Sample records for binding protein gene

  1. A homeodomain protein binds to. gamma. -globin gene regulatory sequences

    Lavelle, D.; Ducksworth, J.; Eves, E.; Gomes, G.; Keller, M.; Heller, P.; DeSimone, J. (Univ. of Illinois, Chicago (United States) Veterans Administration Westside Medical Center, Chicago, IL (United States))


    Developmental regulation of {gamma}-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the {gamma} gene promoters and 3{prime}{sup A}{gamma} enhancer located in close proximity to the genes. The authors have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, {gamma} gene promoter, and 3{prime} {sup A}{gamma} enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a {beta}-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.

  2. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    Mandrup, S; Hummel, R; Ravn, S;


    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous...... remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  3. RNA Binding Proteins that Control Human Papillomavirus Gene Expression.

    Naoko Kajitani; Stefan Schwartz


    The human papillomavirus (HPV) life cycle is strictly linked to the differentiation program of the infected mucosal epithelial cell. In the basal and lower levels of the epithelium, early genes coding for pro-mitotic proteins and viral replication factors are expressed, while terminal cell differentiation is required for activation of late gene expression and production of viral particles at the very top of the epithelium. Such productive infections are normally cleared within 18–24 months. I...

  4. Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

    Jönsson, Klas; Guo, Betty P.; Monstein, Hans-Jürg; Mekalanos, John J.; Kronvall, Göran


    Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described; however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A s...

  5. APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression

    Huh, Sung Un; Paek, Kyung-Hee


    Background A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening. Results APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection su...

  6. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders; Bolund, Lars; Nielsen, Anders Lade


    Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins......IP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins....

  7. Identification of a binding protein to the X gene promoter region of hepatitis B virus.

    Nakamura, I; Koike, K


    The X protein of hepatitis B virus (HBV) is a transactivator to homologous and heterologous viral and cellular transcriptional regulatory elements. One sequence-specific binding protein, whose binding site located from nt 1102 to nt 1117 of HBV DNA, was identified by mobility shift assay and DNase I foot-printing analysis. A CAT assay experiment demonstrated this 16-bp binding site to have a promoter activity in the X gene transcription. The 58-bp DNA fragment (nt 1085 to nt 1142), which contains the above binding site, could be enhanced by the HBV enhancer. Mobility shift assay using the mutated 58-bp DNA fragments as probes, showed that the mutation, which damaged the palindrome structure between nt 1105 and nt 1112, resulted in loss of the binding activity. This mutation also remarkably reduced the promoter activity. The binding site differed from the target sequences of known transcriptional factors. This factor was thus concluded to be a binding protein to the X gene promoter (X-PBP) of HBV. A homology search demonstrated the binding site to be highly homologous to the promoter elements of human laminin receptor (2H5epitope) and lipoprotein receptor-related protein (LRP) genes. PMID:1448911

  8. Role of the RNA-binding Protein Tristetraprolin in Glucocorticoid-mediated Gene Regulation1

    Ishmael, Faoud T.; Fang, Xi; Galdiero, Maria Rosaria; Atasoy, Ulus; Rigby, William F C; Gorospe, Myriam; Cheadle, Chris; Stellato, Cristiana


    Glucocorticoids (GCs) are the mainstay of anti-inflammatory therapy. Modulation of post-transcriptional regulation (PTR) of gene expression by GCs is a relevant yet poorly characterized mechanism of their action. The RNA-binding protein tristetraprolin (TTP) plays a central role in PTR by binding to AU-rich elements in the 3’untranslated region of proinflammatory transcripts and accelerating their decay. We found that GCs induce TTP expression in primary and immortalized human bronchial epith...

  9. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    Nordlund Henri R


    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  10. Cloning and Sequence Analysis of Y-box Binding Protein Gene in Min Pig

    Zhang Dong-jie; Liu Di; Wang Liang; He Xin-miao; Wang Wen-tao


    In order to study the gene sequence of Min pig Y-box binding protein (YB-1) gene, the complete coding sequence of Min pig YB-1 gene was cloned by RT-PCR, the sequence features were analyzed by some software and online website. The results showed that the complete CDS of Min pig Y-box was found to be 975 bp long, encoding 324 amino acids. It contained a conserved cold shock domain and several phosphorylation sites, but had no transmembrane domains, and was consistent with a protein found in the cytoplasm. Min pig YB-1 nucleotides shared high similarity (61.37%-97.66%) with other mammals.

  11. Characterization of two genes encoding distinct transferrin-binding proteins in different Actinobacillus pleuropneumoniae isolates.

    Gerlach, G F; Klashinsky, S; Anderson, C.; Potter, A A; Willson, P.J.


    The gene encoding the Actinobacillus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70% of the protein was expressed as an aggregate protein in Escherichia coli. The nucleotide sequences of the tfbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The de...

  12. Isolation of osRACD gene encoding a small GTP-binding protein from rice


    Using an improved version of mRNA differential display technology, we have obtained a differentially displayed fragment RDP-8. Homologous comparison indicated that the fragment RDP-8 has high homology with the gene encoding maize small GTP-binding protein. By screening cDNA library of the rice Nongken 58N pan icle using the newly obtained fragment RDP-8 as probe, we further found the full-length cDNA of osRACD gene that encodes a rice small GTP-binding protein. Asco mpared with maize RACD gene, the osRACD of rice shows remarkable homology in both nucleotide sequence and amino acid sequence, 88% and 97% respectively. Evidence from RT-PCR study indicates that osRACD gene is related to photoperiod fertility conversion of photoperiod sensitive genic male sterility (PSGMS) rice.

  13. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. (Weizmann Institute, Rehovoth (Israel))


    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  14. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R


    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  15. The androgen-binding protein gene is expressed in male and female rat brain.

    Wang, Y M; Bayliss, D A; Millhorn, D E; Petrusz, P; Joseph, D R


    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

  16. Nuclear proteins from lactating mammary glands bind to the promoter of a milk protein gene.

    LUBON, H.; Hennighausen, L


    The gene for the whey acidic protein (WAP) is expressed specifically in the lactating mammary glands of rodents. We present evidence that nuclear proteins from mammary epithelial cells form a multiple nucleoprotein complex with the WAP gene promoter/upstream region. As monitored by mobility shifts, nuclear proteins from lactating mammary glands and from the mammary cell line MCF-7 form four high affinity complexes with a fragment spanning the region between nucleotides -175 and -88. Nuclear p...

  17. Common variants of the vitamin D binding protein gene and adverse health outcomes

    Malik, Suneil; Fu, Lei; Juras, David James; Karmali, Mohamed; Wong, Betty Y. L.; Gozdzik, Agnes; Cole, David E.C.


    The vitamin D binding protein (DBP) is the major plasma carrier for vitamin D and its metabolites, but it is also an actin scavenger, and is the precursor to the immunomodulatory protein, Gc-MAF. Two missense variants of the DBP gene – rs7041 encoding Asp432Glu and rs4588 encoding Thr436Lys – change the amino acid sequence and alter the protein function. They are common enough to generate population-wide constitutive differences in vitamin D status, based on assay of the serum metabolite, 25-...

  18. Phylogenetic Analysis of the Insulin-like Growth Factor Binding Protein (IGFBP) and IGFBP-related Protein Gene Families

    Rodgers, Buel D.; Roalson, Eric H.; Thompson, Cullen


    Insulin-like growth factor (IGF) activity is regulated by six high affinity binding proteins (IGFBPs) and possibly by some of the nine IGFBP-related proteins (IGFBP-rPs). To determine the phylogenetic relationship of this proposed gene superfamily, we conducted maximum likelihood (ML) and Bayesian inference analyses on a matrix of amino acid sequences from a diversity of vertebrate species. A single most likely phylogram, ML bootstrap, and Bayesian consensus tree of 10,000,000 generations rev...

  19. The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains

    Brueggeman, R.; Druka, A.; Nirmala, J.; Cavileer, T.; Drader, T.; Rostoks, N.; Mirlohi, A.; Bennypaul, H.; Gill, U; Kudrna, D.; Whitelaw, C.; Kilian, A.; Han, F.; Sun, Y; Gill, K.


    We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed ...

  20. Identification of steroid-sensitive gene-1/Ccdc80 as a JAK2-binding protein.

    O'Leary, Erin E; Mazurkiewicz-Muñoz, Anna M; Argetsinger, Lawrence S; Maures, Travis J; Huynh, Hung T; Carter-Su, Christin


    The tyrosine kinase Janus kinase 2 (JAK2) is activated by many cytokine receptors, including receptors for GH, leptin, and erythropoietin. However, very few proteins have been identified as binding partners for JAK2. Using a yeast 2-hybrid screen, we identified steroid-sensitive gene-1 (SSG1)/coiled-coil domain-containing protein 80 (Ccdc80) as a JAK2-binding partner. We demonstrate that Ccdc80 preferentially binds activated, tyrosyl-phosphorylated JAK2 but not kinase-inactive JAK2 (K882E) in both yeast and mammalian systems. Ccdc80 is tyrosyl phosphorylated in the presence of JAK2. The binding of Ccdc80 to JAK2 occurs via 1 or more of the 3 DUDES/SRPX (DRO1-URB-DRS-Equarin-SRPUL/sushi repeat containing protein, x-linked) domain 5 domains of Ccdc80. Mutagenesis of the second DUDES domain suggests that the N-terminal third of the DUDES domain is sufficient for JAK2 binding. Ccdc80 does not alter the kinase activity of JAK2. However, Ccdc80 increases GH-dependent phosphorylation of Stat (signal transducer and activator of transcription) 5b on Tyr699 and substantially enhances both basal and GH-dependent phosphorylation/activation of Stat3 on Tyr705. Furthermore, Ccdc80 belongs to the group of proteins that function both in the intracellular compartment and are secreted. Secreted Ccdc80 associates with the extracellular matrix and is also found in the medium. A substantial portion of the Ccdc80 detected in the medium is cleaved. Finally, consistent with the DUDES domain serving as a JAK2-binding domain, we also demonstrate that another protein that contains a DUDES domain, SRPX2, binds preferentially to the activated tyrosyl-phosphorylated form of JAK2. PMID:23449887

  1. Uncovering the functional constraints underlying the genomic organisation of the Odorant-Binding Protein genes

    Librado, Pablo; Rozas, Julio


    Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. Here, we conducted a comprehensive study aiming to g...

  2. Molecular pathology of familial hypertrophic cardiomyopathy caused by mutations in the cardiac myosin binding protein C gene.

    Yu, B.; French, J. A.; Carrier, L.; Jeremy, R W; McTaggart, D R; Nicholson, M R; Hambly, B; Semsarian, C; Richmond, D R; Schwartz, K.; Trent, R.J.


    DNA studies in familial hypertrophic cardiomyopathy (FHC) have shown that it is caused by mutations in genes coding for proteins which make up the muscle sarcomere. The majority of mutations in the FHC genes result from missense changes, although one of the most recent genes to be identified (cardiac myosin binding protein C gene, MYBPC3) has predominantly DNA mutations which produce truncated proteins. Both dominant negative and haploinsufficiency models have been proposed to explain the mol...

  3. Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt.

    Banas, J A; Russell, R R; Ferretti, J J


    The nucleotide sequence of the gbp gene, which encodes the glucan-binding protein (GBP) of Streptococcus mutans, was determined. The reading frame for gbp was 1,689 bases. A ribosome-binding site and putative promoter preceded the start codon, and potential stem-loop structures were identified downstream from the termination codon. The deduced amino acid sequence of the GBP revealed the presence of a signal peptide of 35 amino acids. The molecular weight of the processed protein was calculated to be 59,039. Two series of repeats spanned three-quarters of the carboxy-terminal end of the protein. The repeats were 32 to 34 and 17 to 20 amino acids in length and shared partial identity within each series. The repeats were found to be homologous to sequences hypothesized to be involved in glucan binding in the GTF-I of S. downei and to sequences within the protein products encoded by gtfB and gtfC of S. mutans. The repeated sequences may represent peptide segments that are important to glucan binding and may be distributed among GBPs from other bacterial inhabitants of plaque or the oral cavity. PMID:2307516

  4. Fine tuning gene expression through short DNA-protein binding cycles.

    Michel, Denis


    Certain transcription factors have recently been shown to interact with DNA in living cells, through very short binding cycles, contrasting with the data previously obtained in vitro, and with the view of a stepwise building of transcription initiation complexes. These short cycles are triggered by active dissociation mechanisms, suggesting that they ensure important biological functions. Various interpretations of these observations have been proposed, including a mechanism allowing the cell to switch off gene expression after removal of the inducer, or increasing the availability of free transcription factors. The interpretation examined here is that the brevity of the transcription factor turnovers favors the determinism of gene expression. For the genes with open chromatin and subject to this mode of interaction, the differential dynamics between promoter occupancy and the following processes mediating protein accumulation, can be essential for the dosage of gene expression. Biological activities and quantitative conditions allowing to increase the frequency of DNA-protein binding cycles are proposed. The unexpected dynamics of certain DNA-protein interactions can provide a concrete example of the notion of apparent gradation of single-site occupancy, which is a general solution allowing to extend the mass action determinism to low copy number molecules. PMID:19376190

  5. Molecular mechanisms of gene activation and gene expression mediated by CCAAT/enhancer binding proteins

    Zaragoza Dörr, Katrin


    Der Transkriptionsfaktor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) koordiniert Proliferationshemmung und Differenzierung von myeloiden VorlŠuferzellen und Adipozyten. C/EBPa ist ein transkriptioneller Aktivator von abstammungspezifischen Genen und blockiert den Zellzyklus durch Repression von proliferationsfšrdernden E2F Zielgenen. Die hier gezeigten Daten zeigen, dass auch umgekehrt E2F die transkriptionelle und differenzierungsfšrdernde AktivitŠt von C/EBPa entgegenwirkt. Somit besitzen...

  6. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K


    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

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

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


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

  8. t(8;21)(q22;q22) fusion proteins preferentially bind to duplicated AML1/RUNX1 DNA-binding sequences to differentially regulate gene expression

    Okumura, Akiko J.; Peterson, Luke F.; Okumura, Fumihiko; Boyapati, Anita; Zhang, Dong-Er


    Chromosome abnormalities are frequently associated with cancer development. The 8;21(q22;q22) chromosomal translocation is one of the most common chromosome abnormalities identified in leukemia. It generates fusion proteins between AML1 and ETO. Since AML1 is a well-defined DNA-binding protein, AML1-ETO fusion proteins have been recognized as DNA-binding proteins interacting with the same consensus DNA-binding site as AML1. The alteration of AML1 target gene expression due to the presence of ...

  9. Molecular Identification and Sequencing of Mannose Binding Protein (MBP Gene of Acanthamoeba palestinensis

    M Rezaeian


    Full Text Available "nBackground: Acanthamoeba keratitis develops by pathogenic Acanthamoeba such as A. pal­es­tinen­sis. Indeed this species is one of the known causative agents of amoebic keratitis in Iran. Mannose Binding Protein (MBP is the main pathogenicity factors for developing this sight threatening disease. We aimed to characterize MBP gene in pathogenic Acanthamoeba isolates such as A. palestinensis."nMethods: This experimental research was performed in the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2007-2008.  A. palestinensis was grown on 2% non-nutrient agar overlaid with Escherichia coli. DNA extraction was performed using phenol-chloroform method. PCR reaction and amplification were done using specific primer pairs of MBP. The amplified fragment were purified and sequenced. Finally, the obtained fragment was deposited in the gene data bank."nResults: A 900 bp PCR-product was recovered after PCR reaction. Sequence analysis of the purified PCR product revealed a gene with 943 nucleotides. Homology analysis of the ob­tained sequence showed 81% similarity with the available MBP gene in the gene data bank. The fragment was deposited in the gene data bank under accession number EU678895"nConclusion: MBP is known as the most important factor in Acanthamoeba pathogenesis cas­cade. Therefore, characterization of this gene can aid in developing better therapeutic agents and even immunization of high-risk people.

  10. Rat prostatic steroid binding protein: characterisation of the Alu element upstream of the C3 genes.

    Hurst, H C; Parker, M G


    We have characterised an Alu-like repetitive element found about 400 bp upstream of the gene encoding the C3 component of rat prostatic steroid binding protein and suggest, from comparisons with other published sequences, that it is an example of a third class of rodent Alu-equivalent sequences. Members of this class are 80-90 bp long, share greater than 90% sequence homology, and contain sequences resembling the RNA polymerase III bipartite promoter. The Alu type III element within the C3 ge...

  11. Liver Fatty Acid Binding Protein Gene Ablation Enhances Age-Dependent Weight Gain in Male Mice

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


    Although studies performed in vitro and with transfected cells in culture suggest a role for liver fatty acid binding protein (L-FABP) in regulating fatty acid oxidation and fat deposition, the physiological significance of this possibility is not completely clear. To begin to address this question, the effect of L-FABP gene ablation on phenotype of standard rodent chow-fed male mice was examined with increasing age up to 18 mo. While young (2-3 mo) L-FABP null mice displayed no visually obvi...

  12. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A


    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  13. RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells

    Penalva Luiz OF


    Full Text Available Abstract Background Tumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one another's gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes. Results In order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A binding protein (PABP and cap-binding protein (eIF-4E as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells. Conclusion RNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors.

  14. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.

    Norris, M L; Millhorn, D E


    We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift-Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID:7559551

  15. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.


    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  16. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors.

    Treisman, R


    Transient transcriptional activation of the c-fos gene following serum stimulation of susceptible cells requires a conserved DNA element located 300 bp 5' to the mRNA cap site. A DNA-binding gel electrophoresis assay was used to detect a protein(s) in HeLa cell nuclear extracts that specifically binds to the 5' activating element. The protein recognizes a region of dyad symmetry within the 5' activating element, defined by binding competition, dimethylsulphate (DMS) interference and DNAase I and DMS protection studies. A single 22 bp synthetic copy of the dyad symmetry element will both compete efficiently for protein binding and restore serum regulation to c-fosH genes that lack the 5' activating element. PMID:3524858

  17. hgpB, a Gene Encoding a Second Haemophilus influenzae Hemoglobin- and Hemoglobin-Haptoglobin-Binding Protein

    Ren, Zhen; Jin, Hongfan; Morton, Daniel J.; Stull, Terrence L.


    Haemophilus influenzae requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in H. influenzae HI689. Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of...

  18. Genomic binding and regulation of gene expression by the thyroid carcinoma-associated PAX8-PPARG fusion protein

    Zhang, Yanxiao; Yu, Jingcheng; Lee, Chee; Xu, Bin; Sartor, Maureen A.; Koenig, Ronald J.


    A chromosomal translocation results in production of an oncogenic PAX8-PPARG fusion protein (PPFP) in thyroid carcinomas. PAX8 is a thyroid transcription factor, and PPARG is a transcription factor that plays important roles in adipocytes and macrophages. PPFP retains the DNA binding domains of both proteins; however, the genomic binding sites of PPFP have not been identified, and only limited data exist to characterize gene expression in PPFP thyroid carcinomas. Therefore, the oncogenic func...

  19. Activation of Prophage eib Genes for Immunoglobulin-Binding Proteins by Genes from the IbrAB Genetic Island of Escherichia coli ECOR-9

    Sandt, Carol H.; Hopper, James E.; Hill, Charles W.


    Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9...

  20. The human renin-binding protein gene (RENBP) maps in Xq28

    Ouweland, A.M.W. van der; Verdijk, M.; Oost, B.A. van (Univ. Hospital Nijmegen (Netherlands)); Kiochis, P.; Poustka, A. (Deutsches Krebsforschungszemtrum, Heidelberg (Germany))


    The authors report here the successful application of the method by which cDNA libraries are screened with positionally identified genomic clones. Human cosmid clones were selected from a cosmid library derived from the Q1Z cell line. This Q1Z cell line is a hamster-human somatic cell hybrid that contains the Xq28 region as its sole human component. To search for kidney-expressed genes, they screened a kidney cDNA library purchased from Clontech with cosmid-derived probes. Based on the physical mapping of the vasopressin V2 receptor gene close to the L1CAM gene, they analyzed cosmids derived from this region. One of the cosmids was 12B2, located 50 kb from the L1CAM gene. A 20-kb EcoRI subclone from the 12B2 cosmid was used as probe. This fragment did not hybridize to the probe 2-55 in contrast to the whole cosmid 12B2. Screening of 200,000 cDNA clones resulted in the identification of two positive clones. After sequence determination, it appeared that one of the positive cDNA clones contained Escherichia coli DNA as insert (data not shown). The other cDNA (pMV24) contained an open reading frame corresponding to the 243 amino-terminal amino acids of the human renin binding protein. The RENBP gene maps to interval 3 between the loci for DX52 and G-6-PD. This is the same interval as that for the color blindness gene, DXS707, and the AVPR2, L1CAM, and QM genes. This result confirms that the isolated RENBP cDNA originates from the same location as that from which the parental cosmid clone was derived. 28 refs., 1 fig.

  1. Distribution of transferrin binding protein B gene (tbpB variants among Neisseria species

    Maiden Martin CJ


    Full Text Available Abstract Background Transferrin binding protein B (tbpB, an outer membrane lipoprotein, is required for the acquisition of iron from human transferrin. Two tbpB families have been documented in Neisseria meningitidis: an isotype I tbpB gene of 1.8 kb and an isotype II tbpB gene of 2.1 kb, the former expressed by meningococci in the disease-associated ST-11 clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including ST-8, ST-18, ST-32, ST-41/44 as well as N. gonorrhoeae isolates. The origin of the isotype I tbpB gene is unknown, however several features in common with non-pathogenic Neisseria and the ST-11 clonal complex N. meningitidis isolate FAM18 have been documented leading to the hypothesis that the isotype I tbpB gene may also be shared between non-pathogenic Neisseria and ST-11 meningococci. As a result, the diversity of the tbpB gene was investigated in a defined collection of Neisseria species. Results Two families of isotype I tbpB were identified: family A containing conserved genes belonging to ST-11 meningococci, N. polysaccharea and N. lactamica isolates and family B including more diverse isotype I tbpB genes from N. sicca, N. mucosa, N. flava, N. subflava as well as N. cinerea, N. flavescens and N. polysaccharea isolates. Three isotype II tbpB families were identified with: family C containing diverse tbpB genes belonging to N. polysaccharea, N. lactamica, N. gonorrhoeae and N. meningitidis isolates, family D including another subset of isotype II tbpB genes from N. lactamica isolates and family E solely composed of N. gonorrhoeae tbpB genes. Conclusion This study reveals another instance of similarity between meningococci of the ST-11 clonal complex and non-pathogenic Neisseria with the origin of the isotype I tbpB gene resulting from a horizontal genetic transfer event occurring between these two populations.

  2. Molecular analysis of the murine C4b-binding protein gene. Chromosome assignment and partial gene organization

    Barum, Scott B; Kristensen, Torsten; Chaplin, David D; Seldin, Michal F; Tack, Brian F


    Murine C4b-binding protein (C4BP) is a regulatory molecule in the classical pathway of complement. C4BP is composed predominantly of short consensus repeats (SCRs) approximately 60 amino acids in length, which contain a framework of conserved residues. The SCRs are found in many complement...... molecules and a growing number of noncomplement molecules as well and are a major structural feature of some of these molecules. To characterize the structure of the murine C4BP gene, a cosmid library constructed from Balb/c liver DNA was screened. Several nearly identical, overlapping clones were...... identified; however, none of the clones, alone or in combination, covered the entire C4BP gene. One clone (D26) was chosen for detailed analysis and found to contain all but the leader region, the first SCR, and the first half of the second SCR. The SCRs three through six were each encoded by single exons...

  3. Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of Sitobion avenae.

    Xue, Wenxin; Fan, Jia; Zhang, Yong; Xu, Qingxuan; Han, Zongli; Sun, Jingrui; Chen, Julian


    Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) of aphids are thought to be responsible for the initial molecular interactions during olfaction that mediate detection of chemical signals. Analysis of the diversity of proteins involved comprises critical basic research work that will facilitate the development of sustainable pest control strategies. To help us better understand differences in the olfactory system between winged and wingless grain aphids, we constructed an antennal transcriptome from winged and wingless Sitobion avenae (Fabricius), one of the most serious pests of cereal fields worldwide. Among the 133,331 unigenes in the antennal assembly, 13 OBP and 5 CSP putative transcripts were identified with 6 OBP and 3 CSP sequences representing new S. avenae annotations. We used qPCR to examine the expression profile of these genes sets across S. avenae development and in various tissues. We found 7 SaveOBPs and 1 SaveCSP were specifically or significantly elevated in antennae compared with other tissues, and that some transcripts (SaveOBP8, SaveCSP2 and SaveCSP5) were abundantly expressed in the legs of winged or wingless aphids. The expression levels of the SaveOBPs and SaveCSPs varied depending on the developmental stage. Possible physiological functions of these genes are discussed. Further molecular and functional studies of these olfactory related genes will explore their potential as novel targets for controlling S. avenae. PMID:27561107

  4. A scallop IGF binding protein gene: molecular characterization and association of variants with growth traits.

    Liying Feng

    Full Text Available BACKGROUND: Scallops represent economically important aquaculture shellfish. The identification of genes and genetic variants related to scallop growth could benefit high-yielding scallop breeding. The insulin-like growth factor (IGF system is essential for growth and development, with IGF binding proteins (IGFBPs serving as the major regulators of IGF actions. Although an effect of IGF on growth was detected in bivalve, IGFBP has not been reported, and members of the IGF system have not been characterized in scallop. RESULTS: We cloned and characterized an IGFBP (PyIGFBP gene from the aquaculture bivalve species, Yesso scallop (Patinopecten yessoensis, Jay, 1857. Its full-length cDNA sequence was 1,445 bp, with an open reading frame of 378 bp, encoding 125 amino acids, and its genomic sequence was 10,193 bp, consisting of three exons and two introns. The amino acid sequence exhibited the characteristics of IGFBPs, including multiple cysteine residues and relatively conserved motifs in the N-terminal and C-terminal domains. Expression analysis indicated that PyIGFBP was expressed in all the tissues and developmental stages examined, with a significantly higher level in the mantle than in other tissues and a significantly higher level in gastrulae and trochophore larvae than in other stages. Furthermore, three single nucleotide polymorphisms (SNPs were identified in this gene. SNP c.1054A>G was significantly associated with both shell and soft body traits in two populations, with the highest trait values in GG type scallops and lowest in AG type ones. CONCLUSION: We cloned and characterized an IGFBP gene in a bivalve, and this report also represents the first characterizing an IGF system gene in scallops. A SNP associated with scallop growth for both the shell and soft body was identified in this gene. In addition to providing a candidate marker for scallop breeding, our results also suggest the role of PyIGFBP in scallop growth.

  5. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Andreas Bitter


    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  6. Transcriptional stimulation via SC site of Bombyx sericin-1 gene through an interaction with a DNA binding protein SGF-3.

    Matsuno, K.; Takiya, S; Hui, C C; Suzuki, T.; Fukuta, M.; Ueno, K.; Suzuki, Y


    Three protein binding sites have been identified in the upstream region of the sericin-1 gene. Two of them, SA and SC sites, have been known as putative cis-acting elements. Using synthetic oligonucleotides of these binding sites, it was found that silk gland factor-1 (SGF-1) binds to the SA site, and silk gland factor-3 (SGF-3) binds to the SC site but not to a mutated SC site, SCM. Tissue distribution of the two factors was different. SGF-3 is present abundantly in the middle silk gland (MS...

  7. Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

    Tsang, J S; Henry, Y A; Chambers, A.; Kingsman, A J; Kingsman, S M


    Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but...

  8. Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo

    Eileen McNeill; Iqbal, Asif J; White, Gemma E.; Jyoti Patel; Greaves, David R.; CHANNON, KEITH M


    Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detec...

  9. Structure and expression of major histocompatibility complex-binding protein 2, a 275-kDa zinc finger protein that binds to an enhancer of major histocompatibility complex class I genes

    Veer, L.J. van 't; Lutz, P.M.; Isselbacher, K.J.; Bernards, R.A.


    We have isolated a cDNA encoding a transcription factor that binds to the enhancer of major histocompatibility complex (MHC) class I genes. MHC-binding protein 2 (MBP-2) is a 275-kDa protein, containing two sets of widely separated zinc fingers and a stretch of highly acidic amino acids, a putative

  10. Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha

    Hwang, C S; Mandrup, S; MacDougald, O A;


    /EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together...

  11. Cloning of a down-regulated gene encoding small GTP binding protein in hybrid wheat

    YAO Yingyin; NI Zhongfu; CHEN Rongmin; WU Limin; SUN Qixin


    Previous studies showed that differential gene expression between wheat hybrids and their parents was responsible for the heterosis. To provide an insight into the molecular basis of wheat heterosis, one cDNA, designated TaRab, was identified from the cDNA library of wheat seedling leaves. The sequence comparison in GenBank revealed that TaRab is homologous to a group of genes encoding Rab-GTP binding protein. Semi-quantitative RT-PCR analysis indicated that TaRab was expressed in all plant tissues examined,but at slightly higher level in leaves. Further analysis exhibited that TaRab displayed lower expression in hybrid than in its patents in both roots and leaves, which was in agreement with the original results of suppression subtractive hybridization. TaRab was located on chromosome 7B and C-7DS5-0.36 by in silico mapping. The relationship between differential expression of TaRab and the molecular basis of wheat heterosis was also discussed.

  12. Identification of a TAL1 Target Gene Reveals a Positive Role for the LIM Domain-Binding Protein Ldb1 in Erythroid Gene Expression and Differentiation

    Xu, Zhixiong; Huang, Suming; Chang, Long-Sheng; Agulnick, Alan D.; Brandt, Stephen J.


    The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2)...

  13. The correlation of vitamin D level and vitamin D-binding protein gene polymorphism in chronic obstructive pulmonary disease



    Objective To assess the correlation of serum 25-hydroxyvitamin D(25-OHD)levels with vitamin D-binding protein(the group-specific component,GC)gene polymorphism in chronic obstructive pulmonary disease(COPD).Methods In a cross-sectional case-control study,250 participants,including 116 COPD patients with smoking history and 134 healthy smokers,were in-

  14. SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae.

    Saxe, S A; Kimmel, A R


    We have identified two novel, very closely related genes, SAS1 and SAS2, from Dictyostelium discoideum. These encode small, approximately 20-kilodaton proteins with amino acid sequences thought to be involved in interaction with guanine nucleotides. The protein sizes, spacings of GTP-binding domains, and carboxyl-terminal sequences suggest their relationship to the ubiquitous ras-type proteins. Their sequences, however, are sufficiently different to indicate that they are not true ras protein...

  15. Role of CCAA Nucleotide Repeats in Regulation of Hemoglobin and Hemoglobin-Haptoglobin Binding Protein Genes of Haemophilus influenzae

    Ren, Zhen; Jin, Hongfan; Whitby, Paul W.; Morton, Daniel J.; Stull, Terrence L.


    Haemophilus influenzae utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The H. influenzae hemoglobin- and hemoglobin-haptoglobin binding protein genes, hgpA, hgpB, and hgpC, contain lengths of tetrameric CCAA repeats. Using an hgpA-lacZ translational gene fusion, we demonstrate phase-variable expression of lacZ associated with alteration in the length of the CCAA repeat region.

  16. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

    Lee, Chang Hoon; Melchers, Mark; Wang, Hongsheng; Torrey, Ted A.; Slota, Rebecca; Qi, Chen-Feng; Kim, Ji Young; Lugar, Patricia; Kong, Hee Jeong; Farrington, Lila; van der Zouwen, Boris; ZHOU, JEFF X.; Lougaris, Vassilios; Lipsky, Peter E.; Grammer, Amrie C.


    Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphoma...

  17. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C. (MIT); (UT-Australia); (Macquarie); (Toronto); (New South)


    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  18. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

    Chandrika N Deshpande

    Full Text Available The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  19. Obesity risk gene TMEM18 encodes a sequence-specific DNA-binding protein.

    Jaana M Jurvansuu

    Full Text Available Transmembrane protein 18 (TMEM18 has previously been connected to cell migration and obesity. However, the molecular function of the protein has not yet been described. Here we show that TMEM18 localises to the nuclear membrane and binds to DNA in a sequence-specific manner. The protein binds DNA with its positively charged C-terminus that contains also a nuclear localisation signal. Increase in the amount of TMEM18 in cells suppresses expression from a reporter vector with the TMEM18 target sequence. TMEM18 is a small protein of 140 residues and is predicted to be mostly alpha-helical with three transmembrane parts. As a consequence the DNA binding by TMEM18 would bring the chromatin very near to nuclear membrane. We speculate that this closed perinuclear localisation of TMEM18-bound DNA might repress transcription from it.

  20. Mutations in the selenocysteine insertion sequence–binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

    Schoenmakers, Erik; Agostini, Maura; Mitchell, Catherine; Schoenmakers, Nadia; Papp, Laura; Rajanayagam, Odelia; Padidela, Raja; Ceron-Gutierrez, Lourdes; Doffinger, Rainer; Prevosto, Claudia; Luan, Jian’an; Montano, Sergio; Lu, Jun; Castanet, Mireille; Clemons, Nick


    Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence–binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a comple...

  1. Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

    In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory β-subunit of calcineurin (Ca2+-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression

  2. The ice-binding proteins of a snow alga, Chloromonas brevispina: probable acquisition by horizontal gene transfer.

    Raymond, James A


    All ice-and snow-related unicellular algae examined so far secrete ice-binding proteins (IBPs) to mitigate freezing damage. Two types of IBP have been identified in chlorophytes. Type 1 IBPs are members of a large family of proteins that share a large domain of unknown function (DUF3494). Previous studies have suggested that the type 1 algal IBP genes were acquired by horizontal gene transfer. To test this hypothesis I sequenced the IBP genes of a snow alga, Chloromonas brevispina. The IBPs were identified by ice affinity purification, de novo sequencing of a tryptic peptide and large-scale sequencing of the transcriptome and genome. C. brevispina has genes for over 20 IBP isoforms, which strongly indicates their importance. The IBPs are all of type 1 and match fungal and bacterial proteins more closely than they match known algal IBPs, providing further evidence that the genes were acquired by horizontal transfer. Modeling of the 3D structures of the IBPs based on the known structure of a homologous protein suggests that the ice-binding site has characteristics that are shared by all DUF3494 proteins. PMID:25081506

  3. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site.

    Mohun, T; Garrett, N; Treisman, R


    Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution. PMID:3582369

  4. Characterization of upstream sequences of the LIM2 gene that bind developmentally regulated and lens-specific proteins

    HSU Heng; Robert L. CHURCH


    During lens development, lens epithelial cells differentiate into fiber cells. To date, four major lens fiber cell intrinsic membrane proteins (MIP) ranging in size from 70 kD to 19 kD have been characterized. The second most abundant lens fiber cell intrinsic membrane protein is MP19. This protein probably is involved with lens cell communication and relates with cataractogenesis. The aim of this research is to characterize upstream sequences of the MP19 (also called LIM2) gene that bind developmentally regulated and lens-specific proteins. We have used the gel mobility assays and corresponding competition experiments to identify and characterize cis elements within approximately 500 bases of LIM2 upstream sequences. Our studies locate the positions of some cis elements, including a "CA" repeat, a methylation Hha I island, an FnuD II site, an Ap1 and an Ap2 consensus sequences, and identify some specific cis elements which relate to lens-specific transcription of LIM2. Our experiments also preliminarily identify trans factors which bind to specific cis elements of the LIM2 promoter and/or regulate transcription of LIM2. We conclude that developmental regulation and coordination of the MP 19 gene in ocular lens fiber cells is controlled by the presence of specific cis elements that bind regulatory trans factors that affect LIM2 gene expression. DNA methylation is one mechanism of controlling LIM2 gene expression during lens development.

  5. CCAAT/enhancer binding protein a gene expression in Egyptian patients with acute myeloid leukemia

    Background: Transcription factors play a crucial role in myeloid differentiation and lineage determination. Tumor suppressor protein C/EBPa is a key regulator of granulocytic differentiation whose functional inactivation has become a pathophysiological signature of myeloid leukemia. Given the role that CCAAT/enhancer binding protein α (C/EBP α) plays in myelopoiesis, we anticipated that their expression might be disrupted in myeloid neoplasms. Purpose: To estimate the expression of C/EBP α mRNA in patients with acute myeloid leukemia and correlate its expression with the pathogenesis of the disease. Patients and methods: Forty AML patients and 20 age and sex matched healthy controls were included in the study. Blood samples of patients and controls were analyzed for CEBP α mRNA expression by quantitative RT-real time PCR using TaqMan technology and δδct method for calculation of gene expression. Results: Twenty-nine (72.5%) patients out of the 40 showed low expression levels of CEBP α mRNA below the cutoff value with median of 0.19 (range:0-0.87). While eleven (27.5%) patients out of the 40 showed higher expression levels of CEBP α above the cutoff value with median of 1.52 (range: 1.07-2). Seven patients out of the 11 showed higher expression levels of CEBP α mRNA belong to the M3 subtype of AML harboring the t(15;17) PML-RARa translocation. Conclusion: We conclude that the majority of the AML patients analyzed, express low levels of C/EBPa mRN. However, a subset of patients represented by the M3 subtype, express higher levels of C/EBPa


    Mathew Zachariah


    Full Text Available Objectives: Our objective was to study mannose binding protein (MBP polymorphisms in exonic and promoter region and correlate associated infections and vasoocculsive (VOC episodes, since MBP plays an important role in innate immunity by activating the complement system. Methods: We studied the genetic polymorphisms in the Exon 1 (alleles A/O and promoter region (alleles Y/X; H/L, P/Q of the MBL2 gene, in sickle cell disease (SCD patients as increased incidence of infections is seen in these patients. A PCR-based, targeted genomic DNA sequencing of MBL2 was used to study 68 SCD Omani patients and 44 controls (voluntary blood donors. Results: The observed frequencies of MBL2 promoter polymorphism (-221, Y/X were 44.4% and 20.5% for the heterozygous genotype Y/X and 3.2% and 2.2% for the homozygous (X/X respectively between SCD patients and controls. MBL2 Exon1 gene mutations were 29.4% and 50% for the heterozygous genotype A/O and 5.9% and 6.8% respectively for the homozygous (O/O genotype between SCD patients and controls. The distribution of variant MBL2 polymorphisms did not show any correlation in SCD patients with or without vasoocculsive crisis (VOC attacks (p=0.162; OR-0.486; CI=0.177 -1.33, however, it was correlated with infections (p=0.0162; OR-3.55; CI 1.25-10.04. Conclusions: Although the frequency of the genotypes and haplotypes of MBL2 in SCD patients did not differ from controls, overall in the SCD patient cohort the increased representation of variant alleles was significantly correlated with infections (p<0.05. However, these variant MBL2 polymorphisms did not seem to play a significant role in the VOC episodes in this SCD cohort. Keywords: Mannose-binding lectin, polymorphism, promoter, Sickle cell disease, MBL2, MBP

  7. Update of the human secretoglobin (SCGB gene superfamily and an example of 'evolutionary bloom' of androgen-binding protein genes within the mouse Scgb gene superfamily

    Jackson Brian C


    Full Text Available Abstract The secretoglobins (SCGBs comprise a family of small, secreted proteins found in animals exclusively of mammalian lineage. There are 11 human SCGB genes and five pseudogenes. Interestingly, mice have 68 Scgb genes, four of which are highly orthologous to human SCGB genes; the remainder represent an 'evolutionary bloom' and make up a large gene family represented by only six counterparts in humans. SCGBs are found in high concentrations in many mammalian secretions, including fluids of the lung, lacrimal gland, salivary gland, prostate and uterus. Whereas the biological activities of most individual SCGBs have not been fully characterised, what already has been discovered suggests that this family has an important role in the modulation of inflammation, tissue repair and tumorigenesis. In mice, the large Scgb1b and Scgb2b gene families encode the androgen-binding proteins, which have been shown to play a role in mate selection. Although much has been learned about SCGBs in recent years, clearly more research remains to be done to allow a better understanding of the roles of these proteins in human health and disease. Such information is predicted to reveal valuable novel drug targets for the treatment of inflammation, as well as designing biomarkers that might identify tissue damage or cancer.

  8. The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

    CHEN; Weiwen; (陈蔚文); ZHANG; Jianye; (张建业); Charles; Y; F; Young; ZHANG; Lianying; (张莲英); CHEN; Liucun; (陈留存); ZHAO; Jian; (赵健)


    To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound specifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunctional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding proteins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA promoter.

  9. Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding

    Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work, we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability

  10. Characterization and expression of a new class of zinc finger protein that binds to silencer region of ascorbate oxidase gene.

    Kisu, Y; Ono, T; Shimofurutani, N; Suzuki, M; Esaka, M


    A unique A/T-rich sequence (5'-AAAAAGTAAAAA-GTAAAAAAGTAAAAAG-3), referred to as the AGTA repeat, is found in the silencer region of the pumpkin ascorbate oxidase gene. A cDNA for protein (AOBP) that binds to the AGTA repeat was isolated from pumpkin by the southwestern method. The AOBP protein has a new class of zinc/DNA-binding domain named Dof/MOA domain that is highly conserved in many plant proteins and is significantly related to those of steroid hormone receptors and GATA1. Gel retardation analysis indicated that AOBP bound to the AGTA repeat through the Dof/MOA domain. Metal chelators, 1,10-phenanthroline and EDTA, specifically inhibited the DNA binding of AOBP, indicating that metal coordination plays an important role in DNA binding of AOBP. Thus, the Dof/MOA domain acts as a zinc/DNA-binding domain in AOBP. Gel retardation analysis with mutated oligonucleotides suggested that the Dof/MOA domain recognized the AGTA core sequence. AOBP mRNA was expressed in mature tissues of pumpkin, but was expressed only in small amounts or was not expressed in growing tissues. Furthermore, the expression was auxin-independent. The expression pattern of AOBP and that of ascorbate oxidase did not show a positive correlation. PMID:9871365

  11. The trp RNA-binding attenuation protein of Bacillus subtilis regulates translation of the tryptophan transport gene trpP (yhaG) by blocking ribosome binding.

    Yakhnin, Helen; Zhang, Hong; Yakhnin, Alexander V; Babitzke, Paul


    Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA. RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts. PMID:14702295

  12. Cell-type-specific repression by methyl-CpG-binding protein 2 is biased toward long genes.

    Sugino, Ken; Hempel, Chris M; Okaty, Benjamin W; Arnson, Hannah A; Kato, Saori; Dani, Vardhan S; Nelson, Sacha B


    Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders (Amir et al., 1999). MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knock-out mice using brain tissue homogenates have revealed only subtle changes in gene expression (Tudor et al., 2002; Nuber et al., 2005; Jordan et al., 2007; Chahrour et al., 2008). Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the "dilution problem" associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes upregulated following loss of MeCP2 are biased toward longer genes but this is not true for downregulated genes, suggesting MeCP2 may selectively repress long genes. Because genes involved in neuronal connectivity and communication, such as cell adhesion and cell-cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome. PMID:25232122

  13. A case of Rubinstein-Taybi Syndrome with a CREB-binding protein gene mutation

    Kim, Se Hee; Lim, Byung Chan; Chae, Jong Hee; Kim, Ki Joong; Hwang, Yong Seung


    Rubinstein-Taybi syndrome (RTS) is a congenital disorder characterized by typical facial features, broad thumbs and toes, with mental retardation. Additionally, tumors, keloids and various congenital anomalies including congenital heart defects have been reported in RTS patients. In about 50% of the patients, mutations in the CREB binding protein (CREBBP) have been found, which are understood to be associated with cell growth and proliferation. Here, we describe a typical RTS patient with Arn...

  14. Hepatic phenotype of liver fatty acid binding protein gene-ablated mice

    Martin, Gregory G.; Atshaves, Barbara P.; Huang, Huan; McIntosh, Avery L.; Williams, Brad J.; Pai, Pei-Jing; Russell, David H.; Kier, Ann B.; Schroeder, Friedhelm


    Although the function of liver fatty acid binding protein in hepatic fatty acid metabolism has been extensively studied, its potential role in hepatic cholesterol homeostasis is less clear. Although hepatic cholesterol accumulation was initially reported in L-FABP-null female mice, that study was performed with early N2 backcross generation mice. To resolve whether the hepatic cholesterol phenotype in these L-FABP−/− mice was attributable to genetic inhomogeneity, these L-FABP−/− mice were fu...

  15. Molecular characterization of a defense-related AMP-binding protein gene, OsBIABP1, from rice

    Xin-chun ZHANG; Xin YU; Hui-juan ZHANG; Feng-ming SONG


    We cloned and characterized a rice gene OsBIABPI encoding an AMP-binding protein. The full-length cDNA of OsBIABP1 is 1912-bp long and is predicted to encode a 558-aa protein. OsBIABP1 contains a typical AMP-binding signature motif and shows high similarity to members of AMP-binding protein family. OsBIABP1 is expressed in stems, leaves and flowers of rice plants, but is not expressed, or expressed at a very low level, in rice roots. The expression of OsBIABP1 was induced by some defense-related signal molecules, e.g., salicylic acid (SA), benzothiadiazole, jasmonic acid (JA), and 1-amino cyclopropane-1-carboxylic acid, which mediate SA-and JA/ethylene (ET)-dependent defense signaling pathways, respectively. Furthermore, the expression of OsBIABP1 is activated by the infection of Magnaporthe oryzae, and the induced expression is quicker and stronger during early stages of pathogenesis in incompatible interaction than that in compatible interaction between rice and M. oryzae. Our results suggest that OsBIABP1 may be a defense-related AMP-binding protein that is involved in the regulation of defense re-sponse through SA and/or JA/ET signaling pathways.

  16. Transcriptional regulation of the HMGA1 gene by octamer-binding proteins Oct-1 and Oct-2.

    Eusebio Chiefari

    Full Text Available The High-Mobility Group AT-Hook 1 (HMGA1 protein is an architectural transcription factor that binds to AT-rich sequences in the promoter region of DNA and functions as a specific cofactor for gene activation. Previously, we demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR gene and an important downstream target of the INSR signaling cascade. Moreover, from a pathogenic point of view, overexpression of HMGA1 has been associated with human cancer, whereas functional variants of the HMGA1 gene have been recently linked to type 2 diabetes mellitus and metabolic syndrome. However, despite of this biological and pathological relevance, the mechanisms that control HMGA1 gene expression remain unknown. In this study, to define the molecular mechanism(s that regulate HMGA1 gene expression, the HMGA1 gene promoter was investigated by transient transfection of different cell lines, either before or after DNA and siRNA cotransfections. An octamer motif was identified as an important element of transcriptional regulation of this gene, the interaction of which with the octamer transcription factors Oct-1 and Oct-2 is crucial in modulating HMGA1 gene and protein expression. Additionally, we demonstrate that HMGA1 binds its own promoter and contributes to its transactivation by Oct-2 (but not Oct-1, supporting its role in an auto-regulatory circuit. Overall, our results provide insight into the transcriptional regulation of the HMGA1 gene, revealing a differential control exerted by both Oct-1 and Oct-2. Furthermore, they consistently support the hypothesis that a putative defect in Oct-1 and/or Oct-2, by affecting HMGA1 expression, may cause INSR dysfunction, leading to defects of the INSR signaling pathway.

  17. Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

    Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C


    B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. PMID:27129202

  18. Gene 33/Mig-6, a Transcriptionally Inducible Adapter Protein That Binds GTP-Cdc42 and Activates SAPK/JNK*

    Makkinje, Anthony; Quinn, Deborah A.; Chen, Ang; Cadilla, Carmen L.; Force, Thomas; Bonventre, Joseph V.; Kyriakis, John M.


    Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extra-cellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH2-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences. PMID:10749885

  19. Isolation and identification of proteins binding to the major breakpoint region(mbr) of bcl2 gene

    Nan Yang; Yujie Sun; Changyan Ma


    Objective: We have previously found that mbr is a regulatory element of the bcl2 gene. The objective of this study is to isolate and identify the proteins binding to the 37 mbr in the 3 '-end of the mbr. Methods: Streptavidin magnetic particles were ligated to concatameric oligonucleofides of 37 mbr and incubated with the nuclear extracts of Jurkat cells. The DNA-binding proteins were eluted and then resolved by SDS-PAGE. After silver staining, the protein bands were excised and subjected to MALDI-TOF MS. Results: Several protein bands were detected after the isolation with magnetic particles, and Splicing factor, proline-and glutamine-rich(SFPQ), Poly(ADP-ribose)polymerase I(PARP), and promyelocytic leukemia protein(PML) were identified by MALDI-TOF MS. Conclusion: Several proteins were isolated and identified from the 37 mbr-protein complex. Results of this study establish a foundation for further study of the mechanisms by which mbr executes its regulatory function.

  20. Disruption of the Acyl-CoA binding protein gene delays hepatic adaptation to metabolic changes at weaning

    Neess, Ditte; Marcher, Ann-Britt; Bloksgaard, Maria; Bek, Signe; Elle, Ida Coordt; Færgeman, Nils J.; Mandrup, Susanne

    -CoA esters between different enzymatic systems. However, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP-/-). These mice are viable and fertile and develop normally. However, around weaning the ACBP-/- mice show decreased growth...... rate and increased levels of plasma cholesterol combined with hepatic accumulation of triglycerides and cholesteryl esters. We show by microarray analysis that the liver of ACBP-/- mice displays a significantly delayed induction of target genes of the sterol regulatory element binding protein (SREBP......) family, around the weaning period. As a result, the hepatic de novo cholesterogenesis is significantly decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP to...

  1. Disruption of the acyl-coa binding protein gene delays hepatic adaptation to metabolic changes at weaning

    Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek; Marcher, Ann-Britt; Elle, Ida C; Helledie, Torben; Due, Marianne; Pagmantidis, Vasileios; Finsen, Bente; Wilbertz, Johannes; Kruhoeffer, Mogens; Faergeman, Nils; Mandrup, Susanne


    , little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP-/-). These mice are viable and fertile and develop normally. However, around weaning the ACBP-/- mice go through a crisis with overall weakness, and a slightly decreased growth...... rate. Using microarray analysis we show that the liver of ACBP-/- mice display a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The...... delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to...

  2. Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif.

    Argüello, G; García-Hernández, E; Sánchez, M; Gariglio, P; Herrera-Estrella, L; Simpson, J


    Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes. PMID:1303797

  3. Cyclic AMP response element binding protein (CREB) par ticipates in the heat-inducible expression of humanhsp90β gene


    Human heat shock protein 90β gene (hsp90β)is a constitutively expressed heat shock gene existing in most of cell types tested that can be further induced by heat shock. Chloramphenical acetyl transferase (CAT) reporter plasmids driven by different regulatory fragments of hsp90β gene were constructed and transfected into Jurkat cells to explore the role of a cAMP response element (CRE) in the upstream of the gene. Results show that, in comparison with the wild type construct, a severe reduction (~2/3) in the increased folds of promoter activity induced by heat shock at 42C for 1 b was observed in a construct with CRE-containing frag ment (-173/-91bp) deleted. Electrophoretic mobility shift assays (EMSA) showed that phosphorylated CRE-binding protein (CREB) in the nuclear extract of heat shocked Jur kat cells is specifically bound to the fragment. Additionally,both of the phosphorylation on CREB and the activity of protein kinase A (PKA) were found in Jurkat cells to be en hanced with extending time of heat shock treatment. Our results indicate that in addition to the intronic HSE/HSF pathway, phosphorylated CREB also participates in the heat shock induced expression of human hsp90β gene via its in teraction with CRE which may be regulated by PKA-sig haling pathway.

  4. Identification and characterization of a novel calcyclin binding protein (CacyBP) gene from Apis cerana cerana.

    Yu, Xiaoli; Lu, Wenjing; Sun, Rujiang; Guo, Xingqi; Xu, Baohua


    Calcyclin binding protein (CacyBP), a homolog of Sgt1, was shown to interact with some S100 proteins, Skp1, tubulin, actin and ERK1/2 kinases. Studies have also shown that CacyBP is a neuronal protein in mammals. Limited information is available regarding the properties and functions of CacyBP in insects. Here, we cloned and characterized a novel CacyBP gene, named AccCacyBP, from honeybee (Apis cerana cerana). Bioinformatic analysis indicated that AccCacyBP was highly conserved and closely related to the CacyBP of other insects. Promoter analysis revealed a number of putative tissue, development and stress-related transcription factor-binding sites. RT-qPCR demonstrated that AccCacyBP was expressed at all of the stages of development, especially in the brains of honeybees. Moreover, immunohistochemistry analysis showed the presence of AccCacyBP in the brain. The transcript levels of AccCacyBP in the brains of honeybees were developmentally induced and upregulated by exposure to oxidative stresses, including UV-light, acetamiprid and HgCl(2). This study demonstrates that the CacyBP gene in honeybees may be a neuronal protein involved in the developmental regulation and the stress-response of the brain of honeybees. PMID:22539186

  5. The lipopolysaccharide-binding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT/3 and other cytokine-inducible nuclear proteins.

    Schumann, R.R.; Kirschning, C.J.; Unbehaun, A; Aberle, H P; Knope, H P; Lamping, N; Ulevitch, R J; Herrmann, F.


    Acute-phase reactants (APRs) are proteins synthesized in the liver following induction by interleukin-1 (IL-1), IL-6, and glucocorticoids, involving transcriptional gene activation. Lipopolysaccharide-binding protein (LBP) is a recently identified hepatic secretory protein potentially involved in the pathogenesis of sepsis, capable of binding the bacterial cell wall product endotoxin and directing it to its cellular receptor, CD14. In order to examine the transcriptional induction mechanisms ...

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

    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


    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

  7. The autism-associated gene chromodomain helicase DNA-binding protein 8 (CHD8) regulates noncoding RNAs and autism-related genes

    Wilkinson, B; Grepo, N; Thompson, B. L.; Kim, J.; K. Wang; Evgrafov, O V; Lu, W; Knowles, J A; Campbell, D B


    Chromodomain helicase DNA-binding protein 8 (CHD8) was identified as a leading autism spectrum disorder (ASD) candidate gene by whole-exome sequencing and subsequent targeted-sequencing studies. De novo loss-of-function mutations were identified in 12 individuals with ASD and zero controls, accounting for a highly significant association. Small interfering RNA-mediated knockdown of CHD8 in human neural progenitor cells followed by RNA sequencing revealed that CHD8 insufficiency results in alt...

  8. Liver Fatty Acid Binding Protein Gene-Ablated Female Mice Exhibit Increased Age-Dependent Obesity123

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


    Previous work done in our laboratory suggested a role for liver fatty acid binding protein (L-FABP) in obesity that develops in aging female L-FABP gene-ablated (−/−) mice. To examine this possibility in more detail, cohorts of wild-type (+/+) and L-FABP (−/−) female mice were fed a standard low-fat nonpurified rodent diet for up to 18 mo. Various obesity-related parameters were examined including body weight and fat and lean tissue mass. Obesity in (−/−) mice was associated with increased ex...

  9. Epigenetic analyses of the insulin-like growth factor binding protein 1 gene in type 1 diabetes and diabetic nephropathy

    Gu, Tianwei; Falhammar, Henrik; Gu, Harvest F.; Brismar, Kerstin


    Background Clinical observations have demonstrated that high levels of circulating insulin-like growth factor binding protein-1 (IGFBP-1) are associated with type 1 diabetes (T1D), whereas low serum IGFBP-1 levels are associated with the risk of type 2 diabetes (T2D). Recently, we reported that increased DNA methylation levels in the IGFBP1 gene were associated with T2D. In the present study, we evaluated the epigenetic changes of IGFBP1 in T1D and diabetic nephropathy (DN). Results In total,...

  10. High Dietary Fat Exacerbates Weight Gain and Obesity in Female Liver Fatty Acid Binding Protein Gene-Ablated Mice

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


    Since liver fatty acid binding protein (L-FABP) facilitates uptake/oxidation of long-chain fatty acids in cultured transfected cells and primary hepatocytes, loss of L-FABP was expected to exacerbate weight gain and/or obesity in response to high dietary fat. Male and female wild-type (WT) and L-FABP gene-ablated mice, pair-fed a defined isocaloric control or high fat diet for 12 weeks, consumed equal amounts of food by weight and kcal. Male WT mice gained weight faster than their female WT c...

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

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


    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 exami...

  12. Double heterozygosity for mutations in the β-myosin heavy chain and in the cardiac myosin binding protein C genes in a family with hypertrophic cardiomyopathy

    Richard, P.; Isnard, R.; Carrier, L.; Dubourg, O.; Donatien, Y.; Mathieu, B.; Bonne, G.; Gary, F; Charron, P; HAGEGE, A.; Komajda, M; Schwartz, K.; Hainque, B


    Familial hypertrophic cardiomyopathy is a genetically heterogeneous autosomal dominant disease, caused by mutations in several sarcomeric protein genes. So far, seven genes have been shown to be associated with the disease with the β-myosin heavy chain (MYH7) and the cardiac myosin binding protein C (MYBPC3) genes being the most frequently involved.
We performed electrocardiography (ECG) and echocardiography in 15 subjects with hypertrophic cardiomyopathy from a French Caribbean family. Genet...

  13. The gene for human TATA-binding-protein-associated factor (TAFII) 170: structure, promoter and chromosomal localization.

    Van Der Knaap, J A; Van Den Boom, V; Kuipers, J; Van Eijk, M J; Van Der Vliet, P C; Timmers, H T


    The TATA-binding protein (TBP) plays a central role in eukaryotic transcription and forms protein complexes with TBP-associated factors (TAFs). The genes encoding TAF(II) proteins frequently map to chromosomal regions altered in human neoplasias. TAF(II)170 of B-TFIID is a member of the SF2 superfamily of putative helicases. Members of this superfamily have also been implicated in several human genetic disorders. In this study we have isolated human genomic clones encoding TAF(II)170 and we show that the gene contains 37 introns. Ribonuclease-protection experiments revealed that TAF(II)170 has multiple transcription start sites, consistent with the observation that the promoter lacks a canonical TATA box and initiator element. Deletion analysis of the promoter region showed that a fragment of 264 bp is sufficient to direct transcription. In addition, we determined the chromosomal localization by two independent methods which mapped the gene to human chromosome 10q22-q23 between the markers D10S185 and WI-1183. The region surrounding these markers has been implicated in several human disorders. PMID:10642510

  14. Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

    Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment

  15. Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases

    The present studies establish that there are specific, significant decreases in the neuronal calcium-binding protein (28-kDa calbindin-D) gene expression in aging and in neurodegenerative diseases. The specificity of the changes observed in calbindin mRNA levels was tested by reprobing blots with calmodulin, cyclophilin, and B-actin cDNAs. Gross brain regions of the aging rat exhibited specific, significant decreases in calbindin·mRNA and protein levels in the cerebellum, corpus striatum, and brain-stem region but not in the cerebral cortex or hippocampus. Discrete areas of the aging human brain exhibited significant decreases in calbindin protein and mRNA in the cerebellum, corpus striatum, and nucleus basalis but not in the neocortex, hippocampus, amygdala, locus ceruleus, or nucleus raphe dorsalis. Comparison of diseased human brain tissue with age- and sex-matched controls yielded significant decreases calbindin protein and mRNA in the substantia nigra (Parkinson disease), in the corpus striatum (Huntington disease), in the nucleus basalis (Alzheimer disease), and in the hippocampus and nucleus raphe dorsalis (Parkinson, Huntington, and Alzheimer diseases) but not in the cerebellum, neocortex, amygdala, or locus ceruleus. These findings suggest that decreased calbindin gene expression may lead to a failure of calcium buffering or intraneuronal calcium homeostasis, which contributes to calcium-mediated cytotoxic events during aging and in the pathogenesis of neurodegenerative diseases

  16. Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).

    Sakmar, T P; Khorana, H G


    To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthet...

  17. Down-regulation of the expression of CCAAT/enhancer binding protein α gene in cervical squamous cell carcinoma

    Cervical carcinoma is the second most common cancer and is an important cause of death in women worldwide. CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that regulate cellular differentiation and proliferation in a variety of tissues. However, the role of C/EBPα gene in cervical cancer is still not clear. We investigated the expression of C/EBPα gene in cervical squamous cell carcinoma. C/EBPα mRNA level was measured by real-time quantitative RT-PCR in cervical cancer tissues and their adjacent normal tissues. C/EBPα protein level was measured by immunohistochemistry. Methylation in the promoter of C/EBPα gene was detected by MALDI TOF MassARRAY. We transfected HeLa cells with C/EBPα expression vector. C/EBPα expression in HeLa cells was examined and HeLa cell proliferation was measured by MTT assay and HeLa cells migration was analyzed by matrigel-coated transwell migration assays. There were significant difference in C/EBPα protein expression between chronic cervicitis and cervical carcinoma (P < 0.001). CEBPα mRNA level was significantly lower in cervical cancer tissues than in normal cervical tissues (P < 0.01). Methylation of the promoter of CEBPα gene in CpG 5, CpG-14.15, CpG-19.20 were significantly higher in cervical cancer than in normal cervical tissues (P < 0.05, P < 0.01, P < 0.05, respectively). CEBPα pcDNA3.1 construct transfected into HeLa cells inhibited cell proliferation and decreased cell migration. Our results indicate that reduced C/EBPα gene expression may play a role in the development of cervical squamous cell carcinoma

  18. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein.

    Ubukata, K; Nonoguchi, R; Matsuhashi, M; Konno, M


    A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become ...

  19. Cardiac myosin binding protein C and MAP-kinase activating death domain-containing gene polymorphisms and diastolic heart failure.

    Cho-Kai Wu

    Full Text Available OBJECTIVE: Myosin binding protein C (MYBPC3 plays a role in ventricular relaxation. The aim of the study was to investigate the association between cardiac myosin binding protein C (MYBPC3 gene polymorphisms and diastolic heart failure (DHF in a human case-control study. METHODS: A total of 352 participants of 1752 consecutive patients from the National Taiwan University Hospital and its affiliated hospital were enrolled. 176 patients diagnosed with DHF confirmed by echocardiography were recruited. Controls were matched 1-to-1 by age, sex, hypertension, diabetes, renal function and medication use. We genotyped 12 single nucleotide polymorphisms (SNPs according to HapMap Han Chinese Beijing databank across a 40 kb genetic region containing the MYBPC3 gene and the neighboring DNA sequences to capture 100% of haplotype variance in all SNPs with minor allele frequencies ≥ 5%. We also analyzed associations of these tagging SNPs and haplotypes with DHF and linkage disequilibrium (LD structure of the MYBPC3 gene. RESULTS: In a single locus analysis, SNP rs2290149 was associated with DHF (allele-specific p = 0.004; permuted p = 0.031. The SNP with a minor allele frequency of 9.4%, had an odds ratio 2.14 (95% CI 1.25-3.66; p = 0.004 for the additive model and 2.06 for the autosomal dominant model (GG+GA : AA, 95% CI 1.17-3.63; p = 0.013, corresponding to a population attributable risk fraction of 12.02%. The haplotypes in a LD block of rs2290149 (C-C-G-C was also significantly associated with DHF (odds ratio 2.10 (1.53-2.89; permuted p = 0.029. CONCLUSIONS: We identified a SNP (rs2290149 among the tagging SNP set that was significantly associated with early DHF in a Chinese population.

  20. Cold-inducible RNA binding protein (CIRP, a novel XTcf-3 specific target gene regulates neural development in Xenopus

    Wedlich Doris


    Full Text Available Abstract Background As nuclear mediators of wnt/β-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/β-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants. Results We identified cold-inducible RNA binding protein (CIRP as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3. Conclusion Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.

  1. Update of the human secretoglobin (SCGB) gene superfamily and an example of 'evolutionary bloom' of androgen-binding protein genes within the mouse Scgb gene superfamily

    Jackson Brian C; Thompson David C; Wright Mathew W; McAndrews Monica; Bernard Alfred; Nebert Daniel W; Vasiliou Vasilis


    Abstract The secretoglobins (SCGBs) comprise a family of small, secreted proteins found in animals exclusively of mammalian lineage. There are 11 human SCGB genes and five pseudogenes. Interestingly, mice have 68 Scgb genes, four of which are highly orthologous to human SCGB genes; the remainder represent an 'evolutionary bloom' and make up a large gene family represented by only six counterparts in humans. SCGBs are found in high concentrations in many mammalian secretions, including fluids ...

  2. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D.; Singh, Ravinder


    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5’ ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis. PMID:26942929

  3. Association of polymorphisms in adipocyte fatty acid binding protein gene with fat-related traits in chicken

    Manhong YE; Jie WEN; Honghe CAO; Hongbin LI; Jilan CHEN; Guiping ZHAO; Maiqing ZHENG


    PCR-SSCP analysis was used to detect poly-morphic sites in chicken adipocyte fatty acid binding pro-tein (A-FABP) gene. Six Chinese local breeds, Beijing-You chicken, Dwarf chicken, Taihe silky chicken, Chong-renma chicken, Xiayan chicken, Luyuan chicken and an introduced foreign breed, Arbor Acre broiler, were used as test populations. Three PCR-SSCP loci were detected. Statistical results showed that frequencies of genotypes and alleles were significantly different in the test popula-tions. Sequence analysis revealed that C → T, G → A, and C → T transitions were responsible for the polymorph-isms. Some fat-related traits such as body weight, content of intramuscular fat (IMF) and percentage of abdominal fat (AFP) were measured in Dwarf chickens and male Beijing-You chickens. We found out that chicken quality was significantly related to different genotypes in these two populations.

  4. Smoking and polymorphisms of genes encoding mannose-binding lectin and surfactant protein-D in patients with rheumatoid arthritis

    Kristiansen, Malthe; Frisch, Morten; Madsen, Hans Ole; Garred, Peter; Jacobsen, Søren


    To investigate whether polymorphisms in genes coding for mannose-binding lectin (MBL) and surfactant protein-D (SP-D) are associated directly or by interaction with smoking with rheumatoid arthritis (RA), anti-citrullinated peptide antibody (ACPA) positive RA, and erosive RA. MBL2 genotypes, SFTPD...... genotype at codon 11, and HLA-shared epitope were determined in 456 patients with rheumatoid arthritis and 533 sex- and age-matched controls. Patients were grouped according to the presence of ACPA antibodies and RA-associated bone erosions and sub-stratified according to smoking status as never or ever...... with RA but with erosive RA by interaction with smoking. The genetic disposition for high MBL production was associated with ACPA positive RA irrespective of shared epitope. The findings need to be replicated but do as such offer further explanations for the clinical heterogeneity of RA....

  5. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia


    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  6. Cloning and mutational analysis of the gamma gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization.

    Rubio, Luis M; Rangaraj, Priya; Homer, Mary J; Roberts, Gary P; Ludden, Paul W


    Dinitrogenase is a heterotetrameric (alpha(2)beta(2)) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition alpha(2)beta(2)gamma(2), which can be activated in vitro by the addition of FeMo-co. The gamma protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the gamma subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now gamma. In vitro FeMo-co insertion experiments presented in this work demonstrate that gamma stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter. PMID:11823455

  7. GA-Binding Protein Is Dispensable for Neuromuscular Synapse Formation and Synapse-Specific Gene Expression▿

    Jaworski, Alexander; Smith, Cynthia L.; Burden, Steven J.


    The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and o...

  8. Angiotensinogen gene-inducible enhancer-binding protein 1, a member of a new family of large nuclear proteins that recognize nuclear factor kappa B-binding sites through a zinc finger motif.

    Ron, D; Brasier, A R; Habener, J F


    Transcriptional activation of the rat angiotensinogen gene during the acute-phase response is dependent on a previously characterized acute-phase response element (APRE) that binds at least two types of nuclear proteins: a cytokine-inducible activity indistinguishable from nuclear factor kappa-B (NF kappa B) and a family of C/EBP-like proteins. We screened a rat liver cDNA expression library with a labeled APRE DNA probe and isolated a single clone that encodes a sequence-specific APRE-bindin...

  9. Diverse spatial, temporal, and sexual expression of recently duplicated androgen-binding protein genes in Mus musculus

    Emes Richard D


    Full Text Available Abstract Background The genes for salivary androgen-binding protein (ABP subunits have been evolving rapidly in ancestors of the house mouse Mus musculus, as evidenced both by recent and extensive gene duplication and by high ratios of nonsynonymous to synonymous nucleotide substitution rates. This makes ABP an appropriate model system with which to investigate how recent adaptive evolution of paralogous genes results in functional innovation (neofunctionalization. Results It was our goal to find evidence for the expression of as many of the Abp paralogues in the mouse genome as possible. We observed expression of six Abpa paralogues and five Abpbg paralogues in ten glands and other organs located predominantly in the head and neck (olfactory lobe of the brain, three salivary glands, lacrimal gland, Harderian gland, vomeronasal organ, and major olfactory epithelium. These Abp paralogues differed dramatically in their specific expression in these different glands and in their sexual dimorphism of expression. We also studied the appearance of expression in both late-stage embryos and postnatal animals prior to puberty and found significantly different timing of the onset of expression among the various paralogues. Conclusion The multiple changes in the spatial expression profile of these genes resulting in various combinations of expression in glands and other organs in the head and face of the mouse strongly suggest that neofunctionalization of these genes, driven by adaptive evolution, has occurred following duplication. The extensive diversification in expression of this family of proteins provides two lines of evidence for a pheromonal role for ABP: 1 different patterns of Abpa/Abpbg expression in different glands; and 2 sexual dimorphism in the expression of the paralogues in a subset of those glands. These expression patterns differ dramatically among various glands that are located almost exclusively in the head and neck, where the sensory

  10. Chromosomal localization of the human gene encoding c-myc promoter-binding protein (MPB1) to chromosome 1p35-pter

    White, R.A.; Dowler, L.L. [Univ. of Missouri, Kansas City, MO (United States); Adkison, L.R. [Mercer Univ. School of Medicine, Macon, GA (United States); Ray, R.B. [St. Louis Univ. Health Sciences Center, St. Louis, MO (United States)


    We report the mapping of the human gene MPB1 (c-myc promoter binding protein), a recently identified gene regulatory protein. MPB1 binds to the c-myc P2 promoter and exerts a negative regulatory role on c-myc transcription. Since exogenous expression from transfection of the MPB1 gene suppresses the tumorigenic property of breast cancer cells, there was interest in determining the chromosomal location of this gene. The human MPB1 gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent-human somatic hybrid cell lines. A specific human genomic fragment was observed only in the somatic cell lines containing human chromosome 1 or the p35-pter region of the chromosome. 10 refs., 2 figs.

  11. Telomere binding protein TRB1 is associated with promoters of translation machinery genes in vivo

    Schrumpfová, P.; Vychodilová, I.; Hapala, J.; Schorová, Š.; Dvořáček, Vojtěch; Fajkus, Jiří


    Roč. 90, 1-2 (2016), s. 189-206. ISSN 0167-4412 R&D Projects: GA ČR(CZ) GA13-06943S Institutional support: RVO:68081707 Keywords : Telomere repeat binding * ChIP-seq * Arabidopsis thaliana Subject RIV: BO - Biophysics Impact factor: 4.257, year: 2014

  12. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

    Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.


    This study provides new insight into the requirements for observed silencing of RNA polymerase II transcription near tRNA genes. Mod5 is a conserved tRNA modification enzyme found in both the nucleus and cytoplasm, although it only modifies tRNAs in the cytoplasm. Mod5 is required for silencing near tRNA genes, and it is bound to both nuclear tRNA gene complexes and nuclear pre-tRNA transcripts. Possible mechanisms for this form of RNA-mediated transcriptional silencing are discussed.

  13. A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis.

    Lee, A I; Sarsero, J P; Yanofsky, C


    In Bacillus subtilis, the tryptophan-activated trp RNA-binding attenuation protein (TRAP) regulates expression of the seven tryptophan biosynthetic genes by binding to specific repeat sequences in the transcripts of the trp operon and of the folate operon, the operon containing trpG. Steinberg observed that strains containing a temperature-sensitive mutant form of tryptophanyl-tRNA synthetase, encoded by the trpS1 allele, produced elevated levels of the tryptophan pathway enzymes, when grown ...

  14. Drosophila polypyrimidine tract-binding protein (DmPTB regulates dorso-ventral patterning genes in embryos.

    Joseph Heimiller

    Full Text Available The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus plays an important role during embryogenesis. A loss of function mutation, heph(03429, results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. We have used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5' and 3' splice site switching, alternative 5' ends, and mRNA level changes (up and down regulation. These findings are independently supported by reverse-transcription-polymerase chain reaction (RT-PCR analysis and in situ hybridization. We show that a group of genes, such as Zerknüllt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral patterning and/or dorsal closure processes. Thus, loss of dmPTB function results in specific misregulated transcripts, including those that provide the missing link between the loss of dmPTB function and observed developmental defects in embryogenesis. This study provides the first comprehensive repertoire of genes affected in vivo in the heph mutant in Drosophila and offers insight into the role of dmPTB during embryonic development.

  15. The yeast CDP1 gene encodes a triple-helical DNA-binding protein

    Musso, Marco; Bianchi-Scarrà, Giovanna; Van Dyke, Michael W.


    The formation of triple-helical DNA has been implicated in several cellular processes, including transcription, replication and recombination. While there is no direct evidence for triplexes in vivo, cellular proteins that specifically recognize triplex DNA have been described. Using a purine-motif triplex probe and southwestern library screening, we isolated five independent clones expressing the same C-terminal 210 amino acids of the Saccharomyces cerevisiae protein Cdp1p fused with β-galac...

  16. Mutations in the selenocysteine insertion sequence–binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

    Schoenmakers, Erik; Agostini, Maura; Mitchell, Catherine; Schoenmakers, Nadia; Papp, Laura; Rajanayagam, Odelia; Padidela, Raja; Ceron-Gutierrez, Lourdes; Doffinger, Rainer; Prevosto, Claudia; Luan, Jian’an; Montano, Sergio; Lu, Jun; Castanet, Mireille; Clemons, Nick; Groeneveld, Matthijs; Castets, Perrine; Karbaschi, Mahsa; Aitken, Sri; Dixon, Adrian; Williams, Jane; Campi, Irene; Blount, Margaret; Burton, Hannah; Muntoni, Francesco; O’Donovan, Dominic; Dean, Andrew; Warren, Anne; Brierley, Charlotte; Baguley, David; Guicheney, Pascale; Fitzgerald, Rebecca; Coles, Alasdair; Gaston, Hill; Todd, Pamela; Holmgren, Arne; Khanna, Kum Kum; Cooke, Marcus; Semple, Robert; Halsall, David; Wareham, Nicholas; Schwabe, John; Grasso, Lucia; Beck-Peccoz, Paolo; Ogunko, Arthur; Dattani, Mehul; Gurnell, Mark; Chatterjee, Krishna


    Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence–binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a complex phenotype. Azoospermia, with failure of the latter stages of spermatogenesis, was associated with a lack of testis-enriched selenoproteins. An axial muscular dystrophy was also present, with features similar to myopathies caused by mutations in selenoprotein N (SEPN1). Cutaneous deficiencies of antioxidant selenoenzymes, increased cellular ROS, and susceptibility to ultraviolet radiation–induced oxidative damage may mediate the observed photosensitivity. Reduced levels of selenoproteins in peripheral blood cells were associated with impaired T lymphocyte proliferation, abnormal mononuclear cell cytokine secretion, and telomere shortening. Paradoxically, raised ROS in affected subjects was associated with enhanced systemic and cellular insulin sensitivity, similar to findings in mice lacking the antioxidant selenoenzyme glutathione peroxidase 1 (GPx1). Thus, mutation of SECISBP2 is associated with a multisystem disorder with defective biosynthesis of many selenoproteins, highlighting their role in diverse biological processes. PMID:21084748

  17. Differential stimulation by CCAAT/enhancer-binding protein alpha isoforms of the estrogen-activated promoter of the very-low-density apolipoprotein II gene

    Calkhoven, CF; Snippe, L; Ab, G


    The transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBP alpha and C/EBP beta) are highly expressed in liver and are believed to function in maintaining the differentiated state of the hepatocytes, C/EBP alpha appears to be a critical regulator of genes involved in metabolic p

  18. Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation

    Kalscheuer, VM; Freude, K; Musante, L; Jensen, LR; Yntema, HG; Gecz, J; Sefiani, A; Hoffmann, K; Moser, B; Haas, S; Gurok, U; Haesler, S; Aranda, B; Nshedjan, A; Tzschach, A; Hartmann, N; Roloff, TC; Shoichet, S; Hagens, O; Tao, J; van Bokhoven, H; Turner, G; Chelly, J; Moraine, C; Fryns, JP; Nuber, U; Hoeltzenbein, M; Scharff, C; Scherthan, H; Lenzner, S; Hamel, BCJ; Schweiger, S; Ropers, HH


    We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously

  19. Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation

    Kalscheuer, Vera M; Freude, Kristine; Musante, Luciana;


    We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has...

  20. Fatty-acid binding protein 4 gene variants and childhood obesity: potential implications for insulin sensitivity and CRP levels

    Bhattacharjee Rakesh


    Full Text Available Abstract Introduction Obesity increases the risk for insulin resistance and metabolic syndrome in both adults and children. FABP4 is a member of the intracellular lipid-binding protein family that is predominantly expressed in adipose tissue, and plays an important role in maintaining glucose and lipid homeostasis. The purpose of this study was to measure FABP4 plasma levels, assess FABP4 allelic variants, and explore potential associations with fasting glucose and insulin levels in young school-age children with and without obesity. Methods A total of 309 consecutive children ages 5-7 years were recruited. Children were divided based on BMI z score into Obese (OB; BMI z score >1.65 and non-obese (NOB. Fasting plasma glucose, lipids, insulin, hsCRP, and FABP4 levels were measured. HOMA was used as correlate of insulin sensitivity. Four SNPs of the human FABP4 gene (rs1051231, rs2303519, rs16909233 and rs1054135, corresponding to several critical regions of the encoding FABP4 gene sequence were genotyped. Results Compared to NOB, circulating FABP4 levels were increased in OB, as were LDL, hsCRP and HOMA. FABP4 levels correlated with BMI, and also contributed to the variance of HOMA and hsCRP, but not serum lipids. The frequency of rs1054135 allelic variant was increased in OB, and was associated with increased FABP4 levels, while the presence of rs16909233 variant allele, although similar in OB and NOB, was associated with increased HOMA values. Conclusions Childhood obesity is associated with higher FABP4 levels that may promote cardiometabolic risk. The presence of selective SNPs in the FABP4 gene may account for increased risk for insulin resistance or systemic inflammation in the context of obesity.

  1. Calmodulin Binding Proteins and Alzheimer's Disease.

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A


    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  2. Molecular cloning and expression of the carbohydrate response element binding protein gene and related genes involved in hepatic lipogenesis during post-hatch development of broiler chickens

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Responding to changes in the level of glucose (ChREBP) and insulin (SREBP-1c), these two transcripti...

  3. The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway.

    Jing, Yu-Pu; Wang, Di; Han, Xiao-Lin; Dong, Du-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan


    Animal steroid hormones regulate gene transcription through genomic pathways by binding to nuclear receptors. These steroid hormones also rapidly increase intracellular calcium and cyclic adenosine monophosphate (cAMP) levels and activate the protein kinase C (PKC) and protein kinase A (PKA) nongenomic pathways. However, the function and mechanism of the nongenomic pathways of the steroid hormones are unclear, and the relationship between the PKC and PKA pathways is also unclear. We propose that the steroid hormone 20-hydroxyecdysone (20E) activates the PKA pathway to enhance 20E-induced gene transcription in the lepidopteran insect Helicoverpa armigera The expression of the catalytic subunit 1 of PKA (PKAC1) increased during metamorphosis, and PKAC1 knockdown blocked pupation and repressed 20E-responsive gene expression. 20E regulated PKAC1 phosphorylation at threonine 200 and nuclear translocation through an ecdysone-responsive G-protein-coupled receptor 2. PKAC1 induced cAMP response element-binding protein (CREB) phosphorylation at serine 143, which bound to the cAMP response element on DNA to enhance 20E-responsive gene transcription. Through ecdysone-responsive G-protein-coupled receptor 2, 20E increased cAMP levels, which induced CREB PKA phosphorylation and 20E-responsive gene expression. This study demonstrates that the PKA/CREB pathway tightly and critically regulates 20E-induced gene transcription as well as its relationship with the 20E-induced PKC pathway. PMID:27129227

  4. Dystrobrevin-binding protein 1 gene (DTNBP1) variants associated with cerebrospinal fluid homovanillic acid and 5-hydroxyindoleacetic acid concentrations in healthy volunteers

    Andreou, Dimitrios; Saetre, Peter; Kähler, Anna K;


    The dystrobrevin binding protein-1 (DTNBP1) gene encodes dysbindin-1, a protein involved in neurodevelopmental and neurochemical processes related mainly to the monoamine dopamine. We investigated possible associations between eleven DTNBP1 polymorphisms and cerebrospinal fluid (CSF) concentrations...... of the major dopamine metabolite homovanillic acid (HVA), the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), and the major noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) in healthy human subjects (n=132). Two polymorphisms, rs2619538 and rs760666, were nominally...

  5. Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein.

    Swan, D G; Cortes, J; Hale, R S; Leadlay, P F


    The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ...

  6. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

    Rustgi, A.K.; Dyson, N.; Bernards, R.A.


    The proteins encoded by the myc gene family are involved is the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/

  7. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

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

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


    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,...

  9. Improving the MVA vaccine potential by deleting the viral gene coding for the IL-18 binding protein.

    Juliana Falivene

    Full Text Available BACKGROUND: Modified Vaccinia Ankara (MVA is an attenuated strain of Vaccinia virus (VACV currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR induced by an IL-18 binding protein gene (C12L deleted vector (MVAΔC12L. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt, then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively. Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8(+ and CD4(+ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8(+ T-cells (CD107a/b(+ during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal. Potentiation of MVA's CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.

  10. Developmental pathways from child maltreatment to adolescent marijuana dependence: Examining moderation by FK506 binding protein 5 gene (FKBP5).

    Handley, Elizabeth D; Rogosch, Fred A; Cicchetti, Dante


    The current study examined the prospective association between child maltreatment and the development of substance use disorder in adolescence with the aim of investigating pathways underlying this relation, as well as genetic moderation of these developmental mechanisms. Specifically, we tested whether youth who experienced maltreatment prior to age 8 were at risk for the development of marijuana dependence in adolescence by way of a childhood externalizing pathway and a childhood internalizing pathway. Moreover, we tested whether variation in FK506 binding protein 5 gene (FKBP5) CATT haplotype moderated these pathways. The participants were 326 children (n =179 maltreated; n = 147 nonmaltreated) assessed across two waves of data collection (childhood: ages 7-9 and adolescence: ages 15-18). Results indicated that higher levels of child externalizing symptoms significantly mediated the effect of child maltreatment on adolescent marijuana dependence symptoms for individuals with one or two copies of the FKBP5 CATT haplotype only. We did not find support for an internalizing pathway from child maltreatment to adolescent marijuana dependence, nor did we find evidence of moderation of the internalizing pathway by FKBP5 haplotype variation. Findings extend previous research by demonstrating that whether a maltreated child will traverse an externalizing pathway toward substance use disorder in adolescence is dependent on FKBP5 genetic variation. PMID:26535939

  11. Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

    Huang, B L; Zhang, X K; Li, Y Y; Li, D Y; Ma, M Y; Cai, D T; Wu, W H; Huang, B Q


    Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops. PMID:27525923

  12. Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli

    Yang, Tsung-Yeh; Sung, Yun-Min; Lei, Guang-Sheng; Romeo, Tony; Chak, Kin-Fu


    Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ∼5-fold increase of the lysis protein. Gel mobility shift assays established that both the single...

  13. Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein.

    Krauel, V; Heller, K J


    Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5. In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene. In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome. This analysis also showed that oad has little or no homology with h...

  14. Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells.

    Karsten Jürchott

    Full Text Available Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1 by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

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

    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


    Background: A multiprotein complex designated as lipolysome degrades intracellular triglycerides and contains proteins such as adipose triglyceride lipase (Atgl) and its co-activator Cgi-58. Results: Cgi-58 interacts with fatty acid-binding proteins (Fabps), which impact Atgl-mediated lipolysis and lipid signaling. Conclusion: Fabps modulate Atgl-mediated TG hydrolysis and link lipolysis with intracellular lipid ligand shuttling. Significance: Novel mechanistic insights into the regulation of...

  16. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene

  17. Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family from Citrus and the effect of fruit load on their expression

    Shalom, Liron; Shlizerman, Lyudmila; Zur, Naftali; Doron-Faigenboim, Adi; Blumwald, Eduardo; Sadka, Avi


    We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees), known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting),...

  18. Mutational analysis of a predicted zinc-binding motif in the 26-kilodalton protein encoded by the vaccinia virus A2L gene: correlation of zinc binding with late transcriptional transactivation activity.

    Keck, J G; Feigenbaum, F; B. Moss


    Transient transfection assays indicated that A2L is one of three vaccinia virus intermediate genes that are required for the transcriptional transactivation of viral late genes. We have expressed the A2L open reading frame in Escherichia coli and shown by blotting experiments that the 26-kDa protein binds zinc, a property predicted by the presence of a CX2CX13CX2C zinc finger motif. The specificity for zinc binding was demonstrated by competition with other metals. The role of the sequence mo...

  19. Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

    Shevtsov, M. B.; Streeter, S. D.; Thresh, S.-J.; Swiderska, A.; McGeehan, J. E.; Kneale, G. G., E-mail: [University of Portsmouth, Portsmouth PO1 2DY (United Kingdom)


    The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

  20. Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

    The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal

  1. Molecular cloning, sequencing, and mapping of EGR2, a human early growth response gene encoding a protein with zinc-binding finger structure

    Early growth response gene-1 (Egr-1) is a mouse gene displaying fos-like induction kinetics in diverse cell types following mitogenic stimulation. Egr-1 encodes a protein with zinc-binding finger structure. Zinc fingers are a protein structural motif that serve as DNA-binding domains in several transcriptional regulatory proteins. Using low-stringency hybridization with an Egr-1 cDNA probe, the authors identified a distinct human cDNA (designated EGR2 for early growth response gene-2), which is coregulated with EGR1 by fibroblast and lymphocyte mitogens; however, several stimuli that induce Egr-1 mRNA in PC12 (rat pheochromocytoma) cells do not induce Egr-2 mRNA. The cDNA sequence predicts a protein of 406 amino acids, including three tandem zinc fingers of the Cys2-His2 class. Strikingly, the deduced amino acid sequences of human EGR2 and mouse Egr-1 are 92% identical in the zinc finger region but show no similarity elsewhere. EGR2 maps to human chromosome 10 at bands q21-22. Structure-function analysis of EGR2 and EGR1 proteins should provide insight into the mechanisms linking signal transduction and transcriptional regulation of gene expression

  2. Comparison of Lipopolysaccharide binding protein and C-reactive protein gene expression levels in a Rat model of induced Atherosclerosis

    Kholoud S. Ramadan*1; Rasha E. Hassan1: Abd El Rahman B. Abd El Ghaffar1 and Asmaa A. Deghedy1


    Full Text Available Atherosclerosis is an inflammatory disease characterized by the accumulation of lipids within arterial walls that eventually go on to form plaques, which can cause narrowing, hardening, and/or complete blockage of arteries. This study was designed to examine the cholesterol feeding induction of cardiovascular diseases exemplified by atherosclerosis in rat and induction of CRP, LBP, SAP and P4H on the transcriptional activity of the inflammation / related gene expression by a semi-quantitative RT-PCR in liver and heart tissues, and make comparison between CRP and LBP as biomarker for atherosclerosis. Material & Methods: Experimental Rats were fed with cholesterol diet (2.5% pure (wt/wt cholesterol, 1% cholic acid and 5% oil and sacrificed after 18 weeks of feeding. Results: Histopathological examination for heart showed fatty cells deposition in atherogenic rats. Expression pattern of CRP, LBP, SAP & P4H genes were investigated, in liver and heart, these genes were highly expressed while some of them showed no expression pattern in heart tissues.

  3. Cloning and expression of retinoblastoma-binding protein 4 gene in embryo diapause termination and in response to salinity stress from brine shrimp Artemia sinica.

    Wang, Xiaolu; Yao, Feng; Liang, Xiaoyu; Zhu, Xiaolin; Zheng, Ren; Jia, Baolin; Hou, Lin; Zou, Xiangyang


    Retinoblastoma binding protein 4 (RBBP4) is a nuclear protein with four WD-repeat sequences and thus belongs to a highly conserved subfamily of proteins with such domains. This retinoblastoma-binding protein plays an important role in nucleosome assembly and histone modification, which influences gene transcription and regulates cell cycle and proliferation. Artemia sinica (brine shrimp) undergoes an unusual diapause process under stress conditions of high salinity and low temperature. However, the role of RBBP4 in diapause termination of embryo development in A. sinica remains unknown. Here, the full-length cDNA of the As-rbbp4 gene was obtained from A. sinica and found to contain 1411 nucleotides, including a 1281 bp open reading frame (ORF), 63 bp 5'-untranslated region (UTR) and a 67-bp 3'-UTR, which encodes a 427 amino acid (48 kDa) protein. Bioinformatic analysis indicated As-RBBP4 to be mainly located in the nucleus, with a theoretical isoelectric point of 4.79. Protein sequence domain analysis showed that As-RBBP4 is a conserved protein, especially in the WD40 domain. No specificity in expression of this gene was observed in tissues or organs by in situ hybridization. Real-time quantitative PCR and Western blot analyses of As-RBBP4 gene and protein expression, respectively, showed notably high levels at 10 h and a subsequent downward trend. Obvious trends in upregulation of As-RBBP4 were observed under conditions of low temperature and high salinity stress. As-E2F1 and As-CyclinE also presented similar trends as that of As-RBBP4 in Western blots. Analysis of the RBBP4 expression in early embryonic development of A. sinica indicated that this protein plays an important role in diapause termination and cell cycle regulation. PMID:27267406

  4. Transcriptional modulation of hepatic lipoprotein assembly and secretion : coordinate regulation of the liver-fatty acid binding protein and microsomal triglyceride transfer protein genes

    Spann, Nathanael J.


    Hepatic production of apolipoprotein (apo) B-containing lipoproteins provides a means to transport essential lipids and fat-soluble nutrients to peripheral tissues for utilization and storage. Liver-fatty acid binding protein (L-FABP) and microsomal triglyceride transfer protein (MTP) bind fatty acids and glycerolipids, respectively and facilitate their transfer into the VLDL assembly and secretion pathway. Sequence analysis reveals that the proximal promoter regions of L-FABP and MTP contain...

  5. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Jingna Si


    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  6. A KH Domain-Containing Putative RNA-Binding Protein Is Critical for Heat Stress-Responsive Gene Regulation and Thermotolerance in Arabidopsis

    Qingmei Guan; Changlong Wen; Haitao Zeng; Jianhua Zhu


    Heat stress is a severe environmental factor that significantly reduces plant growth and delays development.Heat stress factors (HSFs) are a class of transcription factors that are synthesized rapidly in response to elevations in temperature and are responsible for the transcription of many heat stress-responsive genes including those encoding heat shock proteins (HSPs).There are 21 HSFs in Arabidopsis,and recent studies have established that the HSFA1 family members are master regulators for the remaining HSFs.However,very little is known about upstream molecular factors that control the expression of HSFA1 genes and other HSF genes under heat stress.Through a forward genetic analysis,we identified RCF3,a K homology (KH) domain-containing nuclear-localized putative RNA-binding protein.RCF3 is a negative regulator of most HSFs,including HSFAla,HSFAlb,and HSFAld.In contrast,RCF3 positively controls the expression of HSFAle,HSFA3,HSFA9,HSFB3,and DREB2C.Consistently with the overall increased accumulation of heat-responsive genes,the rcf3 mutant plants are more tolerant than the wild-type to heat stress.Together,our results suggest that a KH domain-containing putative RNA-binding protein RCF3 is an important upstream regulator for heat stress-responsive gene expression and thermotolerance in Arabidopsis.

  7. The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

    Springer, Mark S.; Burk, Angela; Kavanagh, John R.; Waddell, Victor G.; Stanhope, Michael J.


    The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Pa...

  8. Molecular Characterization and Sequencing of a Gene Encoding Mannose Binding Protein in an Iranian Isolate of Acanthamoeba castellanii as a Major Agent of Acanthamoeba Keratitis

    SH Farnia


    Full Text Available Background: Acanthamoeba castellanii is the important cause of amoebic keratitis in Iran. The key molecule in pathogenesis of Acanthamoeba keratitis is Mannose Binding Protein (MBP led to adhesion of amoeba to corneal epithelium. Subsequent to adhesion other cytopathic effects occur. The goal of this study was to identify the molecular characterization of a gene encoding MBP in an Iranian isolate of A.castellanii in order to pave the way for further investigations such as new therapeutic advances or immunization. Methods: A.castellanii was cultured on non nutrient agar. Extraction of DNA was performed by phenol-chloroform method. After designing a pair of primer for the gene encoding MBP, PCR analysis was performed. Finally, the PCR product has been sequenced and the result submitted to the gene data banks. Results: An MBP gene of 1081 nucleotides was sequenced. This fragment contained three introns and encodes a protein with 194 amino acids. Homology search by Blast program showed a significant homology with the MBP gene in gene data banks (96%. Besides, the identity of amino acids with the other MBPs in gene data banks was about 86%. Conclusion: We isolated and sequenced a gene fragment encoding MBP in an Iranian isolate of A.castellanii. Molecular characterization of this important gene is the first step in pursuing researches such as developing better therapeutic agents, immunization of population at risk or even developing a diagnostic tool by PCR techniques.

  9. Interleukin 6 induces a liver-specific nuclear protein that binds to the promoter of acute-phase genes.

    Poli, V.; Cortese, R


    Interleukin 6 (IL-6) is responsible for a variety of biological effects related to the activation of defenses against infection or inflammation, including the immune response and the acute-phase reaction. Its mechanism of action is unknown. It has recently been shown to induce transcription of several genes encoding acute-phase proteins. Here we describe the identification of an IL-6 responsive element (IL-6RE) present in the promoter of the human hemopexin gene. This element is necessary and...


    Elena Irina Moater; Cristiana Radulescu; Ionica Ionita


    The interactions between surfactants and proteins shows some similarities with interactions between surfactants and polymers, but the hydrophobic amphoteric nature of proteins and their secondary and tertiary structure components make them different from conventional polymer systems. Many studies from the past about surfactant - proteins bonding used the dialysis techniques. Other techniques used to determine the binding isotherm, included ultrafiltration, ultracentrifugation, potentiometry, ...

  11. A possible role of DNA methylation in functional divergence of a fast evolving duplicate gene encoding odorant binding protein 11 in the honeybee.

    Kucharski, R; Maleszka, J; Maleszka, R


    Although gene duplication is seen as the main path to evolution of new functions, molecular mechanisms by which selection favours the gain versus loss of newly duplicated genes and minimizes the fixation of pseudo-genes are not well understood. Here, we investigate in detail a duplicate honeybee gene obp11 belonging to a fast evolving insect gene family encoding odorant binding proteins (OBPs). We report that obp11 is expressed only in female bees in rare antennal sensilla basiconica in contrast to its tandem partner obp10 that is expressed in the brain in both females and males (drones). Unlike all other obp genes in the honeybee, obp11 is methylated suggesting that functional diversification of obp11 and obp10 may have been driven by an epigenetic mechanism. We also show that increased methylation in drones near one donor splice site that correlates with higher abundance of a transcript variant encoding a truncated OBP11 protein is one way of controlling its contrasting expression. Our data suggest that like in mammals and plants, DNA methylation in insects may contribute to functional diversification of proteins produced from duplicated genes, in particular to their subfunctionalization by generating complementary patterns of expression. PMID:27358363

  12. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Stefano Luisa


    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  13. Protein Dynamics in an RNA Binding Protein

    Hall, Kathleen


    Using ^15N NMR relaxation measurements, analyzed with the Lipari-Szabo formalism, we have found that the human U1A RNA binding protein has ps-ns motions in those loops that make contact with RNA. Specific mutations can alter the extent and pattern of motions, and those proteins inevitably lose RNA binding affinity. Proteins with enhanced mobility of loops and termini presumably lose affinity due to increased conformational sampling by those parts of the protein that interact directly with RNA. There is an entropic penalty associated with locking down those elements upon RNA binding, in addition to a loss of binding efficiency caused by the increased number of conformations adopted by the protein. However, in addition to local conformational heterogeneity, analysis of molecular dynamics trajectories by Reorientational Eigenmode Dynamics reveals that loops of the wild type protein undergo correlated motions that link distal sites across the binding surface. Mutations that disrupt correlated motions result in weaker RNA binding, implying that there is a network of interactions across the surface of the protein. (KBH was a Postdoctoral Fellow with Al Redfield from 1985-1990). This work was supported by the NIH (to KBH) and NSF (SAS).


    McIntosh, Avery L.; Atshaves, Barbara P.; Hostetler, Heather A.; Huang, Huan; Davis, Jason; Lyuksyutova, Olga I.; Landrock, Danilo; Kier, Ann B.; Schroeder, Friedhelm


    The effect of liver type fatty acid binding protein (L-FABP) gene ablation on the uptake and distribution of long chain fatty acids (LCFA) to the nucleus by real-time laser scanning confocal imaging and peroxisome proliferator activated receptor-α (PPARα) activity was examined in cultured primary hepatocytes from livers wild-type L-FABP+/+ and gene ablated L-FABP−/− mice. Cultured primary hepatocytes from livers of L-FABP−/− mice exhibited: (i) reduced oxidation of palmitic acid, a common die...

  15. Enhanced expression of lmb gene encoding laminin-binding protein in Streptococcus agalactiae strains harboring IS1548 in scpB-lmb intergenic region.

    Rim Al Safadi

    Full Text Available Group B streptococcus (GBS is the main cause of neonatal sepsis and meningitis. Bacterial surface proteins play a major role in GBS binding to and invasion of different host surfaces. The scpB and lmb genes, coding for fibronectin-binding and laminin-binding surface proteins, are present in almost all human GBS isolates. The scpB-lmb intergenic region is a hot spot for integration of two mobile genetic elements (MGEs: the insertion element IS1548 or the group II intron GBSi1. We studied the structure of scpB-lmb intergenic region in 111 GBS isolates belonging to the intraspecies major clonal complexes (CCs. IS1548 was mostly found (72.2% in CC19 serotype III strains recovered more specifically (92.3% from neonatal meningitis. GBSi1 was principally found (70.6% in CC17 strains, mostly (94.4% of serotype III, but also (15.7% in CC19 strains, mostly (87.5% of serotype II. No MGE was found in most strains of the other CCs (76.0%, notably CC23, CC10 and CC1. Twenty-six strains representing these three genetic configurations were selected to investigate the transcription and expression levels of scpB and lmb genes. Quantitative RT-PCR showed that lmb transcripts were 5.0- to 9.6-fold higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001. Accordingly, the binding ability to laminin was 3.8- to 6.6-fold higher in these strains (P< or =0.001. Moreover, Lmb amount expressed on the cell surface was 2.4- to 2.7-fold greater in these strains (P<0.001. By contrast, scpB transcript levels and fibronectin binding ability were similar in the three groups of strains. Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold, the binding ability to laminin (6.2-fold, and the expression of Lmb protein (5.0-fold. These data highlight the importance of MGEs in bacterial virulence and demonstrate the up-regulation of lmb gene by IS

  16. Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP

    Silva-Jr FP


    Full Text Available The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

  17. Overlapping protein-binding sites within a negative regulatory element modulate the brain-preferential expression of the human HPRT gene

    Rincon-Limas, D.E.; Amaya-Manzanares, E.; Nino-Rosales, M.L. [Baylor College of Medicine, Houston, TX (United States)] [and others


    The hypoxanthine phosphoribosyltransferase (HPRT) gene, whose deficiency in humans causes the Lesch-Nyhan syndrome, is constitutively expressed at low levels in all tissues but at higher levels in the brain, the significance and mechanism of which is unknown. Towards dissecting this molecular mechanism, we have previously identified a 182 bp element (hHPRT-NE) within the 5{prime}-flanking region of the human HPRT gene which is involved not only in conferring neuronal specificity but also in repressing gene expression in non-neuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation, as demonstrated by RT-PCR and RNAase protection assays. We also mapped the binding sites for both complexes to a 60 bp region which, when tested by transient transfections in cultured fibroblasts, functioned as a repressor element. Methylation interference footprinting revealed a minimal unique DNA motif as the binding site for nuclear proteins from both neuronal and non-neuronal sources. Moreover, UV-crosslinking experiments showed that both complexes are formed by the association of several distinct proteins. Strikingly, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the association of these two complexes. These data suggest that differential formation of DNA-protein complexes at this regulatory domain could be a major determinant in the brain-preferential expression of the human HPRT gene.

  18. Grafting of protein-protein binding sites


    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  19. Particularity and universality of a putative Gram-negative bacteria-binding protein (GNBP) gene from amphioxus (Branchiostoma belcheri): insights into the function and evolution of GNBP.

    Jin, Ping; Zhou, Lu; Song, Xiaojun; Qian, Jinjun; Chen, Liming; Ma, Fei


    Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition proteins (PRPs), which can initiate host defense in response to pathogen surface molecules. The roles of GNBP in innate immunity of arthropods and molluscs have recently been reported. However, the GNBP gene has not been characterized in the species of higher evolutionary status yet. In this study, we identified and characterized an amphioxus GNBP gene (designated as AmphiGNBP). First, we identified and cloned the AmphiGNBP and found that the AmphiGNBP encodes a putative protein with 558 amino acids, which contains a conserved β-1, 3-glucan recognizing and binding domain. Second, we found that the AmphiGNBP encodes two extra WSC (cell Wall integrity and Stress response Component) domains, which are unique in AmphiGNBP protein. The two WSC domains of AmphiGNBP protein coupled with the expansion of amphioxus immunity repertoire might undergo intensive domain shuffling during the age of the Cambrian explosion. Finally, we found that the AmphiGNBP was mainly expressed in immune tissues, such as hepatic cecum and intestine, and the expression of AmphiGNBP was affected after LPS stimulation. In conclusion, our findings disclose the particularity and universality of AmphiGNBP and provide profound insights into the function and evolution of GNBP. PMID:22986589

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

    Xianglan Zeng


    Full Text Available 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.

  1. The relationship of sterol regulatory element-binding protein cleavage-activation protein and apolipoprotein E gene polymorphisms with metabolic changes during weight reduction.

    Nieminen, Tuomo; Matinheikki, Jussi; Nenonen, Arja; Kukkonen-Harjula, Katriina; Lindi, Virpi; Hämelahti, Päivi; Laaksonen, Reijo; Fan, Yue-Mei; Kähönen, Mika; Fogelholm, Mikael; Lehtimäki, Terho


    Sterol regulatory element-binding protein cleavage-activating protein (SCAP) and apolipoprotein E (apo E) regulate cellular and plasma lipid metabolism. Therefore, variations in the corresponding genes might influence weight reduction and obesity-associated metabolic changes. We investigated the relationships of SCAP (Ile796Val) and apo E polymorphisms on metabolic changes during weight reduction by using a 12-week very low-energy diet. Body composition, serum lipids, plasma glucose, and insulin were assessed in 78 healthy premenopausal women (initial body mass index, 34 +/- 4 kg/m(2); age, 40 +/- 4 years) before and after the intervention. The SCAP genotype groups did not differ in the responses of any parameters measured during weight reduction. Apo E did not differentiate the weight loss, but the changes in total and low-density lipoprotein cholesterol for the genotype groups apo E epsilon2/3, epsilon3/3, as well as epsilon3/4 and epsilon4/4 combined were -0.94 +/- 0.56 and -0.59 +/- 0.32, -0.71 +/- 0.49 and -0.49 +/- 0.45, and -0.55 +/- 0.47 and -0.37 +/- 0.39 mmol/L, respectively (P < .05 for both). In conclusion, neither the SCAP Ile796Val nor the apo E polymorphism was associated with weight loss in obese premenopausal women. However, the apo E-but not SCAP genotype-seems to be one of the modifying factors for serum cholesterol concentrations during very low-energy diet in obese premenopausal women. PMID:17570245

  2. Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene.

    Toshiyuki Hamada

    Full Text Available BACKGROUND: In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element and AIE (age-related increase element as a stem-loop forming RNA, play critical roles in producing specific age-related expression patterns of genes. PRINCIPAL FINDING: We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3 as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX as well as mouse factor IX (mFIX genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser(359, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser(359. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable. CONCLUSIONS: We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

  3. Comparison of cooperative and isolated site binding of T4 gene 32 protein to ssDNA by 1H NMR

    Deuteriation of all aromatic protons of gene 32 protein (g32P) from phage T4, followed by selective introduction of specific protons, has allowed the precise identification of the number and magnitude of the chemical shift changes induced in the aromatic protons when g32P binds noncooperatively or cooperatively to nucleotides. Signals from five Tyr residues are shifted by binding of g32P to d(pA)8 or d(pA)40-60; however, the change from noncooperative, d(pA)8, to cooperative, d(pA)40-60, binding causes significant increases in the magnitudes of the shifts for only two of these Tyr signals. These two Tyr residues may interact directly with the nucleotide bases, while the shifts associated with the other three Tyr may be due to conformational changes in g32P upon ssDNA binding. Similar conclusions can be drawn for two of the six Phe residues whose protons undergo shifts upon nucleotide binding. Observation of selected proton signals allows for the first time detection by 1H NMR of changes in the proton signals from two Trp residues upon nucleotide binding. The side chains of two Tyr, one or two Phe, and one Trp are probably directly involved in nucleotide base-protein interactions. As assayed by the signals from the H2 and H8 protons of adenine, the bases of a bound nucleotide are undergoing a fast chemical exchange in the noncooperative mode of binding, but shift to slow exchange upon assuming the cooperative mode of ssDNA interaction. When bound to a polynucleotide, the A domain of g32P (residues 254-301) becomes more mobile, as reflected in sharpening of the 1H NMR signals from the A domain

  4. Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta

    Huckert, Mathilde; Stoetzel, Corinne; Morkmued, Supawich; Laugel-Haushalter, Virginie; Geoffroy, Véronique; Muller, Jean; Clauss, François; Prasad, Megana K.; Obry, Frédéric; Raymond, Jean Louis; Switala, Marzena; Alembik, Yves; Soskin, Sylvie; Mathieu, Eric; Hemmerlé, Joseph; Weickert, Jean-Luc; Dabovic, Branka Brukner; Rifkin, Daniel B.; Dheedene, Annelies; Boudin, Eveline; Caluseriu, Oana; Cholette, Marie-Claude; Mcleod, Ross; Antequera, Reynaldo; Gellé, Marie-Paule; Coeuriot, Jean-Louis; Jacquelin, Louis-Frédéric; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Van Hul, Wim; Bertola, Debora; Dollé, Pascal; Verloes, Alain; Mortier, Geert; Dollfus, Hélène; Bloch-Zupan, Agnès


    Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder. PMID:25669657

  5. Identification of light-harvesting chlorophyll a/b-binding protein genes of Zostera marina L. and their expression under different environmental conditions

    Kong, Fanna; Zhou, Yang; Sun, Peipei; Cao, Min; Li, Hong; Mao, Yunxiang


    Photosynthesis includes the collection of light and the transfer of solar energy using light-harvesting chlorophyll a/b-binding (LHC) proteins. In high plants, the LHC gene family includes LHCA and LHCB sub-families, which encode proteins constituting the light-harvesting complex of photosystems I and II. Zostera marina L. is a monocotyledonous angiosperm and inhabits submerged marine environments rather than land environments. We characterized the Lhca and Lhcb gene families of Z. marina from the expressed sequence tags (EST) database. In total, 13 unigenes were annotated as ZmLhc, 6 in Lhca family and 7 in ZmLhcb family. ZmLHCA and ZmLHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll. The average similarity among mature ZmLHCA and ZmLHCB was 48.91% and 48.66%, respectively, which indicated a high degree of divergence within ZmLHChc gene family. The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relationship were similar to those reported in other high plants, suggesting that the Lhc genes were highly conservative and the classification of ZmLhc genes was consistent with the evolutionary position of Z. marina. Real-time reverse transcription (RT) PCR analysis showed that different members of ZmLhca and ZmLhcb responded to a stress in different expression patterns. Salinity, temperature, light intensity and light quality may affect the expression of most ZmLhca and ZmLhcb genes. Inorganic carbon concentration and acidity had no obvious effect on ZmLhca and ZmLhcb gene expression, except for ZmLhca6.

  6. C-terminal binding protein (CtBP activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

    Taichi Q Itoh

    Full Text Available In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes". Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP, a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per, vrille (vri, and PAR domain protein 1ε (Pdp1ε. Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

  7. Multiplex ligation-dependent probe amplification detection of an unknown large deletion of the CREB-binding protein gene in a patient with Rubinstein-Taybi syndrome.

    Calì, F; Failla, P; Chiavetta, V; Ragalmuto, A; Ruggeri, G; Schinocca, P; Schepis, C; Romano, V; Romano, C


    Rubinstein-Taybi syndrome is a rare autosomal dominant congenital disorder characterized by postnatal growth retardation, psychomotor developmental delay, skeletal anomalies, peculiar facial morphology, and tumorigenesis. Mutations in the gene encoding the cAMP response element-binding protein (CREB, also known as CREBBP or CBP) on chromosome 16p13.3 have been identified. In addition, some patients with low intelligence quotients and autistic features bear large deletions. Based on these observations, we used multiplex ligation-dependent probe amplification to search for large deletions affecting the CREBBP gene in a Rubinstein-Taybi syndrome patient. We identified a novel heterozygote deletion removing five exons (exons 17-21), encoding the histone acetyltransferase domain. We propose the use of multiplex ligation-dependent probe amplification as a fast, accurate and cheap test for detecting large deletions in the CREBBP gene in the sub-group of Rubinstein-Taybi syndrome patients with low intelligence quotients and autistic features. PMID:23315884

  8. Analysis of the DNA-Binding Profile and Function of TALE Homeoproteins Reveals Their Specialization and Specific Interactions with Hox Genes/Proteins

    Dmitry Penkov


    Full Text Available The interactions of Meis, Prep, and Pbx1 TALE homeoproteins with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA-binding sequences, Prep associating mostly with promoters and housekeeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless coregulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. During evolution, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination.

  9. The gene therapy of collagen-induced arthritis in rats by intramuscular administration of the plasmid encoding TNF-binding domain of variola virus CrmB protein.

    Shchelkunov, S N; Taranov, O S; Tregubchak, T V; Maksyutov, R A; Silkov, A N; Nesterov, A E; Sennikov, S V


    Wistar rats with collagen-induced arthritis were intramuscularly injected with the recombinant plasmid pcDNA/sTNF-BD encoding the sequence of the TNF-binding protein domain of variola virus CrmB protein (VARV sTNF-BD) or the pcDNA3.1 vector. Quantitative analysis showed that the histopathological changes in the hind-limb joints of rats were most severe in the animals injected with pcDNA3.1 and much less severe in the group of rats injected with pcDNA/sTNF-BD, which indicates that gene therapy of rheumatoid arthritis is promising in the case of local administration of plasmids governing the synthesis of VARV immunomodulatory proteins. PMID:27599513

  10. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

    Yang, T.; Poovaiah, B. W.


    35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

  11. The TamA protein fused to a DNA-binding domain can recruit AreA, the major nitrogen regulatory protein, to activate gene expression in Aspergillus nidulans.

    Small, A J; Hynes, M J; Davis, M A


    The areA gene of Aspergillus nidulans encodes a GATA zinc finger transcription factor that activates the expression of a large number of genes subject to nitrogen metabolite repression. The amount and activity of the AreA protein under different nitrogen conditions is modulated by transcriptional, post-transcriptional, and post-translational controls. One of these controls of AreA activity has been proposed to involve the NmrA protein interacting with the DNA-binding domain and the extreme C ...

  12. SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

    Gjerstorff, Morten; Greve, Katrine Buch Vidén; Møller, Jesper Bonnet; Elias, Daniel; Schmidt, Steffen; Mollenhauer, Jan; Volborg, Bjørn; Møllegaard, NIels Erik; Ditzel, Henrik

    Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body formation and derepresses Pc......G target genes. SSX2 further negatively regulates the level of the PcG-associated histone mark H3K27me3 in melanoma cells, and there is a clear inverse correlation between SSX2/3 expression and H3K27me3 in spermatogenesis. SSX2 does not affect the overall composition and stability of PcG complexes, but SSX...

  13. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  14. Identification of Enhancer Binding Proteins Important for Myxococcus xanthus Development▿

    Giglio, Krista M.; Eisenstatt, Jessica; Garza, Anthony G.


    Enhancer binding proteins (EBPs) control the temporal expression of fruiting body development-associated genes in Myxococcus xanthus. Eleven previously uncharacterized EBP genes were inactivated. Six EBP gene mutations produced minor but reproducible defects in fruiting body development. One EBP gene mutation that affected A-motility produced strong developmental defects.

  15. Signal transduction by guanine nucleotide binding proteins.

    Spiegel, A M


    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  16. Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL gene family from Citrus and the effect of fruit load on their expression

    Liron eShalom


    Full Text Available We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees, known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting, inducing next-year flowering, resulted in its up-regulation. In the current work, we set on to study the function of the gene. We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity. In order to find out if fruit load affected the expression of additional genes of the SPL family, we identified and classified all SPL members in the Citrus genome, and studied their seasonal expression patterns in buds and leaves, and in response to de-fruiting. Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load. The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed.

  17. Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family from Citrus and the effect of fruit load on their expression.

    Shalom, Liron; Shlizerman, Lyudmila; Zur, Naftali; Doron-Faigenboim, Adi; Blumwald, Eduardo; Sadka, Avi


    We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees), known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting), inducing next-year flowering, resulted in its up-regulation. In the current work, we set on to study the function of the gene. We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity. In order to find out if fruit load affected the expression of additional genes of the SPL family, we identified and classified all SPL members in the Citrus genome, and studied their seasonal expression patterns in buds and leaves, and in response to de-fruiting. Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load. The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed. PMID:26074947

  18. Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

    Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber-DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-α levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-α, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c

  19. Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein

    Sachleben, Joseph R.; McElroy, Craig A.; Gollnick, Paul; Foster, Mark P.


    Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT12, composed of a tetramer of trimers, AT3. Structural and biochemical studies suggest that only trime...

  20. Homologues of the 24-kDa flagellar Ca(2+)-binding protein gene of Trypanosoma cruzi are present in other members of the Trypanosomatidae family.

    Maldonado, R A; Linss, J; Thomaz, N; Olson, C L; Engman, D M; Goldenberg, S


    A full-length cDNA encoding the 24-kDa flagellar Ca(2+)-binding protein (FCaBP) of the Dm28c clone of Trypanosoma cruzi was cloned and characterized. Comparison of the deduced amino acid sequence with those of the FCaBPs of other T. cruzi strains revealed greater than 97% sequence conservation. FCaBP-like genes are found in Trypanosoma conorhini, Trypanosoma freitasi, Trypanosoma lewisi, Herpetomonas megaseliae, Leptomonas seymouri, and Phytomonas serpens, but not in Crithidia deanei, Leishmania amazonensis, or Endotrypanum schaudinni: Among various T. cruzi strains, FCaBP genes are located on chromosomes of different size, although all strains possess multiple FCaBP genes organized as tandemly arranged gene families. Northern and Western blot analyses revealed that FCaBP mRNAs are produced in all organisms possessing FCaBP-hybridizing sequences, indicating that expression of FCaBP or an FCaBP-like protein is common to a number of trypanosomatid species. PMID:9225770

  1. Probing protein phosphatase substrate binding

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen; Gammeltoft, Steen


    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  2. CERKL, a retinal disease gene, encodes an mRNA-binding protein that localizes in compact and untranslated mRNPs associated with microtubules.

    Fathinajafabadi, Alihamze; Pérez-Jiménez, Eva; Riera, Marina; Knecht, Erwin; Gonzàlez-Duarte, Roser


    The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases. PMID:24498393

  3. A stage—specific protein factor binding to a CACCC motif in both human β—globin gene promoter and 5‘—HS2 region



    The DNaseI hypersensitive site 2 (HS2) of human β-globin locus control region(LCR) is required for the high level expression of human β-globin genes.In the present study,a stage-specific protein factor (LPF-β) was identified in the nuclear extract prepared from mouse fetal liver at d 18 of gestation,which could bind to the HS2 region of human β-globin LCR.We also found that the shift band of LPF-β factor could be competed by human β-globin promoter.However,it couldn't be competed by human ε-globin promoter or by human Aγ-globin promoter.Furthermore,our data demonstrated that the binding-sequence of LPF-β factor is 5'CACACCCTA 3',which is located at the HS2 region of β-LCR(from-10845 to-10853 bp)and human β-globin promoter(from-92 to -84 bp).We speculated that these regions containing the CACCC box in both the human β-globin promoter and HS2 might function as stage selector elements in the regulation of human β-globin switching and the LPF-β factor might be a stage-specific protein factor involved in the regulation of human β-globin gene expression.

  4. Genome-wide identification and characterization of odorant-binding protein (OBP) genes in the malaria vector Anopheles sinensis (Diptera: Culicidae).

    He, Xiu; He, Zheng-Bo; Zhang, Yu-Juan; Zhou, Yong; Xian, Peng-Jie; Qiao, Liang; Chen, Bin


    Anopheles sinensis is a major malaria vector. Insect odorant-binding proteins (OBPs) may function in the reception of odorants in the olfactory system. The classification and characterization of the An. sinensis OBP genes have not been systematically studied. In this study, 64 putative OBP genes were identified at the whole-genome level of An. sinensis based on the comparison between OBP conserved motifs, PBP_GOBP, and phylogenetic analysis with An. gambiae OBPs. The characterization of An. sinensis OBPs, including the motif's conservation, gene structure, genomic organization and classification, were investigated. A new gene, AsOBP73, belonging to the Plus-C subfamily, was identified with the support of transcript and conservative motifs. These An. sinensis OBP genes were classified into three subfamilies with 37, 15 and 12 genes in the subfamily Classic, Atypical and Plus-C, respectively. The genomic organization of An. sinensis OBPs suggests a clustered distribution across nine different scaffolds. Eight genes (OBP23-28, OBP63-64) might originate from a single gene through a series of historic duplication events at least before divergence of Anopheles, Culex and Aedes. The microsynteny analyses indicate a very high synteny between An. sinensis and An. gambiae OBPs. OBP70 and OBP71 earlier classified under Plus-C in An. gambiae are recognized as belonging to the group Obp59a of the Classic subfamily, and OBP69 earlier classified under Plus-C has been moved to the Atypical subfamily in this study. The study established a basic information frame for further study of the OBP genes in insects as well as in An. sinensis. PMID:26970073

  5. Impaired Uptake and/or Utilization of Leucine by Saccharomyces cerevisiae Is Suppressed by the SPT15-300 Allele of the TATA-Binding Protein Gene

    Baerends, RJ; Qiu, Jin-Long; Rasmussen, Simon;


    mutant allele of the SPT15 gene (SPT15-300) corresponding to the three amino acid changes F177S, Y195H, and K218R has been reported (H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, and G. Stephanopoulos, Science 314:1565-1568, 2006). The SPT15 gene codes for the TATA-binding protein. This finding prompted...... us to examine the effect of expression of the SPT15-300 allele in various yeast species of industrial importance. Expression of SPT15-300 in leucine-prototrophic strains of S. cerevisiae, Saccharomyces bayanus, or Saccharomyces pastorianus (lager brewing yeast), however, did not improve tolerance to...... ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing the SPT15-300 mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol was...

  6. CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts

    Umayahara, Y.; Billiard, J.; Ji, C.; Centrella, M.; McCarthy, T. L.; Rotwein, P.


    Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in

  7. Co-ordinate control of synthesis of mitochondrial and non-mitochondrial hemoproteins: a binding site for the HAP1 (CYP1) protein in the UAS region of the yeast catalase T gene (CTT1).

    Winkler, H.; Adam, G.; Mattes, E; Schanz, M.; Hartig, A; Ruis, H


    Control of expression of the Saccharomyces cerevisiae CTT1 (catalase T) gene by the HAP1 (CYP1) gene, a mediator of heme control of mitochondrial cytochromes, was studied. Expression of a CTT1-lacZ fusion in a hap1 mutant showed that the CTT1 promoter is under HAP1 control. As demonstrated by a gel retardation assay, the HAP1 protein binds to a heme control region of the CTT1 gene. This binding in vitro is stimulated by hemin. The HAP1-binding sequence was localized by using DNA fragments spa...

  8. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  9. Microarray analysis of androgen-regulated gene expression in testis: the use of the androgen-binding protein (ABP-transgenic mouse as a model

    Grossman Gail


    Full Text Available Abstract Background Spermatogenesis is an androgen-dependent process, yet the molecular mechanisms of androgens' actions in testis are poorly understood. Transgenic mice overexpressing rat androgen-binding protein (ABP in their testes have reduced levels of intratesticular androgens and, as a result, show a progressive impairment of spermatogenesis. We used this model to characterize changes in global gene expression in testis in response to reduced bioavailability of androgens. Methods Total RNA was extracted from testes of 30-day old transgenic and wild-type control mice, converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays. Microarray results were confirmed by real-time reverse transcription polymerase chain reaction. Results Three-hundred-eighty-one genes (3.05% of all transcripts represented on the chips were up-regulated and 198 genes (1.59% were down-regulated by at least a factor of 2 in the androgen-deficient animals compared to controls. Genes encoding membrane proteins, intracellular signaling molecules, enzymes, proteins participating in the immune response, and those involved in cytoskeleton organization were significantly overrepresented in the up-regulated group. Among the down-regulated transcripts, those coding for extracellular proteins were overrepresented most dramatically, followed by those related to proteolysis, cell adhesion, immune response, and growth factor, cytokine, and ion channel activities. Transcripts with the greatest potential impact on cellular activities included several transcription factors, intracellular signal transducers, secreted signaling molecules and enzymes, and various cell surface molecules. Major nodes in the up-regulated network were IL-6, AGT, MYC, and A2M, those in the down-regulated network were IL-2, -4, and -10, MAPK8, SOCS1, and CREB1. Conclusion Microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional

  10. Polymorphism rs11085226 in the gene encoding polypyrimidine tract-binding protein 1 negatively affects glucose-stimulated insulin secretion.

    Martin Heni

    Full Text Available OBJECTIVE: Polypyrimidine tract-binding protein 1 (PTBP1 promotes stability and translation of mRNAs coding for insulin secretion granule proteins and thereby plays a role in β-cells function. We studied whether common genetic variations within the PTBP1 locus influence insulin secretion, and/or proinsulin conversion. METHODS: We genotyped 1,502 healthy German subjects for four tagging single nucleotide polymorphisms (SNPs within the PTBP1 locus (rs351974, rs11085226, rs736926, and rs123698 covering 100% of genetic variation with an r(2≥0.8. The subjects were metabolically characterized by an oral glucose tolerance test with insulin, proinsulin, and C-peptide measurements. A subgroup of 320 subjects also underwent an IVGTT. RESULTS: PTBP1 SNP rs11085226 was nominally associated with lower insulinogenic index and lower cleared insulin response in the OGTT (p≤0.04. The other tested SNPs did not show any association with the analyzed OGTT-derived secretion parameters. In the IVGTT subgroup, SNP rs11085226 was accordingly associated with lower insulin levels within the first ten minutes following glucose injection (p = 0.0103. Furthermore, SNP rs351974 was associated with insulin levels in the IVGTT (p = 0.0108. Upon interrogation of MAGIC HOMA-B data, our rs11085226 result was replicated (MAGIC p = 0.018, but the rs351974 was not. CONCLUSIONS: We conclude that common genetic variation in PTBP1 influences glucose-stimulated insulin secretion. This underlines the importance of PTBP1 for beta cell function in vivo.

  11. Gene variability and degree of expression of vaccine candidate factor H binding protein in clinical isolates of Neisseria meningitidis.

    Kelly, Anne; Jacobsson, Susanne; Hussain, Shahida; Olcén, Per; Mölling, Paula


    The factor H binding protein (fHbp) is currently being evaluated in clinical trials as a vaccine candidate for a meningococcal group B vaccine. We have previously described the prevalence and sequence variation of fHbp (Jacobsson et al., 2009) and here we investigate the expression of the antigen. The present study includes isolates from carriers (n = 62) and patients with invasive Neisseria meningitidis infections (n = 146), of which 62 had a fatal outcome. Among the invasive isolates from patients with fatal and non-fatal infections fHbp allele 1 was most common (42% and 29% respectively), but it was only identified in 3% of the carrier isolates, where allele 16 was most frequent (13%). The Fluorescence-activated cell sorting analysis identified fHbp expression in all except seven isolates and further analysis by Western blot showed that five of these seven samples were indeed negative using a polyclonal anti-fHbp serum. The negative isolates belonged to serogroup B fHbp allele 24, Y allele 104, and W-135 allele 16 (all invasive). Two were non-serogroupable carrier isolates (allele 21 and 101). An interesting finding is that isolates from invasive infections with fatal outcome had lower expression of fHbp or lower affinity for the fHbp antibody compared to isolates from non-fatal invasive infections and carriers. PMID:23030708

  12. Structural analysis of the full-length gene encoding a fibronectin-binding-like protein (CadF and its adjacent genetic loci within Campylobacter lari

    Millar B Cherie


    Full Text Available Abstract Background The combined sequences encoding a partial and putative rpsI open reading frame (ORF, non-coding (NC region, a putative ORF for the Campylobacter adhesin to fibronectin-like protein (cadF, a putative Cla_0387 ORF, NC region and a partial and putative Cla_0388 ORF, were identified in 16 Campylobacter lari isolates, using two novel degenerate primer pairs. Probable consensus sequence at the -35 and -10 regions were identified in all C. lari isolates, as a promoter. Results Thus, cadF (-like gene is highly conserved among C. lari organisms. Transcription of the cadF (-like gene in C. lari cells in vivo was also confirmed and the transcription initiation site was determined. A peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins was completely conserved amongst the putative cadF (-like ORFs from the C. lari isolates. Conclusion The putative cadF (-like ORFs from all C. lari isolates were nine amino acid larger than those from C. jejuni, and showed amino acid residues 137 -140 of FALG (50% identity, instead of the FRLS residues of the maximal fibronectin-binding activity site demonstrated within C. jejuni CadF. A neighbor joining tree constructed based on cadF (-like gene sequence information formed a major cluster consisting of C. lari isolates, separating from the other three thermophilic campylobacters.

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

    He Lin


    Full Text Available Abstract 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.

  14. Cell differentiation by interaction of two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S.pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites

    Kjaerulff, S; Dooijes, D; Clevers, H;


    The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion. This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc. Here we report that the mfm1 promoter contains a single, weak Stell-binding site...

  15. OsDREB4 Genes in Rice Encode AP2-Containing Proteins that Bind Specifically to the Dehydration-Responsive Element

    Xiu-Hong TIAN; Xue-Ping LI; Hua-Lin ZHOU; Jin-Song ZHANG; Zhi-Zhong GONG; Shou-Yi CHEN


    Most dehydration-responsive element-binding (DREB) factors interact specifically with the dehydration-responsive element (DRE) and control the expression of many stress-inducible genes in Arabidopsis. In rice (Oryza sativa L. cv. Lansheng), we cloned three DREB homologs: OsDREB1-1, OsDREB4- 1, and OsDREB4-2. The deduced amino acid sequences revealed that each protein contained a potential nuclear localization signal, an AP2 DNA-binding domain, and a possible acidic activation domain. The yeast one-hybrid assay indicated that both OsDREB4-1 and OsDREB4-2 proteins specifically bound to DRE and activated expression of the dual reporter genes of histidine (HIS3) and galactosidase (LacZ). In rice seedlings,expression of OsDREB4-1 was induced by dehydration and high salt, whereas OsDREB1-1 and OsDREB4-2 were expressed constitutively. Under normal growth conditions, OsDREB1-1 was expressed strongly in the leaf, sheath, and spike, was expressed relatively weak in the stem and only faintly expressed in the roots,whereas expression of transcripts of OsDREB4-1 and OsDREB4-2 was higher in the roots, stem, and spike,lower in the leaf, and undetectable in the sheath. Together, these results imply that expression of the OsDREB genes could be controlled by specific aspects of differentiation or development. Thus, OsDREB4-1 could function as a trans-acting factor in the DRE/DREB regulated stress-responsive pathway.

  16. Activating transcription factor 4 and X box binding protein 1 of Litopenaeus vannamei transcriptional regulated white spot syndrome virus genes Wsv023 and Wsv083.

    Xiao-Yun Li

    Full Text Available In response to endoplasmic reticulum (ER stress, the signaling pathway termed unfolded protein response (UPR is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4 which belonged to a branch of the UPR, the [protein kinase RNA (PKR-like ER kinase, (PERK]-[eukaryotic initiation factor 2 subunit alpha (eIF2α] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element (ATF/CRE. Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1, has a significant function in [inositol-requiring enzyme-1(IRE1 - (XBP1] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE. These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection.

  17. Identification and characterization of a novel corticotropin-releasing hormone-binding protein (CRH-BP) gene from Chinese honeybee (Apis cerana cerana).

    Liu, Li; Yu, Xiaoli; Meng, Fei; Guo, Xingqi; Xu, Baohua


    Corticotropin-releasing hormone-binding protein (CRH-BP) is an essential secreted glycoprotein for coordinating the neuroendocrine responses to stress by binding either CRHs or its related peptides. A novel CRH-BP gene AccCRH-BP from Apis cerana cerana was identified and characterized. Its genomic DNA was consisted of seven exons and six introns, and shared high similarity with the homologous members from other insects and vertebrates. Homologous and phylogenetic analysis indicated that AccCRH-BP was highly conserved, suggesting the maintenance of conservative structure might be necessary for its biological function. Real-time quantitative PCR revealed that AccCRH-BP was highly expressed in pupa and adult, especially in the head of pupa. However, there was no expression in larval stage. Furthermore, the transcripts of AccCRH-BP in the brain of honeybees were induced by exposure to environmental stresses including UV-light, heat, and cold. The expression level of AccCRH-BP in workers or queens was significantly higher than that of drones. Additionally, analysis of 5'-flanking region of AccCRH-BP revealed a number of putative development and stress transcription factor-binding sites. These data suggest that AccCRH-BP may play important roles in the regulation of honeybee development, and in the central nervous system of the brain to regulate the neuroendocrine stress responses. PMID:22006535

  18. Erythropoietin binding protein from mammalian serum

    Clemons, G.K.


    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  19. Erythropoietin binding protein from mammalian serum

    Clemons, Gisela K. (Berkeley, CA)


    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  20. Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

    Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian


    Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further. PMID:25009988

  1. Independent associations of polymorphisms in vitamin D binding protein (GC) and vitamin D receptor (VDR) genes with obesity and plasma 25OHD3 levels demonstrate sex dimorphism.

    Almesri, Norah; Das, Nagalla S; Ali, Muhallab E; Gumaa, Khalid; Giha, Hayder Ahmed


    We investigated a possible association between polymorphisms in vitamin D binding protein (GC) and vitamin D receptor (VDR) genes and obesity in Bahraini adults. For this purpose, 406 subjects with varying body mass indexes (BMIs) were selected. Plasma levels of 25-hydroxyvitamin D3 (25OHD3) were measured by chemiluminescence immunoassay. Six single nucleotide polymorphisms, 2 in the VDR gene (rs731236 TC and rs12721377 AG) and 4 in the GC gene (rs2282679 AC, rs4588 CA, rs7041 GT, and rs2298849 TC), were genotyped by real-time polymerase chain reaction. We found that the rs7041 minor allele (G) and rare genotype (GG) were associated with higher BMI (p = 0.007 and p = 0.012, respectively), but they did not influence 25OHD3 levels. However, the minor alleles of rs2282679 (A) and rs4588 (C) were associated with low 25OHD3 plasma levels (p = 0.039 and p = 0.021, respectively), but not with BMI. Having categorized the subjects based on their sex, we found that (i) rs7041 GG associated with high BMI in females (p = 0.003), (ii) rs4588 CC associated with high BMI in females (p = 0.034) and low 25OHD3 levels in males (p = 0.009), and (iii) rs12721377 AA associated with low 25OHD3 levels in females (p = 0.039). Notably, none of the common haplotypes (6 in the GC gene and 3 in the VDR gene) were associated with BMI. Therefore, polymorphisms in the GC (rs2282679, rs4588, rs7041) and VDR (rs12721377) genes were independently associated with obesity and 25OHD3 levels with a clear sex dimorphism. PMID:26881316

  2. The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes

    Sandberg, Maria B; Bloksgaard, Maria; Duran-Sandoval, Daniel; Duval, Caroline; Staels, Bart; Mandrup, Susanne


    The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular lipid-binding protein that transports acylCoA esters. The protein is expressed in most cell types at low levels; however, expression is particularly high in cells with a high turnover of fatty acids. Here we confirm a previous...... observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target...... that ACBP expression is significantly lower in livers from PPARalpha knock-out mice than in livers from wild type mice. In conclusion, expression of ACBP in rodent hepatocytes is subject to dual metabolic regulation by PPARalpha and SREBP-1c, which may reflect the need for ACBP during lipogenic as well...

  3. In vivo and in vitro protein-DNA interactions at the distal oestrogen response element of the chicken vitellogenin gene: evidence for the same protein binding to this sequence in hen and rooster liver.

    McEwan, I J; Saluz, H P; Jost, J P


    The major egg white protein, vitellogenin, is synthesized in a tissue specific and oestradiol dependent manner in the liver of egg-laying hens. In this paper, we describe a detailed study of the protein-DNA interactions at the distal oestrogen response element (ERED) located 600 bp upstream of the start of transcription. In vivo footprinting of hepatocytes from adult hens and roosters with 0.5-0.0005% dimethylsulphate (DMS) revealed, at critical concentrations of DMS, protection of distinct guanosine residues within the ERED and adjacent downstream sequence in both cases. From this, it was concluded that there were proteins present in both tissues binding to this region in vivo. In vitro studies using missing base contact probing and proteolytic clipping band shift assays with hen and rooster liver nuclear extracts identified the ERE binding protein to be the same or very closely related in both tissues. Furthermore, the protein from rooster nuclear extracts bound to the ERE sequence even when the DNA was methylated at CpG dinucleotides, u.v. cross-linking experiments performed with bromodeoxyuridine substituted ERE, revealed that a nuclear protein with Mr of about 75,000-80,000 bound specifically to this sequence. These studies demonstrate that apart from the oestrogen receptor, at least one other protein can interact specifically with the chicken vitellogenin ERE, independently of hormonal expression of the gene. PMID:2009219

  4. Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

    McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.


    Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

  5. The human insulin-like growth factor-binding protein 4 gene maps to chromosome region 17q12-q21. 1 and is close to the gene for hereditary breast-ovarian cancer

    Tonin, P.; Vivier, A.; Morgan, K.; Narod, S.; Pollack, M. (McGill Univ., Montreal (Canada)); Ehrenborg, E.; Zazzi, H.; Luthman, H.; Larsson, C. (Karolinska Hospital, Stockholm (Sweden)); Lenoir, G. (International Agency for Research on Cancer, Lyon (France)) (and others)


    The gene for insulin-like growth factor-binding protein 4 (IGFBP4) codes for a serum protein that binds to the family of insulin-like growth factors and modulates their activity. It has been mapped by in situ hybridization to chromosome region 17q12-q21.1. The authors have developed a CA-repeat polymorphism from a cosmid clone containing IGFBP4. By linkage analysis, IGFBP4 maps to the chromosome 17q interval THRA1-D17S579. This interval also contains the gene for hereditary breast-ovarian cancer, BRCA1. Genetic recombination between IGFBP4 and BRCA1 places IGFBP4 centromeric to the cancer susceptibility gene and effectively excludes it as a candidate gene for BRCA1. IGFBP4 is, however, one of the closest known centromeric markers for BRCA1; the estimated recombination fraction is 0.015. IGFBP4 and D17S579 together define a 2.8-cM interval that contains BRCA1. 18 refs., 3 figs., 1 tab.

  6. Candidate SNP Markers of Chronopathologies Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

    Ponomarenko, Petr; Rasskazov, Dmitry; Suslov, Valentin; Sharypova, Ekaterina; Savinkova, Ludmila; Podkolodnaya, Olga; Podkolodny, Nikolay L.; Tverdokhleb, Natalya N.; Chadaeva, Irina; Kolchanov, Nikolay


    Variations in human genome (e.g., single nucleotide polymorphisms, SNPs) may be associated with hereditary diseases, their complications, comorbidities, and drug responses. Using Web service SNP_TATA_Comparator presented in our previous paper, here we analyzed immediate surroundings of known SNP markers of diseases and identified several candidate SNP markers that can significantly change the affinity of TATA-binding protein for human gene promoters, with circadian consequences. For example, rs572527200 may be related to asthma, where symptoms are circadian (worse at night), and rs367732974 may be associated with heart attacks that are characterized by a circadian preference (early morning). By the same method, we analyzed the 90 bp proximal promoter region of each protein-coding transcript of each human gene of the circadian clock core. This analysis yielded 53 candidate SNP markers, such as rs181985043 (susceptibility to acute Q fever in male patients), rs192518038 (higher risk of a heart attack in patients with diabetes), and rs374778785 (emphysema and lung cancer in smokers). If they are properly validated according to clinical standards, these candidate SNP markers may turn out to be useful for physicians (to select optimal treatment for each patient) and for the general population (to choose a lifestyle preventing possible circadian complications of diseases).

  7. The gene encoding the Acyl-CoA-binding protein is activated by peroxisome proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents

    Helledie, Torben; Grøntved, Lars; Jensen, Søren S;


    The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various m...... to demonstrate that the intronic PPRE efficiently binds PPARgamma/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPARgamma-response element....

  8. CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

    Hidehito Kuroyanagi

    Full Text Available An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc-32 gene, encoding the a subunit of V0 complex of vacuolar-type H(+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive

  9. Expression of Lactobacillus reuteri Pg4 collagen-binding protein gene in Lactobacillus casei ATCC 393 increases its adhesion ability to Caco-2 cells.

    Hsueh, Hsiang-Yun; Yueh, Pei-Ying; Yu, Bi; Zhao, Xin; Liu, Je-Ruei


    The collagen-binding protein gene cnb was cloned from the probiotic Lactobacillus reuteri strain Pg4. The DNA sequence of the cnb gene (792 bp) has an open reading frame encoding 263 amino acids with a calculated molecular weight of 28.5 kDa. The cnb gene was constructed so as to constitutively express under the control of the Lactococcus lactis lacA promoter and was transformed into Lactobacillus casei ATCC 393, a strain isolated from dairy products with poor ability to adhere to intestinal epithelial cells. Confocal immunofluorescence microscopic and flow cytometric analysis of the transformed strain Lb. casei pNZ-cnb indicated that Cnb was displayed on its cell surface. Lb. casei pNZ-cnb not only showed a higher ability to adhere to Caco-2 cells but also exhibited a higher competition ability against Escherichia coli O157:H7 and Listeria monocytogenes adhesion to Caco-2 cells than Lb. casei ATCC 393. PMID:21070005

  10. Transcriptomic analysis suggests a key role for SQUAMOSA PROMOTER BINDING PROTEIN LIKE, NAC and YUCCA genes in the heteroblastic development of the temperate rainforest tree Gevuina avellana (Proteaceae).

    Ostria-Gallardo, Enrique; Ranjan, Aashish; Chitwood, Daniel H; Kumar, Ravi; Townsley, Brad T; Ichihashi, Yasunori; Corcuera, Luis J; Sinha, Neelima R


    Heteroblasty, the temporal development of the meristem, can produce diverse leaf shapes within a plant. Gevuina avellana, a tree from the South American temperate rainforest shows strong heteroblasty affecting leaf shape, transitioning from juvenile simple leaves to highly pinnate adult leaves. Light availability within the forest canopy also modulates its leaf size and complexity. Here we studied how the interaction between the light environment and the heteroblastic progression of leaves is coordinated in this species. We used RNA-seq on the Illumina platform to compare the range of transcriptional responses in leaf primordia of G. avellana at different heteroblastic stages and growing under different light environments. We found a steady up-regulation of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL), NAC, YUCCA and AGAMOUS-LIKE genes associated with increases in age, leaf complexity, and light availability. In contrast, expression of TCP, TPR and KNOTTED1 homeobox genes showed a sustained down-regulation. Additionally, genes involved in auxin synthesis/transport and jasmonate activity were differentially expressed, indicating an active regulation of processes controlled by these hormones. Our large-scale transcriptional analysis of the leaf primordia of G. avellana sheds light on the integration of internal and external cues during heteroblastic development in this species. PMID:26680017

  11. Reduced function of the RNA-binding protein FPA rescues a T-DNA insertion mutant in the Arabidopsis ZHOUPI gene by promoting transcriptional read-through.

    Zhang, Yaohua; Li, Xin; Goodrich, Justin; Wu, Chunxia; Wei, Haichao; Yang, Suxin; Feng, Xianzhong


    T-DNA insertion mutants have been widely used to investigate plant gene functions. Unexpectedly, in several reported cases, the phenotype of T-DNA insertion mutations can be suppressed because of trans T-DNA interactions associated with epigenetic modification, which indicates that caution is needed when T-DNA mutants are used. In the present study, we characterized a novel process suppressing a T-DNA mutation. The spz2 (suppressor of zou 2) mutant was isolated as a suppressor of the phenotype of the zou-4 mutant caused by a T-DNA insertion in the first intron. The spz2 mutation partially recovered the native ZOU gene expression in the zou-4 background, but not in two other zou alleles, zou-2 and zou-3, with T-DNAs inserted in the exon and intron, respectively. The suppressed phenotype was inherited in a Mendelian fashion and is not associated with epigenetic modification. The recovery of the native ZOU gene expression in the spz2 zou-4 double mutant is caused by transcriptional read-through of the intronic T-DNA as a result of decreased proximal polyadenylation. SPZ2 encodes an RNA-binding protein, FPA, which is known to regulate polyadenylation site selection. This is the first example of FPA rescuing a T-DNA insertion mutation by affecting the polyadenylation site selection. PMID:27164978

  12. Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

    Wehmeyer, B. (Univ. of Pennsylvania, Philadelphia (USA) Albert-Ludwigs-Universitaet, Freiburg (West Germany)); Cashmore, A.R. (Univ. of Pennsylvania, Philadelphia (USA)); Schaefer, E. (Albert-Ludwigs-Universitaet, Freiburg (West Germany))


    Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light. These distinct expression characteristics are shown to reflect differences at the level of transcription.

  13. Molecular cloning and preliminary function study of iron responsive element binding protein 1 gene from cypermethrin-resistant Culex pipiens pallens

    Tan Wenbin


    Full Text Available Abstract Background Insecticide resistance jeopardizes the control of mosquito populations and mosquito-borne disease control, which creates a major public health concern. Two-dimensional electrophoresis identified one protein segment with high sequence homology to part of Aedes aegypti iron-responsive element binding protein (IRE-BP. Method RT-PCR and RACE (rapid amplification of cDNA end were used to clone a cDNA encoding full length IRE-BP 1. Real-time quantitative RT-PCR was used to evaluate the transcriptional level changes in the Cr-IRE strain Aedes aegypti compared to the susceptible strain of Cx. pipiens pallens. The expression profile of the gene was established in the mosquito life cycle. Methyl tritiated thymidine (3H-TdR was used to observe the cypermethrin resistance changes in C6/36 cells containing the stably transfected IRE-BP 1 gene of Cx. pipiens pallens. Results The complete sequence of iron responsive element binding protein 1 (IRE-BP 1 has been cloned from the cypermethrin-resistant strain of Culex pipiens pallens (Cr-IRE strain. Quantitative RT-PCR analysis indicated that the IRE-BP 1 transcription level was 6.7 times higher in the Cr-IRE strain than in the susceptible strain of 4th instar larvae. The IRE-BP 1 expression was also found to be consistently higher throughout the life cycle of the Cr-IRE strain. A protein of predicted size 109.4 kDa has been detected by Western blotting in IRE-BP 1-transfected mosquito C6/36 cells. These IRE-BP 1-transfected cells also showed enhanced cypermethrin resistance compared to null-transfected or plasmid vector-transfected cells as determined by 3H-TdR incorporation. Conclusion IRE-BP 1 is expressed at higher levels in the Cr-IRE strain, and may confer some insecticide resistance in Cx. pipiens pallens.

  14. Regulatory elements in the promoter region of the rat gene encoding the acyl-CoA-binding protein

    Elholm, M; Bjerking, G; Knudsen, J;


    peroxisome proliferator activated receptors (PPAR) and a retinoid X receptor alpha (RXRalpha), we demonstrated that this DR-1 element was capable of binding PPARalpha/RXRalpha, PPARdelta/RXRalpha and PPARgamma2/RXRalpha heterodimers. The PPARgamma2/RXRalpha heterodimer appeared to have the highest affinity...

  15. Identification and Characterization of Cyclic AMP Response Element-Binding Protein H Response Element in the Human Apolipoprotein A5 Gene Promoter

    Kwang Hoon Song


    Full Text Available The cyclic AMP response element-binding protein H (CREBH plays important roles in hepatic lipogenesis, fatty acid oxidation, and lipolysis under metabolic stress. Here, we report CREBH as a novel regulator of human APOA5. Knockdown of endogenous CREBH expression via small interfering RNA resulted in the downregulation of human APOA5 mRNA expression in human hepatoma cells, HepG2. Sequence analysis suggested that putative CREBH response element (CREBHRE is located in the human APOA5 promoter region and is highly conserved in both human and rodent. To clarify whether the human APOA5 promoter is regulated by CREBH, we analyzed the human APOA5 promoter region using a transient transfection assay and determined that transfection of CREBH induced human APOA5 promoter activity. Moreover, it was shown that CREBH directly regulated human APOA5 gene expression by binding to a unique CREBHRE located in the proximal human APOA5 promoter region, using 5′-deletion and mutagenesis of human APOA5 promoter analysis and chromatin immunoprecipitation assay. Taken together, our results demonstrated that human APOA5 is directly regulated by CREBH via CREBHRE and provided a new insight into the role of this liver-specific bZIP transcription factor in lipoprotein metabolism and triglyceride homeostasis.

  16. A Zinc Finger Transcription Factor, αA-Crystallin Binding Protein 1, Is a Negative Regulator of the Chondrocyte-Specific Enhancer of the α1(II) Collagen Gene

    Tanaka, Kazuhiro; Matsumoto, Yoshihiro; Nakatani, Fumihiko; Iwamoto, Yukihide; Yamada, Yoshihiko


    Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, αA-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the ...

  17. Allelic variations of a light harvesting chlorophyll a/b-binding protein gene (Lhcb1 associated with agronomic traits in barley.

    Yanshi Xia

    Full Text Available Light-harvesting chlorophyll a/b-binding protein (LHCP is one of the most abundant chloroplast proteins in plants. Its main function is to collect and transfer light energy to photosynthetic reaction centers. However, the roles of different LHCPs in light-harvesting antenna systems remain obscure. Exploration of nucleotide variation in the genes encoding LHCP can facilitate a better understanding of the functions of LHCP. In this study, nucleotide variations in Lhcb1, a LHCP gene in barley, were investigated across 292 barley accessions collected from 35 different countries using EcoTILLING technology, a variation of the Targeting Induced Local Lesions In Genomes (TILLING. A total of 23 nucleotide variations were detected including three insert/deletions (indels and 20 single nucleotide polymorphisms (SNPs. Among them, 17 SNPs were in the coding region with nine missense changes. Two SNPs with missense changes are predicted to be deleterious to protein function. Seventeen SNP formed 31 distinguishable haplotypes in the barley collection. The levels of nucleotide diversity in the Lhcb1 locus differed markedly with geographic origins and species of accessions. The accessions from Middle East Asia exhibited the highest nucleotide and haplotype diversity. H. spontaneum showed greater nucleotide diversity than H. vulgare. Five SNPs in Lhcb1 were significantly associated with at least one of the six agronomic traits evaluated, namely plant height, spike length, number of grains per spike, thousand grain weight, flag leaf area and leaf color, and these SNPs may be used as potential markers for improvement of these barley traits.

  18. SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

    Gjerstorff, Morten Frier; Relster, Mette Marie; Greve, Katrine Buch Viden;


    Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma and other types of human cancers, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body...

  19. Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.

    S. Hahn; Buratowski, S.; Sharp, P A; Guarente, L


    The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with hig...

  20. Novel Selection for Isoniazid (INH) Resistance Genes Supports a Role for NAD+-Binding Proteins in Mycobacterial INH Resistance

    CHEN, PING; Bishai, William R.


    The genetic basis of isoniazid (INH) resistance remains unknown for a significant proportion of clinical isolates. To identify genes which might confer resistance by detoxifying or sequestering INH, we transformed the Escherichia coli oxyR mutant, which is relatively sensitive to INH, with a Mycobacterium tuberculosis plasmid library and selected for INH-resistant clones. Three genes were identified and called ceo for their ability to complement the Escherichia coli oxyR mutant. ceoA was the ...

  1. Epigenetic Regulation of the IL-13-induced Human Eotaxin-3 Gene by CREB-binding Protein-mediated Histone 3 Acetylation*

    Lim, Eun Jin; Lu, Thomas X.; Blanchard, Carine; Rothenberg, Marc E.


    The etiology of a variety of chronic inflammatory disorders has been attributed to the interaction of genetic and environmental factors. Herein, we identified a link between epigenetic regulation and IL-13-driven eotaxin-3 in the pathogenesis of chronic allergic inflammation. We first demonstrated that the cAMP-responsive element (CRE) site in the eotaxin-3 promoter affects IL-13-induced eotaxin-3 promoter activity. Furthermore, the CRE-binding protein-binding protein (CBP), a histone acetylt...

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

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


    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

  3. Calmodulin Binding Proteins and Alzheimer’s Disease

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.


    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  4. Alternative polyadenylation and RNA-binding proteins.

    Erson-Bensan, Ayse Elif


    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  5. Characterization, Evolution and Tissue-specific Expression of AmphiCalbin, a Novel Gene Encoding EF-hand Calcium-binding Protein in Amphioxus Branchiostoma belcheri

    Jing LUAN; Shicui ZHANG; Zhenhui LIU; Chunxin FAN; Guangdong JI; Lei LI


    An amphioxus full-length cDNA, AmphiCalbin, encoding a novel EF-hand calcium-binding protein (EFCaBP), was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri. It consists of 1321 bp with a 636 bp open reading frame encoding a protein of 211 amino acids with a molecular mass of approximately 24.5 kDa. The phylogenetic analysis offers two interesting inferences. First, AmphiCalbin clusters with a group of unnamed EFCaBPs that are differentiated from other identified EFCaBPs. Second,AmphiCalbin falls at the base of the vertebrate unnamed EFCaBPs clade, probably representing their prototype.This is also corroborated by the fact that AmphiCalbin has an exon-intron organization identical to that of vertebrate unnamed EFCaBP genes. Both tissue-section in situ hybridization and whole-mount in situ hybridization prove a tissue-specific expression pattern of AmphiCalbin, with high levels of expression in the digestive system and gonads. It is proposed that AmphiCalbin might play a role in the digestive system and gonads. These observations lay the foundation for further understanding of the function of the unnamed EFCaBPs.

  6. Isolation of prawn ( Exopalaemon carinicauda) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

    Ge, Qianqian; Li, Jian; Duan, Yafei; Li, Jitao; Sun, Ming; Zhao, Fazhen


    The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn ( Exopalaemon carinicauda) ( EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

  7. Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA 156 Module by the Homeodomain Proteins PENNYWISE and POUND-FOOLISH in Arabidopsis

    Shruti Lal; Leo Bryan Pacis; Harley M.S. Smith


    The morphology of inflorescences is regulated in part by the temporal and spatial events that regulate flower specification.In Arabidopsis,an endogenous flowering time pathway mediated by a subset of SQUAMOSA PROMOTERBINDING PROTEIN-LIKE (SPL) transcription factors,including SPL3,SPL4,and SPL5,function to specify flowers by activating floral meristem identity genes.During shoot development,SPL3,SPL4,and SPL5 are post-transcriptionally regulated by microRNA156 (miR156).The photoperiod regulated florigenic signal,FLOWERING LOCUS T (FT),promotes floral induction,in part by activating SPL3,SPL4,and SPL5.In turn,these SPLs function in parallel with FT to specify flower meristems.Two related BELL1-like homeobox genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) expressed in the shoot apical meristem are absolutely required for the specification of floral meristems.Genetic studies show that the floral specification function of FT depends upon PNY and PNF; however,the interplay between these homeodomain proteins and SPLs is not known.In this manuscript,we show that the photoperiodic floral induction of SPL3,SPL4,and SPL5 is dependent upon PNY and PNF.Further,PNY and PNF also control SPL3,SPL4,and SPL5 expression by negatively regulating miR156.Lastly,ectopic expression of SPL4 partially rescues the pny pnf non-flower-producing phenotype,while overexpression of SPL3 or SPL5 in pny pnf plants was unable to restore flower specification.These results suggest that:(1) SPL3,SPL4,and SPL5 function is dependent upon PNY and PNF,or (2) expression of multiple SPLs is required for floral specification in pny pnf plants.

  8. The 5‘—flanking cis—acting elements of the human ε—globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins



    The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cisacting elements of the human ε-globin gene have been examined.Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII,-446bp- -419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells,indicating that ε-PREII may be an erythroidspecific facultative MAR.In gel mobility shift assay and Southwestern blotting assay,an erythroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (ε-PREII).Furthermore,we demonstrated that the silencer (-392bp- -177bp) upstream of the human ε-globin gene could associate with the nuclear matrices from K562,HEL and Raji cells.In addition,the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer,suggesting that this silencer element might be a constitutive nuclear matrix attachment region(constitutive MAR).Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human ε-globin gene expression.

  9. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I


    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: PMID:25692584

  10. CCAAT/Enhancer-Binding Protein α Is a Crucial Regulator of Human Fat Mass and Obesity Associated Gene Transcription and Expression

    Wei Ren


    Full Text Available Several susceptibility loci have been reported associated with obesity and T2DM in GWAS. Fat mass and obesity associated gene (FTO is the first gene associated with body mass index (BMI and risk for diabetes in diverse patient populations. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. While much is known about the epigenetic mutations contributing to obesity and T2DM, less is certain with the expression regulation of FTO gene. In this study, a highly conserved canonical C/EBPα binding site was located around position −45~−54 bp relative to the human FTO gene transcriptional start site. Site-directed mutagenesis of the putative C/EBPα binding sites decreased FTO promoter activity. Overexpression and RNAi studies also indicated that C/EBPα was required for the expression of FTO. Chromatin immunoprecipitation (ChIP experiment was carried out and the result shows direct binding of C/EBPα to the putative binding regions in the FTO promoter. Collectively, our data suggest that C/EBPα may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

  11. Advances on Plant Pathogenic Mycotoxin Binding Proteins

    WANG Chao-hua; DONG Jin-gao


    Toxin-binding protein is one of the key subjects in plant pathogenic mycotoxin research. In this paper, new advances in toxin-binding proteins of 10 kinds of plant pathogenic mycotoxins belonging to Helminthosporium ,Alternaria ,Fusicoccum ,Verticillium were reviewed, especially the techniques and methods of toxin-binding proteins of HS-toxin, HV-toxin, HMT-toxin, HC-toxin. It was proposed that the isotope-labeling technique and immunological chemistry technique should be combined together in research of toxin-binding protein, which will be significant to study the molecular recognition mechanism between host and pathogenic fungus.

  12. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

    Dou, Q P; Zhao, S; Levin, A H;


    complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a...

  13. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  14. A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

    Jing Liu; Yongze Yuan; Zhi Wu; Na Li; Yuanlei Chen; Tingting Qin; Hui Geng; Li Xiong; Deli Liu


    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved doma...

  15. Time of day and nutrients in feeding govern daily expression rhythms of the gene for sterol regulatory element-binding protein (SREBP)-1 in the mouse liver.

    Matsumoto, Eriko; Ishihara, Akinori; Tamai, Saki; Nemoto, Ayako; Iwase, Katsuro; Hiwasa, Takaki; Shibata, Shigenobu; Takiguchi, Masaki


    Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, "when to eat in a day (the light/dark cycle)," rather than "whenever to eat in a day," is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of "what to eat." Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients. PMID:20720008

  16. Penicillin-Binding Protein Imaging Probes

    Kocaoglu, Ozden; Carlson, Erin E.


    Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5–15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to st...

  17. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

    Elisa E. Figueroa-Angulo


    Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  18. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    Fattaey, A R; Helin, K; Dembski, M S;


    The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one of...

  19. Molecular cloning, characterization, and stress-responsive expression of genes encoding glycine-rich RNA-binding proteins in Camelina sativa L.

    Kwak, Kyung Jin; Kang, Hunseung; Han, Kyung-Hwan; Ahn, Sung-Ju


    Camelina sativa L. is an oil-seed crop that has potential for biofuel applications. Although the importance of C. sativa as a biofuel crop has increased in recent years, reports demonstrating the stress responsiveness of C. sativa and characterizing the genes involved in stress response of C. sativa have never been published. Here, we isolated and characterized three genes encoding glycine-rich RNA-binding proteins (GRPs) from camelina: CsGRP2a, CsGRP2b, and CsGRP2c. The three CsGRP2 proteins were very similar in amino acid sequence and contained a well-conserved RNA-recognition motif at the N-terminal region and glycine-rich domain at the C-terminal region. To understand the functional roles of CsGRP2s under stress conditions, we investigated the expression patterns of CsGRP2s under various environmental stress conditions. The expressions of the three CsGRP2s were highly up-regulated under cold stress. The expression of CsGRP2a was up-regulated under salt or dehydration stress, whereas the transcript levels of CsGRP2b and CsGRP2c were decreased under salt or dehydration stress conditions. The three CsGRP2s had the ability to complement cold-sensitive Escherichia coli mutants at low temperatures and harbored transcription anti-termination and nucleic acid-melting activities, indicating that the CsGRP2s possess RNA chaperone activity. The CsGRP2a protein was localized to both the nucleus and the cytoplasm. Expression of CsGRP2a in cold-sensitive Arabidopsis grp7 mutant plants resulted in decreased electrolyte leakage at freezing temperatures. Collectively, these results suggest that the stress-responsive CsGRP2s play a role as an RNA chaperone during the stress adaptation process in camelina. PMID:23628924

  20. CCAAT/enhancer binding protein β (C/EBPβ isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer cells.

    André Albergaria

    Full Text Available P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

  1. Variation in the maternal corticotrophin releasing hormone-binding protein (CRH-BP gene and birth weight in Blacks, Hispanics and Whites.

    Pathik D Wadhwa

    Full Text Available BACKGROUND: Given the unique role of the corticotrophin-releasing hormone (CRH system in human fetal development, the aim of our study was to estimate the association of birth weight with DNA sequence variation in three maternal genes involved in regulating CRH production, bioavailability and action: CRH, CRH-Binding Protein (CRH-BP, and CRH type 1 receptor (CRH-R1, respectively, in three racial groups (African-Americans, Hispanics, and non-Hispanic Whites. METHODS: Our study was carried out on a population-based sample of 575 mother-child dyads. We resequenced the three genes in mouse-human hybrid somatic cell lines and selected SNPs for genotyping. RESULTS: A significant association was observed in each race between birth weight and maternal CRH-BP SNP genotypes. Estimates of linkage disequilibrium and haplotypes established three common haplotypes marked by the rs1053989 SNP in all three races. This SNP predicted significant birth weight variation after adjustment for gestational age, maternal BMI, parity, and smoking. African American and Hispanic mothers carrying the A allele had infants whose birth weight was on average 254 and 302 grams, respectively, less than infants having C/C mothers. Non-Hispanic White mothers homozygous for the A allele had infants who were on average 148 grams less than those infants having A/C and C/C mothers. CONCLUSIONS: The magnitudes of the estimates of the birth weight effects are comparable to the combined effects of multiple SNPs reported in a recent meta-analysis of 6 GWAS studies and is quantitatively larger than that associated with maternal cigarette smoking. This effect was persistent across subpopulations that vary with respect to ancestry and environment.

  2. Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein.

    Słomińska, Monika; Konopa, Grazyna; Wegrzyn, Grzegorz; Czyz, Agata


    The Vibrio harveyi cgtA gene product belongs to a subfamily of small GTP-binding proteins, called Obg-like proteins. Members of this subfamily are present in diverse organisms ranging from bacteria to humans. On the other hand, the functions of these proteins in the regulation of cellular processes are largely unknown. Genes coding for these proteins are essential in almost all bacteria investigated thus far. However, a viable V. harveyi insertional mutant in the cgtA gene was described recently. Therefore, this mutant gives a unique opportunity to study functions of a member of the subfamily of Obg-like proteins. Here we demonstrate that the mutant cells often form long filaments with expanded, non-partitioned or rarely partitioned chromosomes. Such a phenotype suggests impairment of the mechanism of chromosome partition. Flow cytometric studies revealed that synchronization of chromosome replication initiation is also significantly disturbed in the cgtA mutant. Moreover, in contrast to wild-type V. harveyi, inhibition of chromosome replication and/or of cell division in the mutant bacteria caused significant increase in the number of large cells, suggesting that the cgtA gene product may be involved in the coupling of cell growth to chromosome replication and cell division. These results indicate that CgtA, an Obg-like GTP-binding protein, plays an important role in the regulation of chromosomal functions. PMID:11879184

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

    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


    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

  4. Myristylation alters DNA-binding activity and transactivation of FBR (gag-fos) protein.

    Kamata, N; Jotte, R M; Holt, J. T.


    FBR murine sarcoma virus (gag-fos) protein, a virally transduced Fos protein, exhibits decreased gene transactivation in comparison with the cellular Fos protein. Biochemical analysis suggests that myristylation of the virally encoded N-terminal gag region results in decreased DNA binding and transcriptional activation without affecting heterodimerization with Jun protein. These findings demonstrate that protein myristylation can modulate gene regulation by a DNA-binding protein.

  5. DNA binding sites recognised in vitro by a knotted class 1 homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare)

    Krusell, L; Rasmussen, I; Gausing, K


    The homeodomain of the knotted classes of transcription factors from plants differs from the well characterized Antp/En type homeodomains from Drosophila at key amino acid residues contributing to the DNA binding. A cDNA, Hvh21, derived from the hooded gene and encoding a full length homolog of...

  6. T(lys), a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes.

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta; Contursi, Patrizia


    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (T(lys)) was identified. Transcriptional analysis revealed that T(lys) is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and T(ind)) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and T(ind) transcripts, as well as of its own promoter. Binding sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the T(ind) promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1. PMID:23514883

  7. Identification of a Bipartite Jasmonate-Responsive Promoter Element in the Catharanthus roseus ORCA3 Transcription Factor Gene That Interacts Specifically with AT-Hook DNA-Binding Proteins1[W

    Vom Endt, Débora; Soares e Silva, Marina; Kijne, Jan W.; Pasquali, Giancarlo; Memelink, Johan


    Jasmonates are plant signaling molecules that play key roles in defense against certain pathogens and insects, among others, by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the APETALA2-domain transcription factor ORCA3 is involved in the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter, which contains an autonomous jasmonate-responsive element (JRE). The ORCA3 JRE is composed of two important sequences: a quantitative sequence responsible for a high level of expression and a qualitative sequence that appears to act as an on/off switch in response to methyl jasmonate. We isolated 12 different DNA-binding proteins having one of four different types of DNA-binding domains, using the ORCA3 JRE as bait in a yeast (Saccharomyces cerevisiae) one-hybrid transcription factor screening. The binding of one class of proteins bearing a single AT-hook DNA-binding motif was affected by mutations in the quantitative sequence within the JRE. Two of the AT-hook proteins tested had a weak activating effect on JRE-mediated reporter gene expression, suggesting that AT-hook family members may be involved in determining the level of expression of ORCA3 in response to jasmonate. PMID:17496112

  8. Mercury-binding proteins of Mytilus edulis

    Roesijadi, G.; Morris, J. E.; Calabrese, A.


    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  9. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.

    Marine, J; Winoto, A


    The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein ...

  10. Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss)CCAAT/enhancer binding protein genes and their responses to induction by GH in vitro and in vivo

    CCAAT/enhancer-binding proteins (C/EBPs) are transcription factors consisting of six isoforms and play diverse physiological roles in vertebrates. In rainbow trout (Oncorhynchus mykiss), in addition to the reported C/EBPbeta1,we have isolated cDNA of four other isoforms, C/EBPalpha, C/EBPbeta2, C/E...