudomonas syringae pv. tabaci Regulates Virulence Factors via N-Acyl Homoserine Lactone and Fatty Acid Synthesis▿

    PubMed Central

    Taguchi, Fumiko; Ogawa, Yujiro; Takeuchi, Kasumi; Suzuki, Tomoko; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki

    2006-01-01

    Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Δorf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Δorf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Δorf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Δorf3 mutant. The phenotypes of the Δorf3 mutant and an AHL synthesis (ΔpsyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Δorf3 mutant. The swarming motility of the Δorf3 mutant was greater than that of the ΔpsyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Δorf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605. PMID:17028280

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    1995-06-30

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

  3. Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network

    PubMed Central

    Keith, Benjamin P.; Robertson, David L.; Hentges, Kathryn E.

    2014-01-01

    Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity. PMID:25538735

  4. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    PubMed

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development. PMID:27336588

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

    PubMed

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

    2014-06-11

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

  6. Suppressors of ssy1 and ptr3 null mutations define novel amino acid sensor-independent genes in Saccharomyces cerevisiae.

    PubMed Central

    Forsberg, H; Hammar, M; Andréasson, C; Molinér, A; Ljungdahl, P O

    2001-01-01

    Ssy1p and Ptr3p are components of the yeast plasma membrane SPS amino acid sensor. In response to extracellular amino acids this sensor initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and amino acid permeases (AAPs). As a result of diminished leucine uptake capabilities, ssy1Delta leu2 and ptr3Delta leu2 mutant strains are unable to grow on synthetic complete medium (SC). Genes affecting the functional expression of AAPs were identified by selecting spontaneous suppressing mutations in amino acid sensor-independent (ASI) genes that restore growth on SC. The suppressors define 11 recessive (asi) complementation groups and 5 dominant (ASI) linkage groups. Strains with mutations in genes assigned to these 16 groups fall into two phenotypic classes. Mutations in the class I genes (ASI1, ASI2, ASI3, TUP1, SSN6, ASI13) derepress the transcription of AAP genes. ASI1, ASI2, and ASI3 encode novel membrane proteins, and Asi1p and Asi3p are homologous proteins that have conserved ubiquitin ligase-like RING domains at their extreme C termini. Several of the class II genes (DOA4, UBA1, BRO1, BUL1, RSP5, VPS20, VPS36) encode proteins implicated in controlling aspects of post-Golgi endosomal-vacuolar protein sorting. The results from genetic and phenotypic analysis indicate that SPS sensor-initiated signals function positively to facilitate amino acid uptake and that two independent ubiquitin-mediated processes negatively modulate amino acid uptake. PMID:11454748

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

    PubMed Central

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

    2012-01-01

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

  8. Fatty acid induced remodeling within the human liver fatty acid-binding protein.

    PubMed

    Sharma, Ashwani; Sharma, Amit

    2011-09-01

    We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP. PMID:21757748

  9. Glial Fibrillary acidic protein: From intermediate filament assembly and gliosis to neurobiomarker

    PubMed Central

    Yang, Zhihui; Wang, Kevin K.W.

    2015-01-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament-III protein uniquely found in astrocytes in the CNS, non-myelinating Schwann cells in the PNS and enteric glial cells. GFAP mRNA expressions are regulated by several nuclear-receptor hormones, growth factors and lipopolysaccharides. GFAP is also subjected to a number of post-translational modifications while GFAP mutations result in protein deposits known as Rosenthal fibers in Alexander disease. GFAP gene activation and protein induction appear to play a critical role in astroglia cell activation (astrogliosis) following CNS injuries and neurodegeneration. Emerging evidence also suggests that, following traumatic brain and spinal cord injuries and stroke, GFAP protein and its breakdown products are rapidly released into biofluids, making them strong candidate biomarkers for such neurological disorders. PMID:25975510

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

    PubMed

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

    2013-01-01

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

  11. Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

    PubMed

    Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

    2008-02-15

    Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. PMID:17661353

  12. Protein location prediction using atomic composition and global features of the amino acid sequence

    SciTech Connect

    Cherian, Betsy Sheena; Nair, Achuthsankar S.

    2010-01-22

    Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectively used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.

  13. Characterization of the yellow fever mosquito sterol carrier protein-2 like 3 gene and ligand-bound protein structure

    SciTech Connect

    Dyer, David H.; Vyazunova, Irina; Lorch, Jeffery M.; Forest, Katrina T.; Lan, Que

    2009-06-12

    The sterol carrier protein-2 like 3 gene (AeSCP-2L3), a new member of the SCP-2 protein family, is identified from the yellow fever mosquito, Aedes aegypti. The predicted molecular weight of AeSCP-2L3 is 13.4 kDa with a calculated pI of 4.98. AeSCP-2L3 transcription occurs in the larval feeding stages and the mRNA levels decrease in pupae and adults. The highest levels of AeSCP-2L3 gene expression are found in the body wall, and possibly originated in the fat body. This is the first report of a mosquito SCP-2-like protein with prominent expression in tissue other than the midgut. The X-ray protein crystal structure of AeSCP-2L3 reveals a bound C16 fatty acid whose acyl tail penetrates deeply into a hydrophobic cavity. Interestingly, the ligand-binding cavity is slightly larger than previously described for AeSCP-2 (Dyer et al. J Biol Chem 278:39085-39091, 2003) and AeSCP-2L2 (Dyer et al. J Lipid Res M700460-JLR200, 2007). There are also an additional 10 amino acids in SCP-2L3 that are not present in other characterized mosquito SCP-2s forming an extended loop between {beta}3 and {beta}4. Otherwise, the protein backbone is exceedingly similar to other SCP-2 and SCP-2-like proteins. In contrast to this observed high structural homology of members in the mosquito SCP2 family, the amino acid sequence identity between the members is less than 30%. The results from structural analysis imply that there have been evolutionary constraints that favor the SCP-2 C{alpha} backbone fold while the specificity of ligand binding can be altered.

  14. An essential role of a ferritin-like protein in acid stress tolerance of Listeria monocytogenes.

    PubMed

    Milecka, Dorota; Samluk, Anna; Wasiak, Katarzyna; Krawczyk-Balska, Agata

    2015-03-01

    The expression of ten genes of Listeria monocytogenes previously identified as penicillin G-inducible was transcriptionally analyzed in the presence of 0.5 M KCl, pH 5.0 and 42 °C. This study revealed that all the genes are upregulated by osmotic stress, seven by acid stress and four by temperature stress conditions. The contribution of a gene encoding a ferritin-like protein (fri), a two-component phosphate-response regulator (phoP) and an AraC/XylS family transcription regulator (axyR) to temperature, acid and osmotic stress tolerance was further examined by analysis of nonpolar deletion mutants. This revealed that a lack of PhoP or AxyR does not affect the ability to grow under the tested stress conditions. However, the Δ fri strain showed slightly delayed growth under osmotic and clearly impaired growth under acid stress conditions, indicating an important role of the ferritin-like protein in acid stress tolerance. PMID:25352185

  15. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition.

    PubMed

    Wu, Guoyao

    2014-01-01

    Amino acids are building blocks for proteins in all animals. Based on growth or nitrogen balance, amino acids were traditionally classified as nutritionally essential or nonessential for mammals, birds and fish. It was assumed that all the "nutritionally nonessential amino acids (NEAA)" were synthesized sufficiently in the body to meet the needs for maximal growth and optimal health. However, careful analysis of the scientific literature reveals that over the past century there has not been compelling experimental evidence to support this assumption. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, fertility, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and to protect the integrity of the intestinal mucosa. Thus, diets for animals must contain all NEAA to optimize their survival, growth, development, reproduction, and health. Furthermore, NEAA should be taken into consideration in revising the "ideal protein" concept that is currently used to formulate swine and poultry diets. Adequate provision of all amino acids (including NEAA) in diets enhances the efficiency of animal production. In this regard, amino acids should not be classified as nutritionally essential or nonessential in animal or human nutrition. The new Texas A&M University's optimal ratios of dietary amino acids for swine and chickens are expected to beneficially reduce dietary protein content and improve the efficiency of their nutrient utilization, growth, and production performance. PMID:24999386

  16. Structure, expression, and hormonal control of genes from the mosquito, Aedes aegypti, which encode proteins similar to the vitelline membrane proteins of Drosophila melanogaster.

    PubMed

    Lin, Y; Hamblin, M T; Edwards, M J; Barillas-Mury, C; Kanost, M R; Knipple, D C; Wolfner, M F; Hagedorn, H H

    1993-02-01

    Genomic and cDNA clones of a gene expressed after a blood meal in the mosquito, Aedes aegypti, were identified as having significant similarity to the vitelline membrane protein genes of Drosophila melanogaster. The predicted protein had unusually high contents of alanine, histidine, and proline and contained a region of hydrophobic amino acids that was highly conserved in the predicted protein of the D. melanogaster vitelline membrane protein genes. The 15a gene was expressed from 5 to 40 hr after a blood meal. It was expressed only in the follicle cells of the ovary, particularly in the cells surrounding the oocyte. The 15a gene was expressed in ovaries of the blood-fed, decapitated female in response to an injection of 20-hydroxyecdysone, and in ovaries from non-blood-fed females incubated with the hormone, even in the presence of cycloheximide. A second gene, with weaker homology to 15a, is presumably another member of a family of related genes, as is the case with D. melanogaster vitelline membrane protein genes. This second gene contained a coding sequence similar to a decapeptide recently isolated from mosquito ovaries as an "oostatic factor" (Borovsky et al., FASEB J. 4, 3015-3020, 1990). PMID:8432405

  17. Genes VI, VII, and IX of phage M13 code for minor capsid proteins of the virion.

    PubMed Central

    Simons, G F; Konings, R N; Schoenmakers, J G

    1981-01-01

    The minor capsid proteins C and D from phage M13 have been characterized by differential amino acid labeling and amino-terminal sequence analysis. We demonstrate that D protein (Mr 12,260) is the product of gene VI, whereas the C component is composed of the products of both gene VII (Mr 3580) and gene IX (Mr 3650). Our data further show that the proteins of genes VI, VII, and IX are not subject to proteolytic processing but are packaged into mature virions as their primary translational products. On the basis of incorporation of specific amino acids, the copy numbers of these proteins in M13 virions could be estimated relative to the number of A protein molecules. The M13 phage contains on the average 5 molecules of A protein, 5 molecules of VI protein and 3-4 molecules of both VII protein and IX protein. These copy numbers remained unchanged in M13 recombinant phages of up to two times the length of wild-type phages, a fact that indicates that these minor capsid proteins are located at either one or both ends of the phage filament. Images PMID:6945579

  18. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

    PubMed

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-05-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  19. Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.

    PubMed Central

    Abe, H; Yamaguchi-Shinozaki, K; Urao, T; Iwasaki, T; Hosokawa, D; Shinozaki, K

    1997-01-01

    In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) and requires protein biosynthesis for ABA-dependent gene expression. Previous experiments established that a 67-bp DNA fragment of the rd22 promoter is sufficient for dehydration- and ABA-induced gene expression and that this DNA fragment contains two closely located putative recognition sites for the basic helix-loop-helix protein MYC and one putative recognition site for MYB. We have carefully analyzed the 67-bp region of the rd22 promoter in transgenic tobacco plants and found that both the first MYC site and the MYB recognition site function as cis-acting elements in the dehydration-induced expression of the rd22 gene. A cDNA encoding a MYC-related DNA binding protein was isolated by DNA-ligand binding screening, using the 67-bp region as a probe, and designated rd22BP1. The rd22BP1 cDNA encodes a 68-kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors. The rd22BP1 protein binds specifically to the first MYC recognition site in the 67-bp fragment. RNA gel blot analysis revealed that transcription of the rd22BP1 gene is induced by dehydration stress and ABA treatment, and its induction precedes that of rd22. We have reported a drought- and ABA-inducible gene that encodes the MYB-related protein ATMYB2. In a transient transactivation experiment using Arabidopsis leaf protoplasts, we demonstrated that both the rd22BP1 and ATMYB2 proteins activate transcription of the rd22 promoter fused to the beta-glucuronidase reporter gene. These results indicate that both the rd22BP1 (MYC) and ATMYB2 (MYB) proteins function as transcriptional activators in the dehydration- and ABA-inducible expression of the rd22 gene. PMID:9368419

  20. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    PubMed

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems. PMID:25521817

  1. Expression of genes encoding extracellular matrix proteins: A macroarray study

    PubMed Central

    FUTYMA, KONRAD; MIOTŁA, PAWEŁ; RÓŻYŃSKA, KRYSTYNA; ZDUNEK, MAŁGORZATA; SEMCZUK, ANDRZEJ; RECHBERGER, TOMASZ; WOJCIEROWSKI, JACEK

    2014-01-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs. PMID:25231141

  2. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    PubMed

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  3. Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

    PubMed Central

    Estes, M K; Mason, B B; Crawford, S; Cohen, J

    1984-01-01

    The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic. Images PMID:6322125

  4. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  5. The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

    PubMed Central

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

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

    PubMed

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

    2012-05-16

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

  7. Lists2Networks: Integrated analysis of gene/protein lists

    PubMed Central

    2010-01-01

    Background Systems biologists are faced with the difficultly of analyzing results from large-scale studies that profile the activity of many genes, RNAs and proteins, applied in different experiments, under different conditions, and reported in different publications. To address this challenge it is desirable to compare the results from different related studies such as mRNA expression microarrays, genome-wide ChIP-X, RNAi screens, proteomics and phosphoproteomics experiments in a coherent global framework. In addition, linking high-content multilayered experimental results with prior biological knowledge can be useful for identifying functional themes and form novel hypotheses. Results We present Lists2Networks, a web-based system that allows users to upload lists of mammalian genes/proteins onto a server-based program for integrated analysis. The system includes web-based tools to manipulate lists with different set operations, to expand lists using existing mammalian networks of protein-protein interactions, co-expression correlation, or background knowledge co-annotation correlation, as well as to apply gene-list enrichment analyses against many gene-list libraries of prior biological knowledge such as pathways, gene ontology terms, kinase-substrate, microRNA-mRAN, and protein-protein interactions, metabolites, and protein domains. Such analyses can be applied to several lists at once against many prior knowledge libraries of gene-lists associated with specific annotations. The system also contains features that allow users to export networks and share lists with other users of the system. Conclusions Lists2Networks is a user friendly web-based software system expected to significantly ease the computational analysis process for experimental systems biologists employing high-throughput experiments at multiple layers of regulation. The system is freely available at http://www.lists2networks.org. PMID:20152038

  8. A macromolecular delivery vehicle for protein-based vaccines: Acid-degradable protein-loaded microgels

    PubMed Central

    Murthy, Niren; Xu, Mingcheng; Schuck, Stephany; Kunisawa, Jun; Shastri, Nilabh; Fréchet, Jean M. J.

    2003-01-01

    The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigen-presenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200–500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The acid-degradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity. PMID:12704236

  9. Predicting disease-related genes by topological similarity in human protein-protein interaction network

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Hu, Ke; Tang, Yi

    2010-08-01

    Predicting genes likely to be involved in human diseases is an important task in bioinformatics field. Nowadays, the accumulation of human protein-protein interactions (PPIs) data provides us an unprecedented opportunity to gain insight into human diseases. In this paper, we adopt the topological similarity in human protein-protein interaction network to predict disease-related genes. As a computational algorithm to speed up the identification of disease-related genes, the topological similarity has substantial advantages over previous topology-based algorithms. First of all, it provides a global measurement of similarity between two vertices. Secondly, quantity which can measure new topological feature has been integrated into the notion of topological similarity. Our method is specially designed for predicting disease-related genes of single disease-gene family. The proposed method is applied to human protein-protein interaction and hepatocellular carcinoma (HCC) data. The results show a significant enrichment of disease-related genes that are characterized by higher topological similarity than other genes.

  10. The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes

    SciTech Connect

    Dyer, K.D. |; Handen, J.S.; Rosenberg, H.F.

    1997-03-01

    The Charcot-Leyden crystal (CLC) protein, or eosinophil lysophospholipase, is a characteristic protein of human eosinophils and basophils; recent work has demonstrated that the CLC protein is both structurally and functionally related to the galectin family of {beta}-galactoside binding proteins. The galectins as a group share a number of features in common, including a linear ligand binding site encoded on a single exon. In this work, we demonstrate that the intron-exon structure of the gene encoding CLC is analogous to those encoding the galectins. The coding sequence of the CLC gene is divided into four exons, with the entire {beta}-galactoside binding site encoded by exon III. We have isolated CLC {beta}-galactoside binding sites from both orangutan (Pongo pygmaeus) and murine (Mus musculus) genomic DNAs, both encoded on single exons, and noted conservation of the amino acids shown to interact directly with the {beta}-galactoside ligand. The most likely interpretation of these results suggests the occurrence of one or more exon duplication and insertion events, resulting in the distribution of this lectin domain to CLC as well as to the multiple galectin genes. 35 refs., 3 figs.

  11. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  12. (-)-Hydroxycitric Acid Nourishes Protein Synthesis via Altering Metabolic Directions of Amino Acids in Male Rats.

    PubMed

    Han, Ningning; Li, Longlong; Peng, Mengling; Ma, Haitian

    2016-08-01

    (-)-Hydroxycitric acid (HCA), a major active ingredient of Garcinia Cambogia extracts, had shown to suppress body weight gain and fat accumulation in animals and humans. While, the underlying mechanism of (-)-HCA has not fully understood. Thus, this study was aimed to investigate the effects of long-term supplement with (-)-HCA on body weight gain and variances of amino acid content in rats. Results showed that (-)-HCA treatment reduced body weight gain and increased feed conversion ratio in rats. The content of hepatic glycogen, muscle glycogen, and serum T4 , T3 , insulin, and Leptin were increased in (-)-HCA treatment groups. Protein content in liver and muscle were significantly increased in (-)-HCA treatment groups. Amino acid profile analysis indicated that most of amino acid contents in serum and liver, especially aromatic amino acid and branched amino acid, were higher in (-)-HCA treatment groups. However, most of the amino acid contents in muscle, especially aromatic amino acid and branched amino acid, were reduced in (-)-HCA treatment groups. These results indicated that (-)-HCA treatment could reduce body weight gain through promoting energy expenditure via regulation of thyroid hormone levels. In addition, (-)-HCA treatment could promote protein synthesis by altering the metabolic directions of amino acids. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145492

  13. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction

    PubMed Central

    Teng, Shaolei; Wang, Liangjiang; Srivastava, Anand K.; Schwartz, Charles E.; Alexov, Emil

    2012-01-01

    A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases. PMID:21297231

  16. The herpes simplex virus 1 protein kinase encoded by the US3 gene mediates posttranslational modification of the phosphoprotein encoded by the UL34 gene.

    PubMed Central

    Purves, F C; Spector, D; Roizman, B

    1991-01-01

    Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable. Images PMID:1656069

  17. Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis.

    PubMed

    Seidel, C; Walz, A; Park, S; Cohen, J D; Ludwig-Müller, J

    2006-05-01

    Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates. PMID:16807826

  18. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-05-15

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  19. Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways

    PubMed Central

    Dawson, Deborah V.; Blanchette, Derek R.; Drake, David R.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    ABSTRACT Lipids endogenous to skin and mucosal surfaces exhibit potent antimicrobial activity against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Our previous work demonstrated the antimicrobial activity of the fatty acid sapienic acid (C16:1Δ6) against P. gingivalis and found that sapienic acid treatment alters both protein and lipid composition from those in controls. In this study, we further examined whole-cell protein differences between sapienic acid-treated bacteria and untreated controls, and we utilized open-source functional association and annotation programs to explore potential mechanisms for the antimicrobial activity of sapienic acid. Our analyses indicated that sapienic acid treatment induces a unique stress response in P. gingivalis resulting in differential expression of proteins involved in a variety of metabolic pathways. This network of differentially regulated proteins was enriched in protein-protein interactions (P = 2.98 × 10−8), including six KEGG pathways (P value ranges, 2.30 × 10−5 to 0.05) and four Gene Ontology (GO) molecular functions (P value ranges, 0.02 to 0.04), with multiple suggestive enriched relationships in KEGG pathways and GO molecular functions. Upregulated metabolic pathways suggest increases in energy production, lipid metabolism, iron acquisition and processing, and respiration. Combined with a suggested preferential metabolism of serine, which is necessary for fatty acid biosynthesis, these data support our previous findings that the site of sapienic acid antimicrobial activity is likely at the bacterial membrane. IMPORTANCE P. gingivalis is an important opportunistic pathogen implicated in periodontitis. Affecting nearly 50% of the population, periodontitis is treatable, but the resulting damage is irreversible and eventually progresses to tooth loss. There is a great need for natural products that can be used to treat and/or prevent the overgrowth of

  20. INCAP studies of energy, amino acids, and protein.

    PubMed

    Viteri, Fernando E

    2010-03-01

    This Special Issue summarizes the results of several studies aimed at providing information on a series of questions related to the adequate protein and energy intakes that allow adequate growth and function in children and work performance and productivity in adults. The effect of different sources of protein on nitrogen balance and the requirements of essential amino acids in young children were also explored in fully recovered, previously malnourished children housed in the Metabolic Ward of the Biomedical Division of INCAP. The following are the main results of these investigations: Animal experiments and studies in children recovering from protein-energy malnutrition (PEM) strongly suggest that even when requirements of all nutrients are satisfied, inactivity reduces the rate of linear growth and physical activity improves it as well as lean body mass repletion. The effects of different energy intakes on nitrogen balance demonstrated how energy intake modifies the need to ingest different amounts of protein to satisfy protein requirements. Insensible nitrogen losses in preschool children and their relation to protein intake was demonstrated. The quality of even "good protein sources" modifies the amount needed to satisfy nitrogen requirements, and corn and bean-based diets can satisfy protein needs for health and even growth of young children. Essential amino acid requirements of 2-year-old children was assessed by diverse measurements of nitrogen metabolism and amino acid levels in blood, and were found lower than those recommended by FAO-WHO. In rural adult populations the relationship between energy and protein intake, productivity and body composition, and the impact of environmental hygiene on nitrogen balance was demonstrated and measured. PMID:20461903

  1. The ribosomal protein genes and Minute loci of Drosophila melanogaster

    PubMed Central

    Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

    2007-01-01

    Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

  2. Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage.

    PubMed

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

    2007-10-01

    Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment. PMID:17675438

  3. Immunoreactivity of polyclonal antibodies generated against the carboxy terminus of the predicted amino acid sequence of the Huntington disease gene

    SciTech Connect

    Alkatib, G.; Graham, R.; Pelmear-Telenius, A.

    1994-09-01

    A cDNA fragment spanning the 3{prime}-end of the Huntington disease gene (from 8052 to 9252) was cloned into a prokaryotic expression vector containing the E. Coli lac promoter and a portion of the coding sequence for {beta}-galactosidase. The truncated {beta}-galactosidase gene was cleaved with BamHl and fused in frame to the BamHl fragment of the Huntington disease gene 3{prime}-end. Expression analysis of proteins made in E. Coli revealed that 20-30% of the total cellular proteins was represented by the {beta}-galactosidase-huntingtin fusion protein. The identity of the Huntington disease protein amino acid sequences was confirmed by protein sequence analysis. Affinity chromatography was used to purify large quantities of the fusion protein from bacterial cell lysates. Affinity-purified proteins were used to immunize New Zealand white rabbits for antibody production. The generated polyclonal antibodies were used to immunoprecipitate the Huntington disease gene product expressed in a neuroblastoma cell line. In this cell line the antibodies precipitated two protein bands of apparent gel migrations of 200 and 150 kd which together, correspond to the calculated molecular weight of the Huntington disease gene product (350 kd). Immunoblotting experiments revealed the presence of a large precursor protein in the range of 350-750 kd which is in agreement with the predicted molecular weight of the protein without post-translational modifications. These results indicate that the huntingtin protein is cleaved into two subunits in this neuroblastoma cell line and implicate that cleavage of a large precursor protein may contribute to its biological activity. Experiments are ongoing to determine the precursor-product relationship and to examine the synthesis of the huntingtin protein in freshly isolated rat brains, and to determine cellular and subcellular distribution of the gene product.

  4. The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

    PubMed

    Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

    2016-01-01

    Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis. PMID:26837571

  5. The androgen-binding protein gene is expressed in male and female rat brain.

    PubMed

    Wang, Y M; Bayliss, D A; Millhorn, D E; Petrusz, P; Joseph, D R

    1990-12-01

    Extracellular androgen-binding proteins (ABP) are thought to modulate the regulatory functions of androgens and the trans-acting nuclear androgen receptor. Testicular ABP and plasma sex hormone-binding globulin (SHBG), which is produced in liver, are encoded by the same gene. We have now found that the ABP-SHBG gene is also expressed in male and female rat brain. Immunoreactive ABP was found to be present in neuronal cell bodies throughout the brain as well as in fibers of the hypothalamic median eminence. The highest concentrations of immunoreactive cell bodies were located in the supraoptic and paraventricular nuclei. Likewise, ABP mRNA was present in all brain regions examined. Analysis of cDNA clones representing brain ABP mRNAs revealed amino acid sequence differences in brain and testicular ABPs. The protein encoded by an alternatively processed RNA has sequence characteristics suggesting that the protein could act as a competitior of ABP binding to cell surface receptors. These data and gene-sequencing experiments indicate that a specific ABP gene promoter is used for transcription initiation in brain. ABP may function in brain as an androgen carrier protein; however, in view of the widespread presence of ABP and ABP mRNA in brain, the protein may have a much broader, yet unknown, function. PMID:1701136

  6. Strategy for large scale solubilization of coal - characterization of Neurospora protein and gene

    SciTech Connect

    Patel, A.; Chen, Y.P.; Mishra, N.C.

    1995-12-31

    Low grade coal placed on mycelial mat of Neurospora crassa growing on Petri plate was found to be solubilized by this fungus. A heat stable protein has been purified to near homogeneity which can solubilize low grade coal in in vitro. The biochemical properties of the Neurospora protein will be presented. The nature of the product obtained after solubilization of coal by Neurospora protein in vivo and in vitro will also be presented. The N-terminus sequence of the amino acids of this protein will be used to design primer for possible cloning of gene for Neurospora protein capable of solubilization of coal in order to develop methodology for coal solubilization on a large scale.

  7. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals. PMID:25265085

  8. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    NASA Astrophysics Data System (ADS)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  9. Gene Delivery from Supercharged Coiled-coil Protein and Cationic Lipid Hybrid Complex

    PubMed Central

    More, Haresh T.; Frezzo, Joseph A.; Dai, Jisen; Yamano, Seiichi; Montclare, Jin K.

    2014-01-01

    A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP•DNA(8:1)•FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat•FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP•DNA(8:1)•FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery. PMID:24875765

  10. The proteolipid protein gene: Double, double, . . . and trouble

    SciTech Connect

    Hodes, M.E.; Dlouhy, S.R.

    1996-07-01

    That more of a good thing may be too much has been apparent at least since the discovery that Down syndrome is caused by three copies of chromosome 21 instead of the normal two. Duplications of myelin genes also lead to trouble. An extra dose of PMP22, the gene for a protein of peripheral nervous system myelin, causes Charcot-Marie Tooth type 1A disease (CMT1A). Increased dosage of the proteolipid protein gene, PLP, which encodes the chief protein of CNS myelin, can cause Pelizaeus-Merzbacher disease (PMD). The work of Inoue et al. is of particular importance because they found the duplication in four of five families with {open_quotes}classical{close_quotes} PMD, whereas other changes in PLP, such as missense mutations, are found in no more than one in four or five patients with the disease. 27 refs.

  11. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually “gutted” and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  12. Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1987-01-01

    The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991]. Images Fig. 1. Fig. 3. PMID:3593284

  13. Mutations of the Microsomal Triglyceride-Transfer–Protein Gene in Abetalipoproteinemia

    PubMed Central

    Narcisi, Teresa M. E.; Shoulders, Carol C.; Chester, S. Ann; Read, Jacqueline; Brett, David J.; Harrison, Georgina B.; Grantham, Tamsin T.; Fox, Margaret F.; Povey, Sue; de Bruin, Tjerk W. A.; Erkelens, D. Willem; Muller, David P. R.; Lloyd, June K.; Scott, James

    1995-01-01

    Elevated plasma levels of apolipoprotein B (apoB)–containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. ImagesFigure 1p1304-aFigure 3Figure 4 PMID:8533758

  14. Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia.

    PubMed

    Narcisi, T M; Shoulders, C C; Chester, S A; Read, J; Brett, D J; Harrison, G B; Grantham, T T; Fox, M F; Povey, S; de Bruin, T W

    1995-12-01

    Elevated plasma levels of apolipoprotein B (apoB)-containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. PMID:8533758

  15. Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia

    SciTech Connect

    Narcisi, T.M.E.; Shoulders, C.C.; Chester, S.A.

    1995-12-01

    Elevated plasma levels of apolipoprotein B (apoB)-containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity. 49 refs., 4 figs., 5 tabs.

  16. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis.

    PubMed

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  17. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    PubMed Central

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  18. Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

    PubMed Central

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2014-01-01

    The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens. PMID:25514417

  19. Protein and amino acid metabolism in the human newborn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Birth and adaptation to extrauterine life involve major shifts in the protein and energy metabolism of the human newborn. These include a shift from a state of continuous supply of nutrients including amino acids from the mother to cyclic periodic oral intake, a change in the redox state of organs, ...

  20. Regulation of Brome Mosaic Virus Gene Expression by Restriction of Initiation of Protein Synthesis

    PubMed Central

    Chroboczek, Jadwiga; Puchkova, Ludmiła; Zagórski, Włodzimierz

    1980-01-01

    The translation of total and individual brome mosaic virus (BMV) RNAs was examined in a wheat germ cell-free system in the presence of various inhibitors. Inhibitors of the initiation of polypeptide synthesis, e.g., potassium ions, 7-methylguanosine 5′ -monophosphate, and aurintricarboxylic acid, were shown not only to inhibit overall BMV protein synthesis but also to change the ratio of BMV polypeptides synthesized. Under conditions restrictive for initiation, the translation of nonstructural BMV genes was suppressed, but coat protein synthesis proceeded at a high rate. A similar discrimination among BMV messengers was exerted by a regulatory protein kinase isolated from wheat germ. These results suggest that the regulation of the expression of BMV genes is based on a difference in the mechanism of formation of initiation complexes for individual BMV messages. Images PMID:16789194

  1. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  2. Sequence analysis and expression of the M1 and M2 matrix protein genes of hirame rhabdovirus (HIRRV)

    USGS Publications Warehouse

    Nishizawa, T.; Kurath, G.; Winton, J.R.

    1997-01-01

    We have cloned and sequenced a 2318 nucleotide region of the genomic RNA of hirame rhabdovirus (HIRRV), an important viral pathogen of Japanese flounder Paralichthys olivaceus. This region comprises approximately two-thirds of the 3' end of the nucleocapsid protein (N) gene and the complete matrix protein (M1 and M2) genes with the associated intergenic regions. The partial N gene sequence was 812 nucleotides in length with an open reading frame (ORF) that encoded the carboxyl-terminal 250 amino acids of the N protein. The M1 and M2 genes were 771 and 700 nucleotides in length, respectively, with ORFs encoding proteins of 227 and 193 amino acids. The M1 gene sequence contained an additional small ORF that could encode a highly basic, arginine-rich protein of 25 amino acids. Comparisons of the N, M1, and M2 gene sequences of HIRRV with the corresponding sequences of the fish rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) or viral hemorrhagic septicemia virus (VHSV) indicated that HIRRV was more closely related to IHNV than to VHSV, but was clearly distinct from either. The putative consensus gene termination sequence for IHNV and VHSV, AGAYAG(A)(7), was present in the N-M1, M1-M2, and M2-G intergenic regions of HIRRV as were the putative transcription initiation sequences YGGCAC and AACA. An Escherichia coli expression system was used to produce recombinant proteins from the M1 and M2 genes of HIRRV. These were the same size as the authentic M1 and M2 proteins and reacted with anti-HIRRV rabbit serum in western blots. These reagents can be used for further study of the fish immune response and to test novel control methods.

  3. Identification of Nuclear Genes Encoding Chloroplast-Localized Proteins Required for Embryo Development in Arabidopsis1[W][OA

    PubMed Central

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-01-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function. PMID:21139083

  4. Predicting Abdominal Aortic Aneurysm Target Genes by Level-2 Protein-Protein Interaction

    PubMed Central

    Fu, Yi; Cui, Qinghua; Kong, Wei

    2015-01-01

    Abdominal aortic aneurysm (AAA) is frequently lethal and has no effective pharmaceutical treatment, posing a great threat to human health. Previous bioinformatics studies of the mechanisms underlying AAA relied largely on the detection of direct protein-protein interactions (level-1 PPI) between the products of reported AAA-related genes. Thus, some proteins not suspected to be directly linked to previously reported genes of pivotal importance to AAA might have been missed. In this study, we constructed an indirect protein-protein interaction (level-2 PPI) network based on common interacting proteins encoded by known AAA-related genes and successfully predicted previously unreported AAA-related genes using this network. We used four methods to test and verify the performance of this level-2 PPI network: cross validation, human AAA mRNA chip array comparison, literature mining, and verification in a mouse CaPO4 AAA model. We confirmed that the new level-2 PPI network is superior to the original level-1 PPI network and proved that the top 100 candidate genes predicted by the level-2 PPI network shared similar GO functions and KEGG pathways compared with positive genes. PMID:26496478

  5. Identifying Novel Candidate Genes Related to Apoptosis from a Protein-Protein Interaction Network

    PubMed Central

    Wang, Baoman; Yuan, Fei; Kong, Xiangyin; Hu, Lan-Dian; Cai, Yu-Dong

    2015-01-01

    Apoptosis is the process of programmed cell death (PCD) that occurs in multicellular organisms. This process of normal cell death is required to maintain the balance of homeostasis. In addition, some diseases, such as obesity, cancer, and neurodegenerative diseases, can be cured through apoptosis, which produces few side effects. An effective comprehension of the mechanisms underlying apoptosis will be helpful to prevent and treat some diseases. The identification of genes related to apoptosis is essential to uncover its underlying mechanisms. In this study, a computational method was proposed to identify novel candidate genes related to apoptosis. First, protein-protein interaction information was used to construct a weighted graph. Second, a shortest path algorithm was applied to the graph to search for new candidate genes. Finally, the obtained genes were filtered by a permutation test. As a result, 26 genes were obtained, and we discuss their likelihood of being novel apoptosis-related genes by collecting evidence from published literature. PMID:26543496

  6. Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein.

    PubMed

    Pohlner, J; Meyer, T F; Jalajakumari, M B; Manning, P A

    1986-12-01

    The nucleotide sequence of the ompV gene of Vibrio cholerae was determined. The product of the gene is a 28,000 dalton protein which, after the removal of a 19 amino acid signal sequence, produces a mature outer membrane protein of 26,000 daltons. The cleavage site was determined by amino-terminal amino acid sequencing of the purified mature protein. The DNA upstream of the gene shows the presence of a typical promoter region as judged from the Escherichia coli consensus information; however, the Shine-Dalgarno sequence is associated with a region capable of forming a secondary structure in the mRNA. The formation of this structure would inhibit binding of the mRNA to the ribosome and reduce translation. It is proposed that this structure is recognized by a positive activator in V. cholerae and because of its absence in E. coli ompV is poorly expressed. The distribution of rare codons within ompV suggests that they may serve to slow down the translation of particular domains such that the nascent polypeptide has an opportunity to take up its conformation without interference from the later formed regions. Such a mechanism could aid localization of the protein if export were by a contranslational secretion system. PMID:3031428

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

    PubMed Central

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

    2015-01-01

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

  8. Benzoic Acid-Inducible Gene Expression in Mycobacteria

    PubMed Central

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

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

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

    PubMed

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

    2005-01-01

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

  11. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample. PMID:21762678

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

    PubMed Central

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

    2006-01-01

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

  13. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  14. The WD protein Cpc2p is required for repression of Gcn4 protein activity in yeast in the absence of amino-acid starvation.

    PubMed

    Hoffmann, B; Mösch, H U; Sattlegger, E; Barthelmess, I B; Hinnebusch, A; Braus, G H

    1999-02-01

    The CPC2 gene of the budding yeast Saccharomyces cerevisiae encodes a G beta-like WD protein which is involved in regulating the activity of the general control activator Gcn4p. The CPC2 gene encodes a premRNA which is spliced and constitutively expressed in the presence or absence of amino acids. Loss of CPC2 gene function suppresses a deletion of the GCN2 gene encoding the general control sensor kinase, but not a deletion in the GCN4 gene. The resulting phenotype has resistance against amino-acid analogues. The Neurospora crassa cpc-2 and the rat RACK1 genes are homologues of CPC2 that complement the yeast cpc2 deletion. The cpc2 delta mutation leads to increased transcription of Gcn4p-dependent genes under non-starvation conditions without increasing GCN4 expression or the DNA binding activity of Gcn4p. Cpc2p-mediated transcriptional repression requires the Gcn4p transcriptional activator and a Gcn4p recognition element in the target promoter. Frameshift mutations resulting in a shortened G beta-like protein cause a different phenotype that has sensitivity against amino-acid analogues similar to a gcn2 deletion. Cpc2p seems to be part of an additional control of Gcn4p activity, independent of its translational regulation. PMID:10048025

  15. The BURP domain protein AtUSPL1 of Arabidopsis thaliana is destined to the protein storage vacuoles and overexpression of the cognate gene distorts seed development.

    PubMed

    Van Son, Le; Tiedemann, Jens; Rutten, Twan; Hillmer, Stefan; Hinz, Giselbert; Zank, Thorsten; Manteuffel, Renate; Bäumlein, Helmut

    2009-11-01

    BURP domain proteins comprise a broadly distributed, plant-specific family of functionally poorly understood proteins. VfUSP (Vicia faba Unknown Seed Protein) is the founding member of this family. The BURP proteins are characterized by a highly conserved C-terminal protein domain with a characteristic cysteine-histidine pattern. The Arabidopsis genome contains five BURP-domain encoding genes. Three of them are similar to the non-catalytic beta-subunit of the polygalacturonase of tomato and form a distinct subgroup. The remaining two genes are AtRD22 and AtUSPL1. The deduced product of AtUSPL1 is similar in size and sequence to VfUSP and that of the Brassica napus BNM2 gene which is expressed during microspore-derived embryogenesis. The protein products of BURP genes have not been found, especially that of VfUSP despite a great deal of interest arising from copious transcription of the gene in seeds. Here, we demonstrate that VfUSP and AtUSPL1 occur in cellular compartments essential for seed protein synthesis and storage, like the Golgi cisternae, dense vesicles, prevaculoar vesicles and the protein storage vacuoles in the parenchyma cells of cotyledons. Ectopic expression of AtUSPL1 leads to a shrunken seed phenotype; these seeds show structural alterations in their protein storage vacuoles and lipid vesicles. Furthermore, there is a reduction in the storage protein content and a perturbation in the seed fatty acid composition. However, loss of AtUSP1 gene function due to T-DNA insertions does not lead to a phenotypic change under laboratory conditions even though the seeds have less storage proteins. Thus, USP is pertinent to seed development but its role is likely shared by other proteins that function well enough under the laboratory growth conditions. PMID:19639386

  16. Japanese neuropathy patients with peripheral myelin protein-22 gene aneuploidy

    SciTech Connect

    Lebo, R.V.; Li, L.Y.; Flandermeyer, R.R.

    1994-09-01

    Peripheral myelin protein (PMP-22) gene aneuploidy results in Charcot-Marie-Tooth disease Type 1A (CMT1A) and the Hereditary Neuropathy with Liability to Pressure Palsy (HNPP) in Japanese patients as well as Caucasian Americans. Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, results when expression of one of at least seven genes is defective. CMT1A, about half of all CMT mutations, is usually associated with a duplication spanning the peripheral myelin protein-22 gene on distal chromosome band 17p11.2. Autosomal dominant HNPP (hereditary pressure and sensory neuropathy, HPSN) results from a deletion of the CMT1A gene region. Multicolor in situ hybridization with PMP-22 gene region probe characterized HNPP deletion reliably and detected all different size duplications reported previously. In summary, 72% of 28 Japanese CMT1 (HMSNI) patients tested had the CMT1A duplication, while none of the CMT2 (HMSNII) or CMT3 (HMSNIII) patients had a duplication. Three cases of HNPP were identified by deletion of the CMT1A gene region on chromosome 17p. HNPP and CMT1A have been reported to result simultaneously from the same unequal recombination event. The lower frequency of HNPP compared to CMT1A suggests that HNPP patients have a lower reproductive fitness than CMT1A patients. This result, along with a CMT1A duplication found in an Asian Indian family, demonstrates the broad geographic distribution and high frequency of PMP-22 gene aneuploidy.

  17. A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

    PubMed Central

    Subbarao, E K; London, W; Murphy, B R

    1993-01-01

    The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses. PMID:8445709

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

    PubMed

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

    1998-05-01

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

  19. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition

    PubMed Central

    2014-01-01

    Amino acids are building blocks for proteins in all animals. Based on growth or nitrogen balance, amino acids were traditionally classified as nutritionally essential or nonessential for mammals, birds and fish. It was assumed that all the “nutritionally nonessential amino acids (NEAA)” were synthesized sufficiently in the body to meet the needs for maximal growth and optimal health. However, careful analysis of the scientific literature reveals that over the past century there has not been compelling experimental evidence to support this assumption. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, fertility, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and to protect the integrity of the intestinal mucosa. Thus, diets for animals must contain all NEAA to optimize their survival, growth, development, reproduction, and health. Furthermore, NEAA should be taken into consideration in revising the “ideal protein” concept that is currently used to formulate swine and poultry diets. Adequate provision of all amino acids (including NEAA) in diets enhances the efficiency of animal production. In this regard, amino acids should not be classified as nutritionally essential or nonessential in animal or human nutrition. The new Texas A&M University’s optimal ratios of dietary amino acids for swine and chickens are expected to beneficially reduce dietary protein content and improve the efficiency of their nutrient utilization, growth, and production performance. PMID:24999386

  20. Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering

    PubMed Central

    Liu, Qiulei; Li, Lei; Yang, Liu; Liu, Tianming; Cai, Chenggu; Liu, Bo

    2016-01-01

    Natural sweet protein monellin has a high sweetness and low calorie, suggesting its potential in food applications. However, due to its low heat and acid resistance, the application of monellin is limited. In this study, we show that the thermostability of monellin can be improved with no sweetness decrease by means of sequence, structure analysis, and site-directed mutagenesis. We analyzed residues located in the α-helix as well as an ionizable residue C41. Of the mutants investigated, the effects of E23A and C41A mutants were most remarkable. The former displayed significantly improved thermal stability, while its sweetness was not changed. The mutated protein was stable after 30 min incubation at 85°C. The latter showed increased sweetness and slight improvement of thermostability. Furthermore, we found that most mutants enhancing the thermostability of the protein were distributed at the two ends of α-helix. Molecular biophysics analysis revealed that the state of buried ionizable residues may account for the modulated properties of mutated proteins. Our results prove that the properties of sweet protein monellin can be modified by means of bioinformatics analysis, gene manipulation, and protein modification, highlighting the possibility of designing novel effective sweet proteins based on structure-function relationships. PMID:26881217

  1. Acid extraction and purification of recombinant spider silk proteins.

    PubMed

    Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

    2004-01-01

    A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers. PMID:15360297

  2. FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export

    PubMed Central

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-01-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  3. FAX1, a novel membrane protein mediating plastid fatty acid export.

    PubMed

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-02-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  4. Intramuscular fat content and genetic variants at fatty acid-binding protein loci in Austrian pigs.

    PubMed

    Nechtelberger, D; Pires, V; Söolknet, J; Stur; Brem, G; Mueller, M; Mueller, S

    2001-11-01

    Intramuscular fat is an important meat quality trait in pig production. Previously, genetic variants of the heart fatty acid-binding protein (H-FABP) gene and the adipocyte fatty acid-binding protein (A-FABP) gene were suggested to be associated with intramuscular fat content. The objective of this investigation was to study these associations in the three most important Austrian breeding populations (Piétrain, Large White, and Landrace). Restriction fragment length polymorphism analysis of the H-FABP gene revealed a new MspI polymorphic site and genetic variation in all three breeds. Microsatellite analysis of the A-FABP locus showed up to nine different microsatellite alleles segregating. In Austrian breeds, no significant influence of the A-FABP and H-FABP gene polymorphisms on intramuscular fat could be detected. We also evaluated possible associations between the genetic variations at the H-FABP and A-FABP loci and other growth and carcass traits (average daily gain, feed conversion ratio, lean meat content, pH values, meat color, and drip loss). With regard to the extent of the effects, these genetic markers cannot be recommended for selection on growth and carcass traits in Austrian breeding populations. PMID:11768107

  5. Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover.

    PubMed

    Lynch, Christopher J; Kimball, Scot R; Xu, Yuping; Salzberg, Anna C; Kawasawa, Yuka Imamura

    2015-11-01

    Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes. PMID

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

    PubMed Central

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

    1992-01-01

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

  7. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  8. Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein.

    PubMed

    Long, Dong; Yang, Daiwen

    2009-02-18

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding protein (hLFABP). Rather than affecting the structure of hLFABP, we found that the dynamics of hLFABP, which were previously proposed to be relevant to its functions, were significantly affected by the binding of hLFABP with MES. Buffer interference with protein dynamics was also demonstrated with Bis-Tris buffer, which is quite different from MES and fatty acids in terms of their molecular structures and properties. This result, to our knowledge, is the first published report on buffer interference with protein dynamics on a microsecond to millisecond timescale and could represent a generic problem in the studies of functionally relevant protein dynamics. Although being a fortuity, our finding of buffer-induced changes in protein dynamics offers a clue to how hLFABP accommodates its ligands. PMID:19217864

  9. Computer analysis of protein functional sites projection on exon structure of genes in Metazoa

    PubMed Central

    2015-01-01

    Background Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. Results One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. Conclusions These results characterize the features of the coding exon-intron structure that affect the

  10. Challenges in biotechnology at LLNL: from genes to proteins

    SciTech Connect

    Albala, J S

    1999-03-11

    This effort has undertaken the task of developing a link between the genomics, DNA repair and structural biology efforts within the Biology and Biotechnology Research Program at LLNL. Through the advent of the I.M.A.G.E. (Integrated Molecular Analysis of Genomes and their Expression) Consortium, a world-wide effort to catalog the largest public collection of genes, accepted and maintained within BBRP, it is now possible to systematically express the protein complement of these to further elucidate novel gene function and structure. The work has ensued in four phases, outlined as follows: (1) Gene and System selection; (2) Protein expression and purification; (3) Structural analysis; and (4) biological integration. Proteins to be expressed have been those of high programmatic interest. This includes, in particular, proteins involved in the maintenance of genome integrity, particularly those involved in the repair of DNA damage, including ERCC1, ERCC4, XRCC2, XRCC3, XRCC9, HEX1, APN1, p53, RAD51B, RAD51C, and RAD51. Full-length cDNA cognates of selected genes were isolated, and cloned into baculovirus-based expression vectors. The baculoviral expression system for protein over-expression is now well-established in the Albala laboratory. Procedures have been successfully optimized for full-length cDNA clining into expression vectors for protein expression from recombinant constructs. This includes the reagents, cell lines, techniques necessary for expression of recombinant baculoviral constructs in Spodoptera frugiperda (Sf9) cells. The laboratory has also generated a high-throughput baculoviral expression paradigm for large scale expression and purification of human recombinant proteins amenable to automation.

  11. Advances in protein-amino acid nutrition of poultry.

    PubMed

    Baker, David H

    2009-05-01

    The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn-soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn-soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary L: -cysteine (2.5% or higher) is lethal for young chicks, but a similar level of DL: -methionine, L: -cystine or N-acetyl-L: -cysteine causes no mortality. A supplemental dietary level of 3.0% L: -cysteine (7x requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks. PMID:19009229

  12. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

    PubMed Central

    Dynan, W S; Yoo, S

    1998-01-01

    The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs. PMID:9512523

  13. Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae.

    PubMed

    Misra, Hari S; Khairnar, Nivedita P; Mathur, Manjula; Vijayalakshmi, N; Hire, Ramesh S; Dongre, T K; Mahajan, S K

    2002-04-01

    A sporulating culture of Bacillus thuringiensis subsp. kenyae strain HD549 is toxic to larvae of lepidopteran insect species such as Spodoptera litura, Helicoverpa armigera and Phthorimaea operculella, and a dipteran insect, Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against Helicoverpa armigera, and lower mortality against third-instar larvae of dipteran insects Culex fatigans, Anopheles stephensi and Aedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus thuringiensis var. kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block. PMID:12357073

  14. Relationship of the pelargonium flower break carmovirus (PFBV) coat protein gene with that of other carmoviruses.

    PubMed

    Berthomé, R; Kusiak, C; Renou, J P; Albouy, J; Freire, M A; Dinant, S

    1998-01-01

    The 3'-terminal 1500 nucleotides of the genome of pelargonium flower break carmovirus (PFBV) were sequenced from RT-PCR amplification products. One large ORF was found, encoding a 345 amino acid protein of Mr 37 kDa, which corresponds to the coat protein, as confirmed by immunoprecipitation of products of in vitro transcription and translation. The sequence also included the putative promoter of the coat protein gene subgenomic RNA, as well as its 5' and 3' untranslated regions. The PFBV coat protein was more similar to that of saguaro cactus virus and carnation mottle virus than to that of other carmoviruses. Despite the lower level of similarity of CP gene sequences compared to the RNA dependent RNA polymerase (RdRp) gene sequences of small icosahedral viruses used in taxonomic studies, PFBV CP sequence comparisons and alignments confirmed that PFBV is related to carmoviruses, tombusviruses and a dianthovirus, as previously concluded from the analysis of a PFBV RdRp gene fragment. PMID:9787665

  15. Cloning and sequence of the gene for heat shock protein 60 from Chlamydia trachomatis and immunological reactivity of the protein.

    PubMed Central

    Cerrone, M C; Ma, J J; Stephens, R S

    1991-01-01

    We isolated and sequenced the gene for the chlamydial heat shock protein 60 (HSP-60) from a Chlamydia trachomatis genomic library by molecular genetic methods. The DNA sequence derived revealed an operon-like gene structure with two open reading frames encoding an 11,122- and a 57,956-Da protein. The translated amino acid sequence of the larger open reading frame showed a high degree of homology with known sequences for HSP-60 from several bacterial species as well as with plant and human sequences. By using the determined nucleotide sequence, fragments of the gene were cloned into the plasmid vector pGEX for expression as fusion proteins consisting of glutathione S-transferase and peptide portions of the chlamydial HSP-60. HSP-60 antigenic identity was confirmed by an immunoblot with anti-HSP-60 rabbit serum. Sera from patients that exhibited both high antichlamydial titers and reactivity to chlamydial HSP-60 showed reactivity on immunoblots to two fusion proteins that represented portions of the carboxyl-terminal half of the molecule, whereas fusion proteins defining the amino-terminal half were nonreactive. No reactivity with the fusion proteins was seen with sera from patients that had been previously screened as nonreactive to native chlamydial HSP-60 but which had high antichlamydial titers. Sera from noninfected control subjects also exhibited no reactivity. Definition of recognized HSP-60 epitopes may provide a predictive screen for those patients with C. trachomatis infections who may develop damaging sequelae, as well as providing tools for the study of immunopathogenic mechanisms of Chlamydia-induced disease. Images PMID:1987066

  16. Nucleic Acid Modifications in Regulation of Gene Expression.

    PubMed

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

    2016-01-21

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

  17. Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

    PubMed Central

    2013-01-01

    Background Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important

  18. Mechanism of expression of the rat HCNP precursor protein gene.

    PubMed

    Tohdoh, N; Tojo, S; Kimura, M; Ishii, T; Ojika, K

    1997-04-01

    The hippocampal cholinergic neurostimulating peptide (HCNP), isolated from hippocampal tissue of 10- to 12-day-old rats, enhances the in vitro synthesis of acetylcholine in medial septal tissue explants. The HCNP precursor is a 21 kDa protein that binds hydrophobic ligands and Mg-ATP, and is associated with the opioid-binding protein. We employed an HCNP-precursor cDNA as probe to clone the genomic DNA, used for mapping of the exon-intron structure of the gene. We also determined the nucleotide structure of the promoter region of the rat HCNP precursor protein gene. By using S1 mapping and CAT as a reporter, we found multiple promoters that were aligned in the 5' untranslated region. In addition, the presence of several putative enhancer binding sequences were tested by electrophoresis mobility shift assays. Northern blot analysis revealed that the gene is expressed in a variety of rat tissues and various subregions of the brain. These results suggest that HCNP-precursor gene expression is regulated by a general transactivation factor such as SP1, and that the specific presence of the bioactive HCNP in certain tissues results from post-translational events such as proteolytic processing of the precursor protein, which takes place predominantly in the hippocampus of young rats. PMID:9105667

  19. Expression of heat shock protein genes in insect stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  20. Protein Subcellular Relocalization Increases the Retention of Eukaryotic Duplicate Genes

    PubMed Central

    Byun, S. Ashley; Singh, Sarabdeep

    2013-01-01

    Gene duplication is widely accepted as a key evolutionary process, leading to new genes and novel protein functions. By providing the raw genetic material necessary for functional expansion, the mechanisms that involve the retention and functional diversification of duplicate genes are one of the central topics in evolutionary and comparative genomics. One proposed source of retention and functional diversification is protein subcellular relocalization (PSR). PSR postulates that changes in the subcellular location of eukaryotic duplicate proteins can positively modify function and therefore be beneficial to the organism. As such, PSR would promote retention of those relocalized duplicates and result in significantly lower death rates compared with death rates of nonrelocalized duplicate pairs. We surveyed both relocalized and nonrelocalized duplicate proteins from the available genomes and proteomes of 59 eukaryotic species and compared their relative death rates over a Ks range between 0 and 1. Using the Cox proportional hazard model, we observed that the death rates of relocalized duplicate pairs were significantly lower than the death rates of the duplicates without relocalization in most eukaryotic species examined in this study. These observations suggest that PSR significantly increases retention of duplicate genes and that it plays an important, but currently underappreciated, role in the evolution of eukaryotic genomes. PMID:24265504

  1. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  2. Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

    PubMed Central

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  3. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis

    PubMed Central

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-01-01

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the “recycling” of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  4. Transient expression of proteins by hydrodynamic gene delivery in mice.

    PubMed

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  5. Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin.

    PubMed

    George, Caroline L S; White, Misty L; O'Neill, Marsha E; Thorne, Peter S; Schwartz, David A; Snyder, Jeanne M

    2003-12-01

    Chronically inhaled endotoxin, which is ubiquitous in many occupational and domestic environments, can adversely affect the respiratory system resulting in an inflammatory response and decreased lung function. Surfactant-associated protein A (SP-A) is part of the lung innate immune system and may attenuate the inflammatory response in various types of lung injury. Using a murine model to mimic occupational exposures to endotoxin, we hypothesized that SP-A gene expression and protein would be elevated in response to repeat exposure to inhaled grain dust and to purified lipopolysaccharide (LPS). Our results demonstrate that repeat exposure to inhaled endotoxin, either in the form of grain dust or purified LPS, results in increased whole lung SP-A gene expression and type II alveolar epithelial cell hyperplasia, whereas SP-A protein levels in lung lavage fluid are decreased. Furthermore, these alterations in SP-A gene activity and protein metabolism are dependent on an intact endotoxin signaling system. PMID:12922979

  6. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    PubMed

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  7. Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

    NASA Astrophysics Data System (ADS)

    Zheng, Minggang; Liang, Kepeng; Wang, Bo; Sun, Xiuqin; Yue, Yanyan; Wan, Wenwen; Zheng, Li

    2013-03-01

    In most bacteria, plants and algae, fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase (FAS II) system. In the FAS II system, enoylacyl carrier protein reductase (ENR) acts as a determinant for completing the cycles of fatty acid elongation. In this study, the cDNA sequence of ENR, designated as IgENR, was isolated from the microalga Isochrysis galbana CCMM5001. RACE (rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR (1 503 bp), which contains an open reading frame (ORF) of 1 044 bp and encodes a protein of 347 amino acids. The genomic DNA sequence of IgENR is interrupted by four introns. The putative amino acid sequence is homologous to the ENRs of seed plants and algae, and they contain common coenzymebinding sites and active site motifs. Under different stress conditions, real-time quantitative polymerase chain reaction (RT-qPCR) showed the expression of IgENR was upregulated by high temperature (35°C), and downregulated by depleted nitrogen (0 mol/L). To clarify the mechanism of lipids accumulating lipids, other genes involved in lipids accumulation should be studied.

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

    PubMed Central

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

    1995-01-01

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

  9. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein

    SciTech Connect

    Hickey, E.; Brandon, S.E.; Weber, L.A.; Lloyd, D.

    1989-06-01

    Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89/alpha/ and hspio/beta/) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89/alpha/, is induced by the adenovirus E1A gene product. The authors have isolated a human hsp89/alpha/ gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression n a /beta/-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89/alpha/ protein sequence differed from the human hsp89/beta/ sequence reported elsewhere in at least 99 out of the 732 amino acids. Transcription of the hsp89/alpha/ gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycles. hsp89/alpha/ mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

  10. Candidate genes that affect aging through protein homeostasis.

    PubMed

    Argon, Yair; Gidalevitz, Tali

    2015-01-01

    Because aging is a multifactorial, pleiotropic process where many interacting mechanisms contribute to the organismal decline, the candidate gene approach rarely provides a clear message. This chapter discusses some of the inherent complexity, focusing on aspects that impinge upon protein homeostasis and maintain a healthy proteome. We discuss candidate genes that operate in these pathways, and compare their actions in invertebrates, mice and humans. We highlight several themes that emerge from recent research—the interconnections of pathways that regulate aging, the pleiotropic effects of mutations and other manipulations of the candidate proteins and the tissue specificity in these pleiotropic outcomes. This body of knowledge highlights the need for multiple specific readouts of manipulating longevity genes, beyond measuring lifespan, as well as the need to understand the integrated picture, beyond examining the immediate outputs of individual longevity pathways. PMID:25916585

  11. Improved mutation tagging with gene identifiers applied to membrane protein stability prediction

    PubMed Central

    Winnenburg, Rainer; Plake, Conrad; Schroeder, Michael

    2009-01-01

    Background The automated retrieval and integration of information about protein point mutations in combination with structure, domain and interaction data from literature and databases promises to be a valuable approach to study structure-function relationships in biomedical data sets. Results We developed a rule- and regular expression-based protein point mutation retrieval pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval task on a benchmark dataset. In order to link mutations to their proteins, we utilize a named entity recognition algorithm for the identification of gene names co-occurring in the abstract, and establish links based on sequence checks. Vice versa, we could show that gene recognition improved from 77% to 91% F-measure when considering mutation information given in the text. To demonstrate practical relevance, we utilize mutation information from text to evaluate a novel solvation energy based model for the prediction of stabilizing regions in membrane proteins. For five G protein-coupled receptors we identified 35 relevant single mutations and associated phenotypes, of which none had been annotated in the UniProt or PDB database. In 71% reported phenotypes were in compliance with the model predictions, supporting a relation between mutations and stability issues in membrane proteins. Conclusion We present a reliable approach for the retrieval of protein mutations from PubMed abstracts for any set of genes or proteins of interest. We further demonstrate how amino acid substitution information from text can be utilized for protein structure stability studies on the basis of a novel energy model. PMID:19758467

  12. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene.

    PubMed Central

    Duchêne, M; Schweizer, A; Lottspeich, F; Krauss, G; Marget, M; Vogel, K; von Specht, B U; Domdey, H

    1988-01-01

    Porin F is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. It forms water-filled pores of variable size. Porin F is a candidate for a vaccine against P. aeruginosa because it antigenically cross-reacts in all serotype strains of the International Antigenic Typing Scheme. We have isolated the gene for porin F from a lambda EMBL3 bacteriophage library by using oligodeoxynucleotide hybridization probes and have determined its nucleotide sequence. Different peptide sequences obtained from isolated porin F confirmed the deduced protein sequence. The mature protein consists of 326 amino acid residues and has a molecular weight of 35,250. The precursor contains an N-terminal signal peptide of 24 amino acid residues. S1 protection and primer extension experiments, together with Northern (RNA) blots, indicate that the mRNA coding for porin F is monocistronic with short untranslated regions of about 58 bases at the 5' end and about 47 bases at the 3' end. The sequences in the -10 and -35 regions upstream of the transcriptional start site are closely related to the Escherichia coli promoter consensus sequences, which explains why the porin F gene is expressed in E. coli under the control of its own promoter. The amino acid sequence of porin F is not homologous to the different E. coli porins OmpF, OmpC, LamB, and PhoE. On the other hand, a highly homologous region of 30 amino acids between the OmpA proteins of different enteric bacteria and porin F of P. aeruginosa was detected. The core region of the homology to E. coli OmpA had 11 of 12 amino acid residues in common. Images PMID:2447060

  13. Enhanced Acid Tolerance in Bifidobacterium longum by Adaptive Evolution: Comparison of the Genes between the Acid-Resistant Variant and Wild-Type Strain.

    PubMed

    Jiang, Yunyun; Ren, Fazheng; Liu, Songling; Zhao, Liang; Guo, Huiyuan; Hou, Caiyun

    2016-03-28

    Acid stress can affect the viability of probiotics, especially Bifidobacterium. This study aimed to improve the acid tolerance of Bifidobacterium longum BBMN68 using adaptive evolution. The stress response, and genomic differences of the parental strain and the variant strain were compared by acid stress. The highest acid-resistant mutant strain (BBMN68m) was isolated from more than 100 asexual lines, which were adaptive to the acid stress for 10(th), 20(th), 30(th), 40(th), and 50(th) repeats, respectively. The variant strain showed a significant increase in acid tolerance under conditions of pH 2.5 for 2 h (from 7.92 to 4.44 log CFU/ml) compared with the wildtype strain (WT, from 7.87 to 0 log CFU/ml). The surface of the variant strain was also smoother. Comparative whole-genome analysis showed that the galactosyl transferase D gene (cpsD, bbmn68_1012), a key gene involved in exopolysaccharide (EPS) synthesis, was altered by two nucleotides in the mutant, causing alteration in amino acids, pI (from 8.94 to 9.19), and predicted protein structure. Meanwhile, cpsD expression and EPS production were also reduced in the variant strain (p < 0.05) compared with WT, and the exogenous WT-EPS in the variant strain reduced its acid-resistant ability. These results suggested EPS was related to acid responses of BBMN68. PMID:26608165

  14. Abscisic acid regulation of DC8, a carrot embryonic gene. [Daucus carota

    SciTech Connect

    Hatzopoulos, P.; Fong, F.; Sung, Z.R. Texas A M Univ., College Station )

    1990-10-01

    DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage embryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 {times} 10{sup {minus}7} molar ABA while 10{sup {minus}6} molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.

  15. Arabidopsis contains ancient classes of differentially expressed actin-related protein genes.

    PubMed

    McKinney, Elizabeth Cohen; Kandasamy, Muthugapatti K; Meagher, Richard B

    2002-03-01

    Actin-related proteins (ARPs) share less than 60% amino acid sequence homology with conventional actins and have roles in diverse cytoskeletal processes in the cytoplasm and nucleus. The genome of Arabidopsis was explored for possible ARP gene family members. Eight potential ARP gene sequences were found dispersed on three of the five Arabidopsis chromosomes. AtARP2 and AtARP3 are protein orthologs of their similarly named counterparts in other kingdoms. AtARP4, AtARP5, and AtARP6 are orthologs of two classes of nuclear ARPs previously characterized in animals and fungi, BAF53s and ARP6s. AtARP7 and AtARP8 appear to be novel proteins that are not closely related to any known animal or fungal ARPs, and may be plant specific. The complex Arabidopsis ARP gene structures each contain from five to 20 exons. Expressed transcripts were identified and characterized for AtARP2 through AtARP8, but not for AtARP9, and transcripts representing two splice variants were found for AtARP8. The seven expressed genes are predicted to encode proteins ranging from 146 to 471 amino acids in length. Relative to conventional actin and the other ARPs, AtARP2 and AtARP3 transcripts are expressed at very low levels in all organs. AtARP5, AtARP6, and AtARP8 each have distinct transcript expression patterns in seedlings, roots, leaves, flowers, and siliques. Using isovariant-specific monoclonal antibodies, AtARP4 and AtARP7 proteins were shown to be most highly expressed in flowers. The likely involvement of plant ARPs in actin nucleation, branching of actin filaments, chromatin restructuring, and transcription are briefly discussed. PMID:11891255

  16. Early auxin-induced genes encode short-lived nuclear proteins.

    PubMed Central

    Abel, S; Oeller, P W; Theologis, A

    1994-01-01

    The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses. Images Fig. 2 Fig. 3 Fig. 4 PMID:8278386

  17. [Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

    PubMed

    Luo, Ming; Xiao, Yue-Hua; Hou, Lei; Luo, Xiao-Ying; Li, De-Mou; Pei, Yan

    2003-02-01

    LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development. PMID:12776607

  18. Modeling nucleic acid structure in the presence of single-stranded binding proteins

    NASA Astrophysics Data System (ADS)

    Forties, Robert; Bundschuh, Ralf

    2009-03-01

    There are many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV, the RecA DNA repair protein in bacteria, and all proteins involved in mRNA splicing and translation. We extend the Vienna Package for quantitatively modeling the secondary structure of nucleic acids to include proteins which bind to unpaired portions of the nucleic acid. All parameters needed to model nucleic acid secondary structures in the absence of proteins have been previously measured. This leaves the footprint and sequence dependent binding affinity of the protein as adjustable parameters of our model. Using this model we are able to predict the probability of the protein binding at any pos