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Sample records for nuclear encoded gene

  1. The murine Sry gene encodes a nuclear transcriptional activator

    SciTech Connect

    Dubin, R.A.; Ostrer, H.

    1994-09-01

    The Sry gene functions as a genetic switch in gonadal ridge initiating testis determination. The murine Sry and human SRY open reading frames (ORF) share a conserved 79 amino acid motif, the HMG-box, that binds DNA. Outside this region the two genes share no additional homology. These studies were undertaken to determine whether the Sry/SRY genes encode nuclear transcriptional regulators. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and murine SRY ORFs contain a nuclear localization signal. The murine Sry HMG-box selectively binds the sequence NACAAT in vitro when presented with a random pool of oligonucleotides and binds AACAAT with the highest affinity. The murine Sry ORF, when expressed in HeLa cells, activates transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was observed for a GAL4-responsive gene when the murine Sry ORF was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a C-terminal glutamine/histidine-rich domain. In addition, LexA-Sry fusion genes activated a LexA-responsive gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and mouse SRY ORFs encode nuclear, DNA-binding proteins, and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  2. Nuclear genes encoding plastid proteins expressed early in chloroplast development

    SciTech Connect

    Mullet, J.E.

    1991-01-01

    The overall objective of this grant was to characterize events which occur early in chloroplast biogenesis and to isolate nuclear genes encoding plastid proteins which are expressed during this developmental phase. In addition, the possible requirement of plastid transcription for the expression of the nuclear genes such as rbcS and cab was to be tested. The impetus for this research came from studies of chloroplast biogenesis in barley. We found that plastid DNA copy number was relatively high (120 copies/plastid vs 200 at maximal accumulation) in the meristematic region of the leaf base whereas plastid transcription activity was low in this plastid population. Later in chloroplast development transcription activity increased at least 5 fold per plastid or per template indicating that activation of plastid transcription occurred after most of the build up in plastid DNA per plastid. This suggested that activation of plastid DNA synthesis occurred early in chloroplast development and that nuclear genes involved in this activity must be regulated differently from genes such rbcS or cab which are expressed later in development. 3 refs., 7 figs.

  3. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5' UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  4. (Structure and expression of nuclear genes encoding rubisco activase)

    SciTech Connect

    Zielinski, R.E.

    1990-01-01

    Our activities during the past year have centered around two basic aspects of the project: describing more thoroughly the diurnal and light irradiance effects on activase gene expression in barley; and isolating and structurally characterizing cDNA and genomic DNA sequences encoding activase from barley. Three appendices are included that summarize these activities.

  5. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  6. Gene set of nuclear-encoded mitochondrial regulators is enriched for common inherited variation in obesity.

    PubMed

    Knoll, Nadja; Jarick, Ivonne; Volckmar, Anna-Lena; Klingenspor, Martin; Illig, Thomas; Grallert, Harald; Gieger, Christian; Wichmann, Heinz-Erich; Peters, Annette; Hebebrand, Johannes; Scherag, André; Hinney, Anke

    2013-01-01

    There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50(th) and 95(th) percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50(th) percentile for the set of the 16 nuclear regulators of mitochondrial genes (p(GSEA,50) = 0.0103). This finding was not confirmed in the trios (p(GSEA,50) = 0.5991), but in KORA (p(GSEA,50) = 0.0398). The meta-analysis again indicated a trend for enrichment (p(MAGENTA,50) = 0.1052, p(MAGENTA,75) = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes.

  7. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1990--June 30, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5` UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  8. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems.

  9. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.

    PubMed

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-04-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.

  10. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    PubMed

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  11. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  12. Analyses of nuclearly encoded mitochondrial genes suggest gene duplication as a mechanism for resolving intralocus sexually antagonistic conflict in Drosophila.

    PubMed

    Gallach, Miguel; Chandrasekaran, Chitra; Betrán, Esther

    2010-01-01

    Gene duplication is probably the most important mechanism for generating new gene functions. However, gene duplication has been overlooked as a potentially effective way to resolve genetic conflicts. Here, we analyze the entire set of Drosophila melanogaster nuclearly encoded mitochondrial duplicate genes and show that both RNA- and DNA-mediated mitochondrial gene duplications exhibit an unexpectedly high rate of relocation (change in location between parental and duplicated gene) as well as an extreme tendency to avoid the X chromosome. These trends are likely related to our observation that relocated genes tend to have testis-specific expression. We also infer that these trends hold across the entire Drosophila genus. Importantly, analyses of gene ontology and functional interaction networks show that there is an overrepresentation of energy production-related functions in these mitochondrial duplicates. We discuss different hypotheses to explain our results and conclude that our findings substantiate the hypothesis that gene duplication for male germline function is likely a mechanism to resolve intralocus sexually antagonistic conflicts that we propose are common in testis. In the case of nuclearly encoded mitochondrial duplicates, our hypothesis is that past sexually antagonistic conflict related to mitochondrial energy function in Drosophila was resolved by gene duplication.

  13. Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

    PubMed

    Troll, H; Winckler, T; Lascu, I; Müller, N; Saurin, W; Véron, M; Mutzel, R

    1993-12-05

    We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

  14. Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin

    SciTech Connect

    Kurooka, Hisanori; Kato, Keizo; Minoguchi, Shigeru

    1997-02-01

    In a yeast artificial chromosome contig close to the nude locus on mouse chromosome 11, we identified a novel gene, nucleoredoxin, that encodes a protein with similarity to the active site of thioredoxins. Nucleoredoxin is conserved between mammalian species, and two homologous genes were found in Caenorhabditis elegans. The nucleoredoxin transcripts are expressed in all adult tissues examined, but restricted to the nervous system and the limb buds in Day 10.5-11.5 embryos. The nucleoredoxin protein is predominantly localized in the nucleus of cells transfected with the nucleoredoxin expression construct. Since the bacterially expressed protein of nucleoredoxin showed oxidoreductase activity of the insulin disulfide bonds with kinetics similar to that of thioredoxin, it may be a redox regulator of the nuclear proteins, such as transcription factors. 40 refs., 6 figs.

  15. Parkinson's disease in relation to pesticide exposure and nuclear encoded mitochondrial complex I gene variants.

    PubMed

    Corder, Elizabeth H; Mellick, George D

    2006-01-01

    Parkinson's disease (PD) is a common age-related neurodegenerative disorder thought to result from the integrated effects of genetic background and exposure to neuronal toxins. Certain individual nuclear-encoded mitochondrial complex I gene polymorphisms were found to be associated with approximately 2-fold risk variation in an Australian case-control sample. We further characterized this sample of 306 cases and 321 controls to determine the mutual information contained in the 22 SNPs and, additionally, level of pesticide exposure: five distinct risk sets were identified using grade-of-membership analysis. Of these, one was robust to pesticide exposure (I), three were vulnerable (II, III, IV), and another (V) denoted low risk for unexposed persons. Risk for individual subjects varied > 16-fold according to level of membership in the vulnerable groups. We conclude that inherited variation in mitochondrial complex I genes and pesticide exposure together modulate risk for PD.

  16. Characterization of the nuclear gene encoding mitochondrial aconitase in the marine red alga Gracilaria verrucosa.

    PubMed

    Zhou, Y H; Ragan, M A

    1995-07-01

    We have cloned a nuclear gene from the marine red alga Gracilaria verrucosa that encodes the complete 779 amino-acid mitochondrial aconitase (m-ACN), the first characterized from a photosynthetic organism. The N-terminal 28 deduced amino acids are predicted to constitute the mitochondrial transit peptide, the first described from a red alga. Putative transcriptional cis-acting elements were identified in the upstream untranslated region. The G. verrucosa m-ACN gene (m-ACN) is present in a single copy and is located ca. 1.5 kb upstream from the single-copy polyubiquitin gene. The single spliceosomal intron is located near the 5' end of the region encoding the mature m-ACN in precisely the same location and phase as intron 2 in Caenorhabditis elegans m-ACN; sequences at its 3' and 5' splice junctions and at the predicted lariat branch point conform well to the eukaryote consensus sequences. Multiple protein-sequence alignment of m-ACN, bacterial aconitase (b-ACN) and iron-responsive element-binding protein (IRE-BP), and phylogenetic analyses, revealed that m-ACN does not share a recent common ancestry with either b-ACN or IRE-BP.

  17. Effects of TCDD on the expression of nuclear encoded mitochondrial genes

    SciTech Connect

    Forgacs, Agnes L.; Burgoon, Lyle D.; Lynn, Scott G.; LaPres, John J.; Zacharewski, Timothy

    2010-07-15

    Generation of mitochondrial reactive oxygen species (ROS) can be perturbed following exposure to environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Reports indicate that the aryl hydrocarbon receptor (AhR) mediates TCDD-induced sustained hepatic oxidative stress by decreasing hepatic ATP levels and through hyperpolarization of the inner mitochondrial membrane. To further elucidate the effects of TCDD on the mitochondria, high-throughput quantitative real-time PCR (HTP-QRTPCR) was used to evaluate the expression of 90 nuclear genes encoding mitochondrial proteins involved in electron transport, oxidative phosphorylation, uncoupling, and associated chaperones. HTP-QRTPCR analysis of time course (30 {mu}g/kg TCDD at 2, 4, 8, 12, 18, 24, 72, and 168 h) liver samples obtained from orally gavaged immature, ovariectomized C57BL/6 mice identified 54 differentially expressed genes (|fold change| > 1.5 and P-value < 0.1). Of these, 8 exhibited a sigmoidal or exponential dose-response profile (0.03 to 300 {mu}g/kg TCDD) at 4, 24 or 72 h. Dose-responsive genes encoded proteins associated with electron transport chain (ETC) complexes I (NADH dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) and could be generally categorized as having proton gradient, ATP synthesis, and chaperone activities. In contrast, transcript levels of ETC complex II, succinate dehydrogenase, remained unchanged. Putative dioxin response elements were computationally found in the promoter regions of all 8 dose-responsive genes. This high-throughput approach suggests that TCDD alters the expression of genes associated with mitochondrial function which may contribute to TCDD-elicited mitochondrial toxicity.

  18. Differential expression of nuclear- and organelle-encoded genes during tomato fruit development.

    PubMed

    Piechulla, B

    1988-12-01

    Steady-state mRNA levels of nuclear-and organelle-encoded genes were determined during fruit development and ripening. Transcripts specific for subunits of the mitochondrial and chloroplast ATPase complexes appear simultaneously and reach high levels two to three weeks after anthesis, but follow a different expression pattern during the ripening period. While the chloroplast-specific mRNA levels continuously decrease to low levels in ripe tomato fruits, the transcripts specific for two mitochondrial ATPase subunits continue to be present at relative high levels in red fruits. Transcript levels for the fructose-1,6-bisphosphate aldolase increase significantly during ripening. Structural proteins such as the alpha-subunit of tubulin and the hydroxyproline-rich glycoprotein extensin are expressed during maximal fruit growth. In addition, comparisons of mRNA levels of different genes in several plant organs (leaf, fruit, stem, and root) show characteristic differences. The results presented in this paper demonstrate that changes at the transcriptional or post-transcriptional level during fruit development can be correlated with morphological and physiological alterations.

  19. Changes in mitochondrial DNA alter expression of nuclear encoded genes associated with tumorigenesis

    SciTech Connect

    Jandova, Jana; Janda, Jaroslav; Sligh, James E

    2012-10-15

    We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-{kappa}B (NF-{kappa}B) is a key transcription factor for production of MMPs. An inhibitor of NF-{kappa}B activation, Bay 11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-{kappa}B in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids. -- Highlights: Black-Right-Pointing-Pointer Cybrids are useful models to study the role of mtDNA changes in cancer development. Black-Right-Pointing-Pointer mtDNA changes affect the expression of nuclear

  20. Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

    PubMed Central

    Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

  1. Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae.

    PubMed

    Koyama, Yosuke; Takimoto, Koji; Kojima, Asuka; Asai, Kei; Matsuoka, Satoshi; Mitsui, Toshiaki; Matsumoto, Kouji; Hara, Hiroshi; Ohta, Niji

    2011-01-01

    SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.

  2. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    SciTech Connect

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  3. Hypothalamic and amygdalar cell lines differ markedly in mitochondrial rather than nuclear encoded gene expression

    PubMed Central

    2013-01-01

    Background Corticotropin-releasing hormone (CRH) plays an important role in regulating the mammalian stress response. Two of the most extensively studied neuronal populations that express CRH are in the hypothalamus and amygdala. Both regions are involved in the stress response, but the amygdala is also involved in mediating response to fear and anxiety. Given that both hypothalamus and amygdala have overlapping functions, but their CRH-expressing neurons may respond differently to a given perturbation, we sought to identify differentially expressed genes between two neuronal cell types, amygdalar AR-5 and hypothalamic IVB cells. Thus, we performed a microarray analysis. Our hypothesis was that we would identify differentially expressed transcription factors, coregulators and chromatin-modifying enzymes. Results A total of 31,042 genes were analyzed, 10,572 of which were consistently expressed in both cell lines at a 95% confidence level. Of the 10,572 genes, 2,320 genes in AR-5 were expressed at ≥ 2-fold relative to IVBs, 1,104 genes were expressed at ≥2-fold in IVB relative to AR-5 and 7,148 genes were expressed at similar levels between the two cell lines. The greatest difference was in six mitochondrial DNA-encoded genes, which were highly abundant in AR-5 relative to IVB cells. The relative abundance of these genes ranged from 413 to 885-fold according to the microarray results. Differential expression of these genes was verified by RTqPCR. The differentially expressed mitochondrial genes were cytochrome b (MT-CYB), cytochrome c oxidase subunit 1 and 2 (MT-CO1 and MT-CO2) and NADH-ubiquinone oxidoreductase chain 1, 2, and 3 (MT-ND1, MT-ND2, MT-ND3). Conclusion As expected, the array revealed differential expression of transcription factors and coregulators; however the greatest difference between the two cell lines was in genes encoded by the mitochondrial genome. These genes were abundant in AR-5 relative to IVBs. At present, the reason for the marked

  4. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

    PubMed Central

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene. PMID:25978564

  5. Reconstruction of Family-Level Phylogenetic Relationships within Demospongiae (Porifera) Using Nuclear Encoded Housekeeping Genes

    PubMed Central

    Hill, Malcolm S.; Hill, April L.; Lopez, Jose; Peterson, Kevin J.; Pomponi, Shirley; Diaz, Maria C.; Thacker, Robert W.; Adamska, Maja; Boury-Esnault, Nicole; Cárdenas, Paco; Chaves-Fonnegra, Andia; Danka, Elizabeth; De Laine, Bre-Onna; Formica, Dawn; Hajdu, Eduardo; Lobo-Hajdu, Gisele; Klontz, Sarah; Morrow, Christine C.; Patel, Jignasa; Picton, Bernard; Pisani, Davide; Pohlmann, Deborah; Redmond, Niamh E.; Reed, John; Richey, Stacy; Riesgo, Ana; Rubin, Ewelina; Russell, Zach; Rützler, Klaus; Sperling, Erik A.; di Stefano, Michael; Tarver, James E.; Collins, Allen G.

    2013-01-01

    Background Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. Methodology/Principal Findings We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosap, Myxospongiaep, Spongillidap, Haploscleromorphap (the marine haplosclerids) and Democlaviap. We found conflicting results concerning the relationships of Keratosap and Myxospongiaep to the remaining demosponges, but our results strongly supported a clade of Haploscleromorphap+Spongillidap+Democlaviap. In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillidap) are sister to Haploscleromorphap rather than part of Democlaviap. Within Keratosap, we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiaep, Chondrosida and Verongida were monophyletic. A well-supported clade within Democlaviap, Tetractinellidap, composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlaviap. Within Tetractinellidap, we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. Conclusions/Significance These results, using an

  6. The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development.

    PubMed

    Schoor, M; Schuster-Gossler, K; Gossler, A

    1993-07-01

    Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.

  7. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    PubMed

    Savage, Linda J; Imre, Kathleen M; Hall, David A; Last, Robert L

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

  8. Analysis of Essential Arabidopsis Nuclear Genes Encoding Plastid-Targeted Proteins

    PubMed Central

    Savage, Linda J.; Imre, Kathleen M.; Hall, David A.; Last, Robert L.

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ∼1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified

  9. Reconstruction of family-level phylogenetic relationships within Demospongiae (Porifera) using nuclear encoded housekeeping genes.

    PubMed

    Hill, Malcolm S; Hill, April L; Lopez, Jose; Peterson, Kevin J; Pomponi, Shirley; Diaz, Maria C; Thacker, Robert W; Adamska, Maja; Boury-Esnault, Nicole; Cárdenas, Paco; Chaves-Fonnegra, Andia; Danka, Elizabeth; De Laine, Bre-Onna; Formica, Dawn; Hajdu, Eduardo; Lobo-Hajdu, Gisele; Klontz, Sarah; Morrow, Christine C; Patel, Jignasa; Picton, Bernard; Pisani, Davide; Pohlmann, Deborah; Redmond, Niamh E; Reed, John; Richey, Stacy; Riesgo, Ana; Rubin, Ewelina; Russell, Zach; Rützler, Klaus; Sperling, Erik A; di Stefano, Michael; Tarver, James E; Collins, Allen G

    2013-01-01

    Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosa(p), Myxospongiae(p), Spongillida(p), Haploscleromorpha(p) (the marine haplosclerids) and Democlavia(p). We found conflicting results concerning the relationships of Keratosa(p) and Myxospongiae(p) to the remaining demosponges, but our results strongly supported a clade of Haploscleromorpha(p)+Spongillida(p)+Democlavia(p). In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillida(p)) are sister to Haploscleromorpha(p) rather than part of Democlavia(p). Within Keratosa(p), we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiae(p), Chondrosida and Verongida were monophyletic. A well-supported clade within Democlavia(p), Tetractinellida(p), composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlavia(p). Within Tetractinellida(p), we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. These results, using an independent nuclear gene set

  10. Downregulation of Nuclear-Encoded Genes of Oxidative Metabolism in Dialyzed Chronic Kidney Disease Patients

    PubMed Central

    Masola, Valentina; Rugiu, Carlo; Fantin, Francesco; Gesualdo, Loreto; Schena, Francesco Paolo; Lupo, Antonio

    2013-01-01

    Background Mitochondria, essential eukaryotic cells organelles defined as the “powerhouse of the cell” because of their ability to produce the vast majority of energy necessary for cellular metabolism, may have a primary role in the oxidative stress-related intracellular machinery associated to chronic kidney disease (CKD). Methods To better assess this research assumption, we decided to study the key factors regulating mitochondrial oxidative metabolism in CKD patients in peritoneal dialysis (PD, n = 15) using several bio-molecular methodologies. Results RT-PCR experiments demonstrate that the expression level of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor-1 (NRF-1), two genes primarily involved in mitochondrial biogenesis and functions, were significantly hypo-expressed in peripheral blood mononuclear cells of PD patients compared to healthy subjects (HS, n = 15). Additionally, mRNA levels of several PGC1-α downstream target genes (TFAM, COX6C,COX7C, UQCRH and MCAD) were profoundly down-regulated in PD cells. TFAM protein analysis confirmed gene-expression results. High plasmatic concentration of Malondialdehyde found in PD patients, confirmed the contribution of the oxidative stress to these biological effects. Finally, Nuclear factor erythroid-derived 2-like 2 (NRF2 or NFE2L2), a transcription factor for numerous antioxidant/detoxifying enzymes and one of its target genes, superoxide dismutase-2 mitochondrial (SOD2) were up-regulated in PD compared to HS. Conclusions Our results revealed, for the first time, that CKD-PD patients’ PBMC, through a complex intracellular biochemical machinery, are able to modulate their mitochondrial functions probably in the attempt to reduce oxidative metabolic damage and to turn on a valuable defense cellular strategy against oxidative stress. PMID:24204994

  11. (Structure and expression of nuclear genes encoding rubisco activase): Progress report

    SciTech Connect

    Not Available

    1989-01-01

    Our first year's activities include: (1) completing a survey of the basic characteristics of activase gene expression in barley; and (2) isolating and structurally characterizing cDNA and genomic DNA sequences encoding activase from barley. Our goal was to determine whether activase mRNA and protein accumulation are coordinated with those of the rubisco subunits. We utilized the first leaves of barley as an experimental system for these studies because they can be used in two ways to study the expression of leaf genes: by following the naturally occurring differentiation of leaf cells, which occurs acropetally along the barley leaf; and by following the photomorphogenesis of etiolated barley seedlings. In the acropetal gradient of leaf cell differentiation, activase mRNA and mRNA and polypeptide expression is tightly coordinated with rubisco subunit mRNA and polypeptide expression. Although we have not measured their precise stoichiometry at each stage of leaf differentiation, activase protein is expressed at the level of about one polypeptide per rubisco holoenzyme in mature regions of the leaf. Coordination of the expression of activase mRNAs and polypeptides indicates that in the barley leaf gradient, activase gene expression is largely controlled at the level of transcription. However, translational controls may play a role in regulating activase expression on a short term basis.

  12. Regulation of the nuclear genes encoding the cytoplasmic and mitochondrial leucyl-tRNA synthetases of Neurospora crassa.

    PubMed Central

    Chow, C M; Rajbhandary, U L

    1989-01-01

    We show that the nuclear genes for the cytoplasmic and mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa are distinct in their encoded proteins, codon usage, mRNA levels, and regulation. The 4.2-kilobase-pair region representing the structural gene for cytoplasmic LeuRS and flanking regions has been sequenced. The positions of the 5' and 3' ends of mRNA and of a single 62-base-pair intron have been mapped. The methionine-initiated open reading frame encoded a protein of 1,123 amino acids and displayed a strong codon bias. Although cytoplasmic LeuRS shares with mitochondrial LeuRS some general features common to most aminoacyl-tRNA synthetases, there is little amino acid sequence similarity between them, mRNA levels for cytoplasmic LeuRS were much higher than those for mitochondrial LeuRS. This observation and the strong codon bias in the cytoplasmic LeuRS gene may contribute to a greater abundance of cytoplasmic LeuRS than mitochondrial LeuRS. The genes for cytoplasmic and mitochondrial LeuRS are regulated independently. The cytoplasmic LeuRS gene is regulated by the cross-pathway control system in N. crassa, which is analogous to general amino acid control in Saccharomyces cerevisiae. The cytoplasmic LeuRS mRNA levels are induced by amino acid starvation resulting from the addition of aminotriazole. Part of this increase is due to utilization of new transcription start sites. In contrast, the mitochondrial LeuRS gene is not induced by amino acid limitation. However, the mitochondrial LeuRS mRNA levels did increase dramatically upon inhibition of mitochondrial protein synthesis by chloramphenicol or ethidium bromide or in the temperature-sensitive strain leu-5 carrying a mutation in the mitochondrial LeuRS structural gene. Images PMID:2532300

  13. Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation.

    PubMed

    Fujimori, A; Matsuda, Y; Takemoto, Y; Hashimoto, Y; Kubo, E; Araki, R; Fukumura, R; Mita, K; Tatsumi, K; Muto, M

    1998-12-01

    We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a lambdagt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif "IXCXE". The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2-q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication.

  14. STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs.

    PubMed Central

    Wang, S S; Stanford, D R; Silvers, C D; Hopper, A K

    1992-01-01

    STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing. Images PMID:1588961

  15. AtWRKY40 and AtWRKY63 Modulate the Expression of Stress-Responsive Nuclear Genes Encoding Mitochondrial and Chloroplast Proteins1[W][OA

    PubMed Central

    Van Aken, Olivier; Zhang, Botao; Law, Simon; Narsai, Reena; Whelan, James

    2013-01-01

    The expression of a variety of nuclear genes encoding mitochondrial proteins is known to adapt to changes in environmental conditions and retrograde signaling. The presence of putative WRKY transcription factor binding sites (W-boxes) in the promoters of many of these genes prompted a screen of 72 annotated WRKY factors in the Arabidopsis (Arabidopsis thaliana) genome for regulators of transcripts encoding mitochondrial proteins. A large-scale yeast one-hybrid screen was used to identify WRKY factors that bind the promoters of marker genes (Alternative oxidase1a, NADH dehydrogenaseB2, and the AAA ATPase Ubiquinol-cytochrome c reductase synthesis1), and interactions were confirmed using electromobility shift assays. Transgenic overexpression and knockout lines for 12 binding WRKY factors were generated and tested for altered expression of the marker genes during normal and stress conditions. AtWRKY40 was found to be a repressor of antimycin A-induced mitochondrial retrograde expression and high-light-induced signaling, while AtWRKY63 was identified as an activator. Genome-wide expression analysis following high-light stress in transgenic lines with perturbed AtWRKY40 and AtWRKY63 function revealed that these factors are involved in regulating stress-responsive genes encoding mitochondrial and chloroplast proteins but have little effect on more constitutively expressed genes encoding organellar proteins. Furthermore, it appears that AtWRKY40 and AtWRKY63 are particularly involved in regulating the expression of genes responding commonly to both mitochondrial and chloroplast dysfunction but not of genes responding to either mitochondrial or chloroplast perturbation. In conclusion, this study establishes the role of WRKY transcription factors in the coordination of stress-responsive genes encoding mitochondrial and chloroplast proteins. PMID:23509177

  16. AtWRKY40 and AtWRKY63 modulate the expression of stress-responsive nuclear genes encoding mitochondrial and chloroplast proteins.

    PubMed

    Van Aken, Olivier; Zhang, Botao; Law, Simon; Narsai, Reena; Whelan, James

    2013-05-01

    The expression of a variety of nuclear genes encoding mitochondrial proteins is known to adapt to changes in environmental conditions and retrograde signaling. The presence of putative WRKY transcription factor binding sites (W-boxes) in the promoters of many of these genes prompted a screen of 72 annotated WRKY factors in the Arabidopsis (Arabidopsis thaliana) genome for regulators of transcripts encoding mitochondrial proteins. A large-scale yeast one-hybrid screen was used to identify WRKY factors that bind the promoters of marker genes (Alternative oxidase1a, NADH dehydrogenaseB2, and the AAA ATPase Ubiquinol-cytochrome c reductase synthesis1), and interactions were confirmed using electromobility shift assays. Transgenic overexpression and knockout lines for 12 binding WRKY factors were generated and tested for altered expression of the marker genes during normal and stress conditions. AtWRKY40 was found to be a repressor of antimycin A-induced mitochondrial retrograde expression and high-light-induced signaling, while AtWRKY63 was identified as an activator. Genome-wide expression analysis following high-light stress in transgenic lines with perturbed AtWRKY40 and AtWRKY63 function revealed that these factors are involved in regulating stress-responsive genes encoding mitochondrial and chloroplast proteins but have little effect on more constitutively expressed genes encoding organellar proteins. Furthermore, it appears that AtWRKY40 and AtWRKY63 are particularly involved in regulating the expression of genes responding commonly to both mitochondrial and chloroplast dysfunction but not of genes responding to either mitochondrial or chloroplast perturbation. In conclusion, this study establishes the role of WRKY transcription factors in the coordination of stress-responsive genes encoding mitochondrial and chloroplast proteins.

  17. Modulation of expression of genes encoding nuclear proteins following exposure to JANUS neutrons or {gamma}-rays

    SciTech Connect

    Woloschak, G.E.; Chang-Liu, Chin-Mei

    1994-05-01

    Previous work has shown that exposure of cells to ionizing radiations causes modulation of a variety of genes, including those encoding c-fos, interleukin-1, tumor necrosis factor, and cytoskeletal elements. The experiments reported herein were designed to examine the effects of either JANUS neutron or {gamma}-ray exposure on expression of genes encoding nucleus-associated proteins (H4-histone, c-jun, c-myc, Rb, and p53). Cycling Syrian hamster embryo cells were irradiated with varying doses and dose rates of either JANUS fission-spectrum neutrons or {gamma}-rays; after incubation of the cell cultures for 1 h following radiation exposure, mRNA was harvested and analyzed by Northern blot. Results revealed induction of transcripts for c-jun, H4-histone, and (to a lesser extent) Rb following {gamma}-ray but not following neutron exposure. Expression of p53 and c-myc genes was unaffected by radiation exposure. Radiations at different doses and dose rates were compared for each of the genes studied.

  18. Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

    PubMed

    Brunsch, Melanie; Schubert, Daniela; Gube, Matthias; Ring, Christiane; Hanisch, Lisa; Linde, Jörg; Krause, Katrin; Kothe, Erika

    2015-01-01

    The white-rot fungus Schizophyllum commune (Agaricomycetes) was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

  19. Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune

    PubMed Central

    Gube, Matthias; Ring, Christiane; Hanisch, Lisa; Linde, Jörg; Krause, Katrin; Kothe, Erika

    2015-01-01

    The white-rot fungus Schizophyllum commune (Agaricomycetes) was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing. PMID:26284622

  20. Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: a novel gene related to nuclear envelopathies.

    PubMed

    Kayman-Kurekci, Gulsum; Talim, Beril; Korkusuz, Petek; Sayar, Nilufer; Sarioglu, Turkan; Oncel, Ibrahim; Sharafi, Parisa; Gundesli, Hulya; Balci-Hayta, Burcu; Purali, Nuhan; Serdaroglu-Oflazer, Piraye; Topaloglu, Haluk; Dincer, Pervin

    2014-07-01

    We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patient's muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle.

  1. The nuclear-encoded sigma factor SIG4 directly activates transcription of chloroplast psbA and ycf17 genes in the unicellular red alga Cyanidioschyzon merolae.

    PubMed

    Fujii, Gaku; Imamura, Sousuke; Era, Atsuko; Miyagishima, Shin-ya; Hanaoka, Mitsumasa; Tanaka, Kan

    2015-05-01

    The plant organelle chloroplast originated from the endosymbiosis of a cyanobacterial-like photosynthetic bacterium, and still retains its own genome derived from this ancestor. We have been focusing on a unicellular red alga, Cyanidioschyzon merolae, as a model photosynthetic eukaryote. In this study, we analyzed the transcriptional specificity of SIG4, which is one of four nuclear-encoded chloroplast RNA polymerase sigma factors in this alga. Accumulation of the SIG4 protein was observed in response to nitrogen depletion or high light conditions. By comparing the chloroplast transcriptomes under nitrogen depletion and SIG4-overexpressing conditions, we identified several candidate genes as SIG4 targets. Together with the results of chromatin immunoprecipitation analysis, the promoters of the psbA (encoding the D1 protein of the photosystem II reaction center) and ycf17 (encoding a protein of the early light-inducible protein family) genes were shown to be direct activation targets. The phycobilisome (PBS) CpcB protein was decreased by SIG4 overexpression, which suggests the negative involvement of SIG4 in PBS accumulation. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Autosomal-Dominant Distal Myopathy Associated with a Recurrent Missense Mutation in the Gene Encoding the Nuclear Matrix Protein, Matrin 3

    PubMed Central

    Senderek, Jan; Garvey, Sean M.; Krieger, Michael; Guergueltcheva, Velina; Urtizberea, Andoni; Roos, Andreas; Elbracht, Miriam; Stendel, Claudia; Tournev, Ivailo; Mihailova, Violeta; Feit, Howard; Tramonte, Jeff; Hedera, Peter; Crooks, Kristy; Bergmann, Carsten; Rudnik-Schöneborn, Sabine; Zerres, Klaus; Lochmüller, Hanns; Seboun, Eric; Weis, Joachim; Beckmann, Jacques S.; Hauser, Michael A.; Jackson, Charles E.

    2009-01-01

    Distal myopathies represent a heterogeneous group of inherited skeletal muscle disorders. One type of adult-onset, progressive autosomal-dominant distal myopathy, frequently associated with dysphagia and dysphonia (vocal cord and pharyngeal weakness with distal myopathy [VCPDM]), has been mapped to chromosome 5q31 in a North American pedigree. Here, we report the identification of a second large VCPDM family of Bulgarian descent and fine mapping of the critical interval. Sequencing of positional candidate genes revealed precisely the same nonconservative S85C missense mutation affecting an interspecies conserved residue in the MATR3 gene in both families. MATR3 is expressed in skeletal muscle and encodes matrin 3, a component of the nuclear matrix, which is a proteinaceous network that extends throughout the nucleus. Different disease related haplotype signatures in the two families provided evidence that two independent mutational events at the same position in MATR3 cause VCPDM. Our data establish proof of principle that the nuclear matrix is crucial for normal skeletal muscle structure and function and put VCPDM on the growing list of monogenic disorders associated with the nuclear proteome. PMID:19344878

  3. DELAYED GREENING 238, a Nuclear-Encoded Chloroplast Nucleoid Protein, Is Involved in the Regulation of Early Chloroplast Development and Plastid Gene Expression in Arabidopsis thaliana.

    PubMed

    Wang, Menglong; Jiang, Lan; Da, Qingen; Liu, Jun; Feng, Dongru; Wang, Jinfa; Wang, Hong-Bin; Jin, Hong-Lei

    2016-12-01

    Chloroplast development is an essential process for plant growth that is regulated by numerous proteins. Plastid-encoded plastid RNA polymerase (PEP) is a large complex that regulates plastid gene transcription and chloroplast development. However, many proteins in this complex remain to be identified. Here, through large-scale screening of Arabidopsis mutants by Chl fluorescence imaging, we identified a novel protein, DELAYED GREENING 238 (DG238), which is involved in regulating chloroplast development and plastid gene expression. Loss of DG238 retards plant growth, delays young leaf greening, affects chloroplast development and lowers photosynthetic efficiency. Moreover, blue-native PAGE (BN-PAGE) and Western blot analysis indicated that PSII and PSI protein levels are reduced in dg238 mutants. DG238 is mainly expressed in young tissues and is regulated by light signals. Subcellular localization analysis showed that DG238 is a nuclear-encoded chloroplast nucleoid protein. More interestingly, DG238 was co-expressed with FLN1, which encodes an essential subunit of the PEP complex. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays showed that DG238 can also interact with FLN1. Taken together, these results suggest that DG238 may function as a component of the PEP complex that is important for the early stage of chloroplast development and helps regulate PEP-dependent plastid gene expression. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes.

    PubMed

    Kodama, Susumu; Koike, Chika; Negishi, Masahiko; Yamamoto, Yukio

    2004-09-01

    The nuclear receptors CAR and PXR activate hepatic genes in response to therapeutic drugs and xenobiotics, leading to the induction of drug-metabolizing enzymes, such as cytochrome P450. Insulin inhibits the ability of FOXO1 to express genes encoding gluconeogenic enzymes. Induction by drugs is known to be decreased by insulin, whereas gluconeogenic activity is often repressed by treatment with certain drugs, such as phenobarbital (PB). Performing cell-based transfection assays with drug-responsive and insulin-responsive enhancers, glutathione S-transferase pull down, RNA interference (RNAi), and mouse primary hepatocytes, we examined the molecular mechanism by which nuclear receptors and FOXO1 could coordinately regulate both enzyme pathways. FOXO1 was found to be a coactivator to CAR- and PXR-mediated transcription. In contrast, CAR and PXR, acting as corepressors, downregulated FOXO1-mediated transcription in the presence of their activators, such as 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone 16alpha-carbonitrile, respectively. A constitutively active mutant of the insulin-responsive protein kinase Akt, but not the kinase-negative mutant, effectively blocked FOXO1 activity in cell-based assays. Thus, insulin could repress the receptors by activating the Akt-FOXO1 signal, whereas drugs could interfere with FOXO1-mediated transcription by activating CAR and/or PXR. Treatment with TCPOBOP or PB decreased the levels of phosphoenolpyruvate carboxykinase 1 mRNA in mice but not in Car(-/-) mice. We conclude that FOXO1 and the nuclear receptors reciprocally coregulate their target genes, modulating both drug metabolism and gluconeogenesis.

  5. A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

    PubMed

    Zuo, Dong-Yun; Yi, Shu-Yuan; Liu, Rong-Jing; Qu, Bo; Huang, Tao; He, Wei-Jie; Li, Cheng; Li, He-Ping; Liao, Yu-Cai

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

  6. The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins. Medium chain acyl-CoA dehydrogenase and mitochondrial malate dehydrogenase.

    PubMed

    Kelly, D P; Gordon, J I; Alpers, R; Strauss, A W

    1989-11-15

    To study the regulation of nuclear genes which encode mitochondrial enzymes involved in oxidative metabolism, absolute levels of mRNA encoding rat medium chain acyl-CoA dehydrogenase (MCAD) and rat mitochondrial malate dehydrogenase (mMDH) were determined in developing and adult male rat tissues. MCAD mRNA is expressed in a variety of adult male tissues with highest steady state levels in heart, adrenal, and skeletal muscle and lowest levels in brain, lung, and testes. In comparison, steady state levels of mMDH mRNA in adult male rat tissues were similar to those of MCAD mRNA in heart, small intestine, adrenal, and skeletal muscle but markedly different in brain, stomach, and testes. Thus, the steady-state levels of MCAD and mMDH mRNA are highest in adult tissues with high energy requirements. Dot blot analysis of RNA prepared from late fetal, suckling, and weaning rat heart, liver, and brain demonstrated the presence of MCAD and mMDH mRNA during the fetal period in all three tissues. Both MCAD and mMDH mRNA levels increased 2-2.5-fold at birth followed by a decline during the first postnatal week in heart and liver. The patterns of accumulation of these mRNAs in heart and liver during the weaning and early adult periods were also similar, although the absolute levels were significantly different. Brain MCAD mRNA levels were consistently low (less than 0.1 pg/micrograms total cellular RNA) throughout the developmental stages. However, brain mMDH mRNA levels exhibited a marked increase during the weaning period, reaching a peak concentration which is higher than the level of mMDH mRNA in heart and liver at any point during development. These results indicate that the level of expression of the nuclear genes encoding MCAD and mMDH is tissue-specific and developmentally regulated. The patterns of MCAD and mMDH mRNA accumulation parallel the changes in energy metabolism which occur during development and among adult tissues.

  7. Infectious salmon anaemia virus nuclear export protein is encoded by a spliced gene product of genomic segment 7.

    PubMed

    Ramly, Rimatulhana Binti; Olsen, Christel M; Braaen, Stine; Rimstad, Espen

    2013-10-01

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing anaemia and circulatory disease with high mortality in farmed Atlantic salmon (Salmo salar). Orthomyxoviruses are unusual as RNA viruses as they replicate in the nucleus and some viral transcripts undergo splicing. The nuclear replication necessitates a tightly controlled nuclear import and export of viral proteins. From ISAV genomic segment 7 two known mRNAs are transcribed; one collinear with the genomic segment, coding for the non-structural protein, and one spliced transcript, S7ORF2, coding for a protein with unknown function. Here we report initial functional analysis of the S7ORF2 protein. The results indicate that S7ORF2 protein gradually accumulates in the host cell during virus replication cycle, locates predominantly in the cytoplasm and is a part of purified virus particles. Trapping of S7ORF2 in the nucleus was obtained by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export, indicating that S7ORF2 use CRM1 for the nuclear exit. Immunofluorescent staining of cells over-expressing both S7ORF2 and matrix protein (M) showed co-localization in the nucleus. However, S7ORF2 protein was found to interact with both the viral nucleoprotein (NP) and M proteins in ISAV infected cells as well as in purified viral particles. These results indicate that the S7ORF2 could be called the ISAV nuclear export protein, ISAV/NEP. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. A hypothesis-driven association study of 28 nuclear-encoded mitochondrial genes with antipsychotic-induced weight gain in schizophrenia.

    PubMed

    Gonçalves, Vanessa F; Zai, Clement C; Tiwari, Arun K; Brandl, Eva J; Derkach, Andriy; Meltzer, Herbert Y; Lieberman, Jeffrey A; Müller, Daniel J; Sun, Lei; Kennedy, James L

    2014-05-01

    Mitochondria are the main source of energy for neurons and have a role in many vital neuronal functions. Mitochondrial dysfunction has been described in schizophrenia, and antipsychotics such as clozapine and olanzapine have been associated with differences in gene expression in mitochondria. We investigated the hypothesis that nuclear-encoded mitochondrial genes, particularly those involved in oxidative phosphorylation or involved in oxidative stress, mitochondrial biogenesis, inflammation, and apoptosis, would be associated with antipsychotic-induced weight gain (AIWG). In total, we selected 28 genes and analyzed 60 SNPs (50 are functional), in 283 schizophrenia subjects, treated with atypical medications for up to 14 weeks. Association between AIWG (as measured by the % of weight gain from baseline) and SNP genotypes were tested using linear regression with treatment duration, baseline body weight, and medication type as covariates. We observed a significant association between rs6435326 in the NDUFS1 gene and AIWG in the subset of European patients (N=150, Pcorrected=0.02). The haplotype carrying the risk alleles of rs6435326 and two other SNPs (rs1053517 and rs1801318) in NDUFS1 was also nominally associated with percentage of weight gain (T-C-G vs A-T-A, P=0.005). In addition, stepwise linear regression was performed to select important variables predictive of the outcome, and a gene-gene interaction analysis was carried out. We observed a significant interaction between the TT risk genotype of rs6435326 in NDUFS1 and AG genotype of rs3762883 in COX18 (Pcorrected=0.001). A permutation-based test of all 60 SNPs jointly showed significant association with weight gain (P=0.02). Finally, our replication study of rs6435326, rs1053517 and rs1801318 in NDUFS1 using samples from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) showed that rs1801318 was significantly associated with AIWG (N=200, Pcorrected=0.04), and the three SNPs were

  9. Molecular phylogeny of brachiopods and phoronids based on nuclear-encoded small subunit ribosomal RNA gene sequences

    PubMed Central

    L.Cohen, B.

    1998-01-01

    Brachiopod and phoronid phylogeny is inferred from SSU rDNA sequences of 28 articulate and nine inarticulate brachiopods, three phoronids, two ectoprocts and various outgroups, using gene trees reconstructed by weighted parsimony, distance and maximum likelihood methods. Of these sequences, 33 from brachiopods, two from phoronids and one each from an ectoproct and a priapulan are newly determined. The brachiopod sequences belong to 31 different genera and thus survey about 10% of extant genus-level diversity. Sequences determined in different laboratories and those from closely related taxa agree well, but evidence is presented suggesting that one published phoronid sequence (GenBank accession UO12648) is a brachiopod-phoronid chimaera, and this sequence is excluded from the analyses. The chiton, Acanthopleura, is identified as the phenetically proximal outgroup; other selected outgroups were chosen to allow comparison with recent, non-molecular analyses of brachiopod phylogeny. The different outgroups and methods of phylogenetic reconstruction lead to similar results, with differences mainly in the resolution of weakly supported ancient and recent nodes, including the divergence of inarticulate brachiopod sub-phyla, the position of the rhynchonellids in relation to long- and short-looped articulate brachiopod clades and the relationships of some articulate brachiopod genera and species. Attention is drawn to the problem presented by nodes that are strongly supported by non-molecular evidence but receive only low bootstrap resampling support. Overall, the gene trees agree with morphology-based brachiopod taxonomy, but novel relationships are tentatively suggested for thecideidine and megathyrid brachiopods. Articulate brachiopods are found to be monophyletic in all reconstructions, but monophyly of inarticulate brachiopods and the possible inclusion of phoronids in the inarticulate brachiopod clade are less strongly established. Phoronids are clearly excluded from

  10. Characterization of the gene encoding mouse serum amyloid P component. Comparison with genes encoding other pentraxins.

    PubMed Central

    Whitehead, A S; Rits, M

    1989-01-01

    A CBA/J-strain mouse serum amyloid P component (SAP) genomic clone was isolated and analysed. The clone contains the entire SAP gene and specifies a primary transcript of 1065 nucleotide residues. This comprises a first exon of 206 nucleotide residues containing the mRNA 5'-untranslated region and sequence encoding the pre-SAP leader peptide and the first two amino acid residues of mature SAP separated by a single 110-base intron from a 749-nucleotide-residue second exon containing sequence encoding the bulk of the mature SAP and specifying the mRNA 3'-untranslated region. The overall organization is similar to that of the human SAP gene, and the coding region and intron sequences are highly conserved. The SAP RNA cap site was defined by primer extension analysis of polyadenylated acute-phase liver RNA. The 5'-region of the mouse SAP gene contains modified CAAT and TATA promoter elements preceded by a putative hepatocyte-nuclear-factor-1-recognition site; these structures are in a region that is highly homologous to the corresponding region of the human SAP gene. Comparisons of the mouse SAP gene structure and derived amino acid sequence with those of other mammalian pentraxins were made. Images Fig. 3. PMID:2481440

  11. The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis.

    PubMed

    Tarrío, N; Díaz Prado, S; Cerdán, M E; González Siso, M I

    2005-01-01

    Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

  12. Characterization of four nuclear-encoded plastid RNA polymerase sigma factor genes in the liverwort Marchantia polymorpha: blue-light- and multiple stress-responsive SIG5 was acquired early in the emergence of terrestrial plants.

    PubMed

    Kanazawa, Takehiko; Ishizaki, Kimitsune; Kohchi, Takayuki; Hanaoka, Mitsumasa; Tanaka, Kan

    2013-10-01

    The plastids of plant cells each contain their own genome, and a bacterial-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP) is involved in transcription of this genome. While the catalytic core subunits are encoded by the plastid genome, the specificity subunit of PEP, sigma, is generally encoded by the nuclear genome and imported into plastids from the cytoplasm after translation. In this study, we identified and analyzed four sigma factor genes from the nuclear genome of a liverwort, Marchantia polymorpha. Phylogenetic analysis suggested that three of the four genes were orthologous to vascular plant genes and thus they were named MpSIG1, MpSIG2 and MpSIG5. The remaining gene was named MpSIGX. The gene products were predicted to localize to the plastid, and this prediction was experimentally demonstrated by expressing yellow fluorescent protein fusion genes in vivo. As with SIG5 genes of other plant species, expression of MpSIG5 was induced by blue-light irradiation and also under various stress conditions, indicating that the regulatory mechanism responsible is conserved among divergent plant species. However, while the major role of SIG5 in vascular plants is to repair the damaged PSII reaction center through psbD gene transcription, the relevant blue-light-responsive promoter (psbD-BLRP) was not found in M. polymorpha and psbD transcript accumulation did not occur in conjunction with MpSIG5 induction. Thus, the physiological role of SIG5 is probably divergent among plant phyla.

  13. LOT1 is a growth suppressor gene down-regulated by the epidermal growth factor receptor ligands and encodes a nuclear zinc-finger protein.

    PubMed

    Abdollahi, A; Bao, R; Hamilton, T C

    1999-11-11

    We previously reported cloning the rLot1 gene, and its human homolog (hLOT1), through analysis of differential gene expression in normal and malignant rat ovarian surface epithelial cells. Both human and rat ovarian carcinoma cell lines exhibited lost or decreased expression of this gene. Interestingly, the LOT1 gene localized at band q25 of human chromosome 6 which is a frequent site for LOH in many solid tumors including ovarian cancer. In this report we have further characterized the potential role of LOT1 in malignant transformation and developed evidence that the gene is a novel target of growth factor signaling pathway. Assays using transient transfections showed that LOT1 is a nuclear protein and may act as a transcription factor. In vitro and in vivo studies involving ovarian cancer cell lines revealed that expression of LOT1 is directly associated with inhibition of cellular proliferation and induction of morphological transformations. Additionally, we show that in normal rat ovarian surface epithelial cells Lot1 gene expression is responsive to growth factor stimulation. Its mRNA is strongly down-regulated by epidermal growth factor receptor (EGFR) ligands, namely EGF and TGF-alpha. Blocking the ligand-activated EGFR signal transduction pathway by the specific EGF receptor inhibitor, tyrphostin AG1478, and the MEK inhibitor, PD098059, restores the normal level of Lot1 gene expression. It appears that the regulation of Lot1 gene is unique to these ligands, as well as the growth promoting agent TPA, since other factors either did not affect Lot1 expression, or the effect was modest and transient. Altogether, the results suggest that Lot1 expression is primarily mediated via EGF receptor or a related pathway and it may regulate the growth promoting signals as a zinc-finger motif containing nuclear transcription factor.

  14. SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family

    PubMed Central

    1993-01-01

    The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. We have cloned the wild-type SMC1 gene. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino- terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1- 2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division. PMID:8276886

  15. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  16. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    SciTech Connect

    Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin . E-mail: min-xin.guan@chmcc.org

    2006-04-21

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

  17. A Novel Gene SbSI-2 Encoding Nuclear Protein from a Halophyte Confers Abiotic Stress Tolerance in E. coli and Tobacco

    PubMed Central

    Yadav, Narendra Singh; Singh, Vijay Kumar; Singh, Dinkar; Jha, Bhavanath

    2014-01-01

    Salicornia brachiata is an extreme halophyte that grows luxuriantly in coastal marshes. Previously, we have reported isolation and characterization of ESTs from Salicornia with large number of novel/unknown salt-responsive gene sequences. In this study, we have selected a novel salt-inducible gene SbSI-2 (Salicornia brachiata salt-inducible-2) for functional characterization. Bioinformatics analysis revealed that SbSI-2 protein has predicted nuclear localization signals and a strong protein-protein interaction domain. Transient expression of the RFP:SbSI2 fusion protein confirmed that SbSI-2 is a nuclear-localized protein. Genomic organization study showed that SbSI-2 is intronless and has a single copy in Salicornia genome. Quantitative RT-PCR analysis revealed higher SbSI-2 expression under salt stress and desiccation conditions. The SbSI-2 gene was transformed in E. coli and tobacco for functional characterization. pET28a-SbSI-2 recombinant E. coli cells showed higher tolerance to desiccation and salinity compared to vector alone. Transgenic tobacco plants overexpressing SbSI-2 have improved salt- and osmotic tolerance, accompanied by better growth parameters, higher relative water content, elevated accumulation of compatible osmolytes, lower Na+ and ROS accumulation and lesser electrolyte leakage than the wild-type. Overexpression of the SbSI-2 also enhanced transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results demonstrate that SbSI-2 might play an important positive modulation role in abiotic stress tolerance. This identifies SbSI-2 as a novel determinant of salt/osmotic tolerance and suggests that it could be a potential bioresource for engineering abiotic stress tolerance in crop plants. PMID:24999628

  18. A novel gene SbSI-2 encoding nuclear protein from a halophyte confers abiotic stress tolerance in E. coli and tobacco.

    PubMed

    Yadav, Narendra Singh; Singh, Vijay Kumar; Singh, Dinkar; Jha, Bhavanath

    2014-01-01

    Salicornia brachiata is an extreme halophyte that grows luxuriantly in coastal marshes. Previously, we have reported isolation and characterization of ESTs from Salicornia with large number of novel/unknown salt-responsive gene sequences. In this study, we have selected a novel salt-inducible gene SbSI-2 (Salicornia brachiata salt-inducible-2) for functional characterization. Bioinformatics analysis revealed that SbSI-2 protein has predicted nuclear localization signals and a strong protein-protein interaction domain. Transient expression of the RFP:SbSI2 fusion protein confirmed that SbSI-2 is a nuclear-localized protein. Genomic organization study showed that SbSI-2 is intronless and has a single copy in Salicornia genome. Quantitative RT-PCR analysis revealed higher SbSI-2 expression under salt stress and desiccation conditions. The SbSI-2 gene was transformed in E. coli and tobacco for functional characterization. pET28a-SbSI-2 recombinant E. coli cells showed higher tolerance to desiccation and salinity compared to vector alone. Transgenic tobacco plants overexpressing SbSI-2 have improved salt- and osmotic tolerance, accompanied by better growth parameters, higher relative water content, elevated accumulation of compatible osmolytes, lower Na+ and ROS accumulation and lesser electrolyte leakage than the wild-type. Overexpression of the SbSI-2 also enhanced transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results demonstrate that SbSI-2 might play an important positive modulation role in abiotic stress tolerance. This identifies SbSI-2 as a novel determinant of salt/osmotic tolerance and suggests that it could be a potential bioresource for engineering abiotic stress tolerance in crop plants.

  19. The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria.

    PubMed

    Sugimoto, Hiroki; Kusumi, Kensuke; Noguchi, Ko; Yano, Masahiro; Yoshimura, Atsushi; Iba, Koh

    2007-11-01

    Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation.

  20. Expression and function of AtMBD4L, the single gene encoding the nuclear DNA glycosylase MBD4L in Arabidopsis.

    PubMed

    Nota, Florencia; Cambiagno, Damián A; Ribone, Pamela; Alvarez, María E

    2015-06-01

    DNA glycosylases recognize and excise damaged or incorrect bases from DNA initiating the base excision repair (BER) pathway. Methyl-binding domain protein 4 (MBD4) is a member of the HhH-GPD DNA glycosylase superfamily, which has been well studied in mammals but not in plants. Our knowledge on the plant enzyme is limited to the activity of the Arabidopsis recombinant protein MBD4L in vitro. To start evaluating MBD4L in its biological context, we here characterized the structure, expression and effects of its gene, AtMBD4L. Phylogenetic analysis indicated that AtMBD4L belongs to one of the seven families of HhH-GPD DNA glycosylase genes existing in plants, and is unique on its family. Two AtMBD4L transcripts coding for active enzymes were detected in leaves and flowers. Transgenic plants expressing the AtMBD4L:GUS gene confined GUS activity to perivascular leaf tissues (usually adjacent to hydathodes), flowers (anthers at particular stages of development), and the apex of immature siliques. MBD4L-GFP fusion proteins showed nuclear localization in planta. Interestingly, overexpression of the full length MBD4L, but not a truncated enzyme lacking the DNA glycosylase domain, induced the BER gene LIG1 and enhanced tolerance to oxidative stress. These results suggest that endogenous MBD4L acts on particular tissues, is capable of activating BER, and may contribute to repair DNA damage caused by oxidative stress. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  2. Gene encoding acetyl-coenzyme A carboxylase

    SciTech Connect

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  3. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  4. The Arabidopsis flowering-time gene LUMINIDEPENDENS is expressed primarily in regions of cell proliferation and encodes a nuclear protein that regulates LEAFY expression.

    PubMed

    Aukerman, M J; Lee, I; Weigel, D; Amasino, R M

    1999-04-01

    Mutations in the LUMINIDEPENDENS (LD) gene of Arabidopsis thaliana (L.) Heynh. (Arabidopsis) confer a late-flowering phenotype, indicating that LD normally functions to promote the floral transition. RNA and protein blot analyses, along with the analysis of transgenic plants containing a fusion between a genomic fragment of LD and the reporter gene uidA (GUS), indicate that LD is expressed primarily ipical proliferative regions of the shoot and root, including the shoot apical meristem and leaf primordia. Subcellular localization studies indicate that LD is a nuclear protein, consistent with its previously proposed transcriptional regulatory role. We have also found that in an apetala1 cauliflower (ap1 cal) background the ld mutation converts the reproductive shoot apex to a more vegetative state, a phenotype that is similar to that seen for the leafy (lfy) mutant. Furthermore, in situ hybridization analysis indicates that LFY levels are drastically reduced at the apex of ld ap1 cal plants after bolting. These data are consistent with the idea that at least one function of LD is to participate in the regulation of LFY.

  5. Disruption of a nuclear gene encoding a mitochondrial gamma carbonic anhydrase reduces complex I and supercomplex I + III2 levels and alters mitochondrial physiology in Arabidopsis.

    PubMed

    Perales, Mariano; Eubel, Holger; Heinemeyer, Jesco; Colaneri, Alejandro; Zabaleta, Eduardo; Braun, Hans-Peter

    2005-07-08

    Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial gamma-carbonic anhydrases. A homozygous Arabidopsis knockout mutant carrying a T-DNA insertion in a gene encoding one of these subunits (At1g47260) was generated to investigate its physiological role. Isolation of mitochondria and separation of mitochondrial protein complexes by Blue-native polyacrylamide gel electrophoresis or sucrose gradient ultracentrifugation revealed drastically reduced complex I levels. Furthermore, the mitochondrial I + III2 supercomplex was very much reduced in mutant plants. Remaining complex I had normal molecular mass, suggesting substitution of the At1g47260 protein by one or several of the structurally related subunits of this respiratory protein complex. Immune-blotting experiments using polyclonal antibodies directed against the At1g47260 protein indicated its presence within complex I, the I + III2 supercomplex and smaller protein complexes, which possibly represent subcomplexes of complex I. Changes within the mitochondrial proteome of mutant cells were systematically monitored by fluorescence difference gel electrophoresis using 2D Blue-native/SDS and 2D isoelectric focussing/SDS polyacrylamide gel electrophoresis. Complex I subunits are largely absent within the mitochondrial proteome. Further mitochondrial proteins are reduced in mutant plants, like mitochondrial ferredoxin, others are increased, like formate dehydrogenase. Development of mutant plants was normal under standard growth conditions. However, a suspension cell culture generated from mutant plants exhibited clearly reduced growth rates and respiration. In summary, At1g47260 is important for complex I assembly in plant mitochondria and respiration. A role of At1g47260 in mitochondrial one-carbon metabolism is supported by micro-array analyses.

  6. Phylogeny and evolutionary history of Leymus (Triticeae; Poaceae) based on a single-copy nuclear gene encoding plastid acetyl-CoA carboxylase.

    PubMed

    Fan, Xing; Sha, Li-Na; Yang, Rui-Wu; Zhang, Hai-Qin; Kang, Hou-Yang; Ding, Cun-Bang; Zhang, Li; Zheng, You-Liang; Zhou, Yong-Hong

    2009-10-08

    Single- and low- copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of polyploid taxa. Leymus is a polyploid genus with a diverse array of morphology, ecology and distribution in Triticeae. The genomic constitution of Leymus was assigned as NsXm, where Ns was presumed to be originated from Psathyrostachys, while Xm represented a genome of unknown origin. In addition, little is known about the evolutionary history of Leymus. Here, we investigate the phylogenetic relationship, genome donor, and evolutionary history of Leymus based on a single-copy nuclear Acc1 gene. Two homoeologues of the Acc1 gene were isolated from nearly all the sampled Leymus species using allele-specific primer and were analyzed with those from 35 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) Leymus is closely related to Psathyrostachys, Agropyron, and Eremopyrum; (2) Psathyrostachys juncea is an ancestral Ns-genome donor of Leymus species; (3) the Xm genome in Leymus may be originated from an ancestral lineage of Agropyron and Eremopyrum triticeum; (4) the Acc1 sequences of Leymus species from the Qinghai-Tibetan plateau are evolutionarily distinct; (5) North America Leymus species might originate from colonization via the Bering land bridge; (6) Leymus originated about 11-12MYA in Eurasia, and adaptive radiation might have occurred in Leymus during the period of 3.7-4.3 MYA and 1.7-2.1 MYA. Leymus species have allopolyploid origin. It is hypothesized that the adaptive radiation of Leymus species might have been triggered by the recent upliftings of the Qinghai-Tibetan plateau and subsequent climatic oscillations. Adaptive radiation may have promoted the rapid speciation, as well as the fixation of unique morphological characters in Leymus. Our results shed new light on our understanding of the origin of Xm genome, the

  7. Coordination of plastid and nuclear gene expression.

    PubMed Central

    Gray, John C; Sullivan, James A; Wang, Jun-Hui; Jerome, Cheryl A; MacLean, Daniel

    2003-01-01

    The coordinated expression of genes distributed between the nuclear and plastid genomes is essential for the assembly of functional chloroplasts. Although the nucleus has a pre-eminent role in controlling chloroplast biogenesis, there is considerable evidence that the expression of nuclear genes encoding photosynthesis-related proteins is regulated by signals from plastids. Perturbation of several plastid-located processes, by inhibitors or in mutants, leads to decreased transcription of a set of nuclear photosynthesis-related genes. Characterization of arabidopsis gun (genomes uncoupled) mutants, which express nuclear genes in the presence of norflurazon or lincomycin, has provided evidence for two separate signalling pathways, one involving tetrapyrrole biosynthesis intermediates and the other requiring plastid protein synthesis. In addition, perturbation of photosynthetic electron transfer produces at least two different redox signals, as part of the acclimation to altered light conditions. The recognition of multiple plastid signals requires a reconsideration of the mechanisms of regulation of transcription of nuclear genes encoding photosynthesis-related proteins. PMID:12594922

  8. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    SciTech Connect

    Ostlund, Cecilia; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Gerace, Larry; Schirmer, Eric C.

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  9. Expression of VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes of Epstein-Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma.

    PubMed

    Hu, Bo; Hong, Guoqiang; Li, Zhaoxia; Xu, Jue; Zhu, Zhenyu; Li, Lin

    2007-05-01

    EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results

  10. The First Gene-encoded Amphibian Neurotoxin*

    PubMed Central

    You, Dewen; Hong, Jing; Rong, Mingqiang; Yu, Haining; Liang, Songping; Ma, Yufang; Yang, Hailong; Wu, Jing; Lin, Donghai; Lai, Ren

    2009-01-01

    Many gene-encoded neurotoxins with various functions have been discovered in fish, reptiles, and mammals. A novel 60-residue neurotoxin peptide (anntoxin) that inhibited tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel (VGSC) was purified and characterized from the skin secretions of the tree frog Hyla annectans (Jerdon). This is the first gene-encoded neurotoxin found in amphibians. The IC50 of anntoxin for the TTX-S channel was about 3.4 μm. Anntoxin shares sequence homology with Kunitz-type toxins but contains only two of three highly conserved cysteine bridges, which are typically found in these small, basic neurotoxin modules, i.e. snake dendrotoxins. Anntoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.025 μm. Anntoxin was distributed in skin, brain, stomach, and liver with a concentration of 25, 7, 3, and 2 μg/g wet tissue, respectively. H. annectans lives on trees or other plants for its entire life cycle, and its skin contains the largest amount of anntoxin, which possibly helps defend against various aggressors or predators. A low dose of anntoxin was found to induce lethal toxicity for several potential predators, including the insect, snake, bird, and mouse. The tissue distribution and functional properties of the current toxin may provide insights into the ecological adaptation of tree-living amphibians. PMID:19535333

  11. SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.

    PubMed Central

    Swanson, M S; Malone, E A; Winston, F

    1991-01-01

    Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae. Images PMID:1840633

  12. Mutations in nuclear genes alter post-transcriptional regulation of mitochondrial genes.

    USDA-ARS?s Scientific Manuscript database

    Nuclear gene products are required for the expression of mitochondrial genes and elaboration of functional mitochondrial protein complexes. To better understand the roles of these nuclear genes, we exploited the mitochondrial encoded S-type of cytoplasmic male sterility (CMS-S) and developed a nove...

  13. ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene.

    PubMed

    Lindsay, E A; Harvey, E L; Scambler, P J; Baldini, A

    1998-04-01

    ES2 is a gene deleted in DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) which has homologs in species as distant as Caenorhabditis elegans and Drosophila . The function of ES2 is unknown, and the predicted protein sequence does not contain motifs which suggest a particular role in the developmental defects present in DGS and VCFS. Here we show that the mouse homolog, Es2 , is transcribed in two forms resulting from the use of alternative polyadenylation signals. Structural analysis programs predict that the Es2 -encoded peptide has a coiled-coil domain, and transfection experiments with an Es2 -green fluorescent protein (GFP) fusion construct show that the peptide is recruited into the nucleus. Es2 is highly expressed during mouse embryogenesis from E7 onwards. In situ hybridization with an RNA probe revealed that the gene is widely expressed; however, relatively higher expression was detected in the nervous system, with a particularly high area of expression in a sub-region of the pons. The Es2 expression domain in the pons is shared with a Goosecoid-like gene ( Gscl) which is located upstream of Es2 , and raises the possibility that the two genes share regulatory elements and/or interact in this region of the developing brain. This finding suggests that different genes in the deleted region may be functionally related and might explain the occurrence of the characteristic phenotype in patients with non-overlapping genetic lesions.

  14. A Continental-Wide Perspective: The Genepool of Nuclear Encoded Ribosomal DNA and Single-Copy Gene Sequences in North American Boechera (Brassicaceae)

    PubMed Central

    Kiefer, Christiane; Koch, Marcus A.

    2012-01-01

    74 of the currently accepted 111 taxa of the North American genus Boechera (Brassicaceae) were subject to pyhlogenetic reconstruction and network analysis. The dataset comprised 911 accessions for which ITS sequences were analyzed. Phylogenetic analyses yielded largely unresolved trees. Together with the network analysis confirming this result this can be interpreted as an indication for multiple, independent, and rapid diversification events. Network analyses were superimposed with datasets describing i) geographical distribution, ii) taxonomy, iii) reproductive mode, and iv) distribution history based on phylogeographic evidence. Our results provide first direct evidence for enormous reticulate evolution in the entire genus and give further insights into the evolutionary history of this complex genus on a continental scale. In addition two novel single-copy gene markers, orthologues of the Arabidopsis thaliana genes At2g25920 and At3g18900, were analyzed for subsets of taxa and confirmed the findings obtained through the ITS data. PMID:22606266

  15. A novel nuclear-encoded mitochondrial poly(A) polymerase PAPD1 is a potential candidate gene for the extreme obesity related phenotypes in mammals.

    PubMed

    Xiao, Qianjun; Wu, Xiao-Lin; Michal, Jennifer J; Reeves, Jerry J; Busboom, Jan R; Thorgaard, Gary H; Jiang, Zhihua

    2006-01-01

    People with obesity, especially extreme obesity, are at risk for many health problems. However, the responsible genes remain unknown in >95% of severe obesity cases. Our previous genome-wide scan of Wagyu x Limousin F2 cattle crosses with extreme phenotypes revealed a molecular marker significantly associated with intramuscular fat deposition. Characterization of this marker showed that it is orthologous to the human gene KIAA1462 located on HSA10p11.23, where a major quantitative trait locus for morbid obesity has been reported. The newly identified mitochondrial poly(A) polymerase associated domain containing 1 (PAPD1) gene, which is located near this marker, is particularly interesting because the polymerase is required for the polyadenylation and stabilization of mammalian mitochondrial mRNAs. In the present study, both cDNA and genomic DNA sequences were annotated for the bovine PAPD1 gene and ten genetic markers were detected in the promoter and exon 1 region. Among seven markers assayed on approximately 250 Wagyu x Limousin F2 animals, two single nucleotide polymorphisms (SNPs) in the promoter region were significantly associated with intramuscular fat (P<0.05). However, there was a significant interaction (P<0.05) between a third SNP, which causes an amino acid change in coding exon 1, and each of these two promoter SNPs on intramuscular fat deposition. In particular, the differences between double heterozygous animals at two polymorphic sites and the slim genotype animals exceeded 2.3 standard deviations for the trait in both cases. Our study provides evidence for a new mechanism--the involvement of compound heterosis in extreme obesity, which warrants further examination.

  16. Rearranged T-cell receptor gene and positive Epstein-Barr virus-encoded nuclear RNA in an extranodal NK/T-cell lymphoma with cutaneous manifestation only: case study.

    PubMed

    Wang, J; Geng, S A; Su, Z; Xu, J; Xu, S; Wang, F; Mo, X; Feng, Z

    2007-11-01

    Natural killer (NK)/cytotoxic T-cell lymphoma, a new type of cutaneous neoplasm, has been described recently in the World Health Organization/European Organization for Research and Treatment of Cancer classification for cutaneous lymphomas. We report an 11-year-old boy who had had erythematous plaques and blisters on his face and hands for 4 years and infiltrating plaques and necrosis on his extremities for 4 months. Routine clinical and laboratory examinations found no primary nasal involvement. Biopsies taken from nasal mucosa and skin showed that the tumour only involved dermis and subcutaneous tissue, and the infiltrated lymphohistiocytic tumour cells were CD56+, TIA+, CD45RO+ and CD30+. In situ hybridization for EBV-encoded nuclear RNA was positive. Clonal T-cell receptor-gamma2 gene rearrangement was positive. A diagnosis of extranodal NK/T-cell lymphoma, nasal type, was made. This is a rare case, with slow course and survival for >51 months with the presentation only occurring in the skin.

  17. The gene encoding proopiomelanocortin in the dog.

    PubMed

    Mol, J A; van Mansfeld, A D; Kwant, M M; van Wolferen, M; Rothuizen, J

    1991-01-01

    The regulation of the synthesis of ACTH in the dog is of interest for studies of the physiology of the pituitary-adrenocortical axis as well as for studies of the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite this broad interest the nucleotide sequence encoding ACTH and its precursor proopiomelanocortin (POMC) is not known, nor is it clear whether there are differences in POMC mRNA from the anterior lobe or the intermediate lobe of the normal pituitary or from pituitary tumours causing ACTH excess. Following the preparation of a cDNA library from the canine intermediate lobe of the pituitary gland, the part of the mRNA that is translated into the proopiomelanocortin prohormone was amplified using a polymerase chain reaction. Sequence analysis revealed the highest homology with the porcine mRNA sequence. Translation in a single reading frame revealed highly homologous areas in the amino-terminal, carboxy-terminal, and ACTH part of the prohormone, whereas a high diversity was noticed at the sequence preceding ACTH and the beginning of beta-lipotropin. Northern blot analysis disclosed the presence of a POMC mRNA of approximately 1300 nucleotides. There were no size differences between the anterior lobe, intermediate lobe, and pituitary tumour derived POMC mRNA. The highest expression levels of POMC mRNA as related to the expression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase were found in the intermediate lobe of the canine pituitary gland. It is concluded that excessive production of ACTH by pituitary tumours is not caused by relatively high expression levels or alterations in the size of mRNA.

  18. Genes Encoding Enzymes Involved in Ethanol Metabolism

    PubMed Central

    Hurley, Thomas D.; Edenberg, Howard J.

    2012-01-01

    The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer. PMID:23134050

  19. 'Green revolution' genes encode mutant gibberellin response modulators.

    PubMed

    Peng, J; Richards, D E; Hartley, N M; Murphy, G P; Devos, K M; Flintham, J E; Beales, J; Fish, L J; Worland, A J; Pelica, F; Sudhakar, D; Christou, P; Snape, J W; Gale, M D; Harberd, N P

    1999-07-15

    World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

  20. Mollusk genes encoding lysine tRNA (UUU) contain introns.

    PubMed

    Matsuo, M; Abe, Y; Saruta, Y; Okada, N

    1995-11-20

    New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

  1. Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

    PubMed

    de Longevialle, Andéol Falcon; Small, Ian D; Lurin, Claire

    2010-07-01

    Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

  2. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  3. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  4. Population-level expression variability of mitochondrial DNA-encoded genes in humans

    PubMed Central

    Wang, Gang; Yang, Ence; Mandhan, Ishita; Brinkmeyer-Langford, Candice L; Cai, James J

    2014-01-01

    Human mitochondria contain multiple copies of a circular genome made up of double-stranded DNA (mtDNA) that encodes proteins involved in cellular respiration. Transcript abundance of mtDNA-encoded genes varies between human individuals, yet the level of variation in the general population has not been systematically assessed. In the present study, we revisited large-scale RNA sequencing data generated from lymphoblastoid cell lines of HapMap samples of European and African ancestry to estimate transcript abundance and quantify expression variation for mtDNA-encoded genes. In both populations, we detected up to over 100-fold difference in mtDNA gene expression between individuals. The marked variation was not due to differences in mtDNA copy number between individuals, but was shaped by the transcription of hundreds of nuclear genes. Many of these nuclear genes were co-expressed with one another, resulting in a module-enriched co-expression network. Significant correlations in expression between genes of the mtDNA and nuclear genomes were used to identify factors involved with the regulation of mitochondrial functions. In conclusion, we determined the baseline amount of variability in mtDNA gene expression in general human populations and cataloged a complete set of nuclear genes whose expression levels are correlated with those of mtDNA-encoded genes. Our findings will enable the integration of information from both mtDNA and nuclear genetic systems, and facilitate the discovery of novel regulatory pathways involving mitochondrial functions. PMID:24398800

  5. Arabidopsis MDA1, a nuclear-encoded protein, functions in chloroplast development and abiotic stress responses.

    PubMed

    Robles, Pedro; Micol, José Luis; Quesada, Víctor

    2012-01-01

    Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis.

  6. In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum

    PubMed Central

    Delage, Ludovic; Dietrich, André; Cosset, Anne; Maréchal-Drouard, Laurence

    2003-01-01

    Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential. PMID:12748301

  7. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide.

    PubMed Central

    Eldridge, R; Li, Y; Miller, L K

    1992-01-01

    We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2+) channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named ctl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extracellular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established. Images PMID:1404603

  8. Coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in mitochondria of a liverwort Marchantia polymorpha.

    PubMed Central

    Akashi, K; Takenaka, M; Yamaoka, S; Suyama, Y; Fukuzawa, H; Ohyama, K

    1998-01-01

    The liverwort Marchantia polymorpha mitochondrial DNA encodes almost all tRNAs required for mitochondrial translation except for the isoleucine (AUU, AUC) and threonine (ACA, ACG) codons, while the missing tRNAs are supplied in part by the nucleus and imported in mitochondria. In this paper, we report a finding of two radically different nuclear tRNAVal(AAC) genes and import of the corresponding tRNA isoacceptors in M.polymorpha mitochondria. This finding is surprising since the mtDNA encodes the gene for tRNAVal(UAC), which alone was considered sufficient for translating all four valine codons GUN by the U/N wobble mechanism. The present results suggest for the first time that the import of ncDNA-encoded tRNAs may result in decoding overlaps in plant mitochondria. The coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in liverwort mitochondria and the significance for the decoding mechanism as well as evolution of tRNA import are discussed. PMID:9547276

  9. Gene encoding herbicide safener binding protein

    SciTech Connect

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

    1999-10-26

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

  10. Nuclear gene indicates coat-color polymorphism in mammoths.

    PubMed

    Römpler, Holger; Rohland, Nadin; Lalueza-Fox, Carles; Willerslev, Eske; Kuznetsova, Tatyana; Rabeder, Gernot; Bertranpetit, Jaume; Schöneberg, Torsten; Hofreiter, Michael

    2006-07-07

    By amplifying the melanocortin type 1 receptor from the woolly mammoth, we can report the complete nucleotide sequence of a nuclear-encoded gene from an extinct species. We found two alleles and show that one allele produces a functional protein whereas the other one encodes a protein with strongly reduced activity. This finding suggests that mammoths may have been polymorphic in coat color, with both dark- and light-haired individuals co-occurring.

  11. The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

    PubMed Central

    Mireau, H; Lancelin, D; Small, I D

    1996-01-01

    In plants, all aminoacyl-tRNA synthetases are nuclearly encoded, despite the fact that their activities are required in the three protein-synthesizing cell compartments (cytosol, mitochondria, and chloroplasts). To investigate targeting of these enzymes, we cloned cDNAs encoding alanyl-tRNA synthetase (AlaRS) and the corresponding nuclear gene, ALATS, from Arabidopsis by using degenerate polymerase chain reaction primers based on highly conserved regions shared between known AlaRSs from other organisms. Analysis of the transcription of the gene showed the presence of two potential translation initiation codons in some ALATS mRNAs. Translation from the upstream AUG would generate an N-terminal extension with features characteristic of mitochondrial targeting peptides. A polyclonal antibody raised against part of the Arabidopsis AlaRS revealed that the Arabidopsis cytosolic and mitochondrial AlaRSs are immunologically similar, suggesting that both isoforms are encoded by the ALATS gene. In vitro experiments confirmed that two polypeptides can be translated from AlATS transcripts, with most ribosomes initiating on the downstream AUG to give the shorter polypeptide corresponding in size to the cytosolic enzyme. The ability of the presequence encoded between the two initiation codons to direct polypeptides to mitochondria was demonstrated by expression of fusion proteins in tobacco protoplasts and in yeast. We conclude that the ALATS gene encodes both the cytosolic and the mitochondrial forms of AlaRS, depending on which of the two AUG codons is used to initiate translation. PMID:8672889

  12. Gene encoding herbicide safener binding protein

    DOEpatents

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

    1999-01-01

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

  13. A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana.

    PubMed

    Nakashima, K; Kiyosue, T; Yamaguchi-Shinozaki, K; Shinozaki, K

    1997-10-01

    A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the beta-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.

  14. Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus.

    PubMed Central

    Sun, R; Lin, S F; Gradoville, L; Miller, G

    1996-01-01

    A newly recognized gamma herpesvirus known as Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is present in Kaposi sarcomas and body-cavity-based lymphomas. Here we identify a novel abundant 1.2-kb RNA, polyadenylated nuclear RNA (PAN RNA), encoded by the virus. The majority of cDNAs produced from poly(A)-selected RNA isolated from a human body cavity lymphoma cell line 48 hr after butyrate induction of KSHV lytic replication represented PAN RNA. Within PAN RNA were two 9 and 16 nt stretches with 89% and 94% identity to U1 RNA. A third stretch of 14 nt was 93% complementary to U1. The 5' upstream region of PAN RNA contained both proximal and distal sequence elements characteristic of regulatory regions of U snRNAs, whereas the 3' end was polyadenylylated. PAN RNA was transcribed by RNA polymerase II, lacked a trimethylguanosine cap, and did not associate with polyribosomes. PAN RNA formed a speckled pattern in the nucleus typical of U snRNAs and colocalized with Sm protein. Therefore, PAN represents a new type of RNA, possessing features of both U snRNA and mRNA. Images Fig. 2 Fig. 3 Fig. 4 PMID:8876232

  15. Parallel loss of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases and mtDNA-encoded tRNAs in Cnidaria.

    PubMed

    Haen, Karri M; Pett, Walker; Lavrov, Dennis V

    2010-10-01

    Unlike most animal mitochondrial (mt) genomes, which encode a set of 22 transfer RNAs (tRNAs) sufficient for mt protein synthesis, those of cnidarians have only retained one or two tRNA genes. Whether the missing cnidarian mt-tRNA genes relocated outside the main mt chromosome or were lost remains unclear. It is also unknown what impact the loss of tRNA genes had on other components of the mt translational machinery. Here, we explored the nuclear genome of the cnidarian Nematostella vectensis for the presence of mt-tRNA genes and their corresponding mt aminoacyl-tRNA synthetases (mt-aaRS). We detected no candidates for mt-tRNA genes and only two mt-aaRS orthologs. At the same time, we found that all but one cytosolic aaRS appear to be targeted to mitochondria. These results indicate that the loss of mt-tRNAs in Cnidaria is genuine and occurred in parallel with the loss of nuclear-encoded mt-aaRS. Our phylogenetic analyses of individual aaRS revealed that although the nearly total loss of mt-aaRS is rare, aaRS gene deletion and replacement have occurred throughout the evolution of Metazoa.

  16. Trichoderma asperellum Chi42 Genes Encode Chitinase

    PubMed Central

    Quang, Hoang Tan; Hung, Nguyen Bao; Huy, Nguyen Duc; Phuong, Truong Thi Bich; Ha, Tran Thi Thu

    2011-01-01

    Four Trichoderma strains (CH2, SH16, PQ34, and TN42) were isolated from soil samples collected from Quang Tri and Thua Thien Hue provinces in Vietnam. The strains exhibited high chitinolytic secretion. Strain PQ34 formed the largest zone of chitinase-mediated clearance (> 4 cm in diameter) in agar containing 1% (w/v) colloidal chitin. Analysis of the internal transcribed spacer regions of these strains indicated that they were Trichoderma asperellum. The molecular weights of the chitinases were approximately 42 kDa. Chitinase genes (chi42) of T. asperellum strains TN42, CH2, SH16, and PQ34 were 98~99% homologous to the ech42 gene of T. harzianum CB-Pin-01 (accession No. DQ166036). The deduced amino acid sequences of both T. asperellum strains SH16 and TN42 shared 100% similarity. PMID:22783101

  17. Bioinformatics analysis and detection of gelatinase encoded gene in Lysinibacillussphaericus

    NASA Astrophysics Data System (ADS)

    Repin, Rul Aisyah Mat; Mutalib, Sahilah Abdul; Shahimi, Safiyyah; Khalid, Rozida Mohd.; Ayob, Mohd. Khan; Bakar, Mohd. Faizal Abu; Isa, Mohd Noor Mat

    2016-11-01

    In this study, we performed bioinformatics analysis toward genome sequence of Lysinibacillussphaericus (L. sphaericus) to determine gene encoded for gelatinase. L. sphaericus was isolated from soil and gelatinase species-specific bacterium to porcine and bovine gelatin. This bacterium offers the possibility of enzymes production which is specific to both species of meat, respectively. The main focus of this research is to identify the gelatinase encoded gene within the bacteria of L. Sphaericus using bioinformatics analysis of partially sequence genome. From the research study, three candidate gene were identified which was, gelatinase candidate gene 1 (P1), NODE_71_length_93919_cov_158.931839_21 which containing 1563 base pair (bp) in size with 520 amino acids sequence; Secondly, gelatinase candidate gene 2 (P2), NODE_23_length_52851_cov_190.061386_17 which containing 1776 bp in size with 591 amino acids sequence; and Thirdly, gelatinase candidate gene 3 (P3), NODE_106_length_32943_cov_169.147919_8 containing 1701 bp in size with 566 amino acids sequence. Three pairs of oligonucleotide primers were designed and namely as, F1, R1, F2, R2, F3 and R3 were targeted short sequences of cDNA by PCR. The amplicons were reliably results in 1563 bp in size for candidate gene P1 and 1701 bp in size for candidate gene P3. Therefore, the results of bioinformatics analysis of L. Sphaericus resulting in gene encoded gelatinase were identified.

  18. Do nuclear-encoded core subunits of mitochondrial complex I confer genetic susceptibility to schizophrenia in Han Chinese populations?

    PubMed

    Li, Xiao; Zhang, Wen; Tang, Jinsong; Tan, Liwen; Luo, Xiong-jian; Chen, Xiaogang; Yao, Yong-Gang

    2015-06-08

    Schizophrenia is one of the most prevalent psychiatric disorders with complex genetic etiology. Accumulating evidence suggests that energy metabolism and oxidative stress play important roles in the pathophysiology of schizophrenia. Dysfunction of mitochondrial respiratory chain and altered expression of complex I subunits were frequently reported in schizophrenia. To investigate whether nuclear-encoded core subunit genes of mitochondrial complex I are associated with schizophrenia, we performed a genetic association study in Han Chinese. In total, 46 tag single nucleotide polymorphisms (SNPs) from 7 nuclear-encoded core genes of mitochondrial complex I were genotyped in 918 schizophrenia patients and 1042 healthy controls. We also analyzed these SNPs in a large sample mainly composed of Europeans through using the available GWAS datasets from the Psychiatric Genomics Consortium (PGC). No significant associations were detected between these SNPs and schizophrenia in Han Chinese and the PGC data set. However, we observed nominal significant associations of 2 SNPs in the NDUFS1 gene and 4 SNPs in the NDUFS2 gene with early onset schizophrenia (EOS), but none of these associations survived the Bonferroni correction. Taken together, our results suggested that common SNPs in the nuclear-encoded core subunit genes of mitochondrial complex I may not confer genetic susceptibility to schizophrenia.

  19. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  20. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  1. Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

    PubMed Central

    Willis, Jordan R.; Briney, Bryan S.; DeLuca, Samuel L.; Crowe, James E.; Meiler, Jens

    2013-01-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding. PMID:23637590

  2. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene.

    PubMed

    Tomkinson, Alan E; Sallmyr, Annahita

    2013-12-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target. © 2013 Elsevier B.V. All rights reserved.

  3. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

    PubMed Central

    Tomkinson, Alan E.; Sallmyr, Annahita

    2013-01-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target. PMID:24013086

  4. A reanalysis of mouse ENCODE comparative gene expression data

    PubMed Central

    Gilad, Yoav; Mizrahi-Man, Orna

    2015-01-01

    Recently, the Mouse ENCODE Consortium reported that comparative gene expression data from human and mouse tend to cluster more by species rather than by tissue. This observation was surprising, as it contradicted much of the comparative gene regulatory data collected previously, as well as the common notion that major developmental pathways are highly conserved across a wide range of species, in particular across mammals. Here we show that the Mouse ENCODE gene expression data were collected using a flawed study design, which confounded sequencing batch (namely, the assignment of samples to sequencing flowcells and lanes) with species. When we account for the batch effect, the corrected comparative gene expression data from human and mouse tend to cluster by tissue, not by species. PMID:26236466

  5. Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp.

    PubMed Central

    Wilbrink, Maarten H.; Casabon, Israël; Stewart, Gordon R.; Liu, Jie; van der Geize, Robert; Eltis, Lindsay D.

    2012-01-01

    Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. PMID:23024343

  6. An Incompatibility between a Mitochondrial tRNA and Its Nuclear-Encoded tRNA Synthetase Compromises Development and Fitness in Drosophila

    PubMed Central

    Meiklejohn, Colin D.; Holmbeck, Marissa A.; Siddiq, Mohammad A.; Abt, Dawn N.; Rand, David M.; Montooth, Kristi L.

    2013-01-01

    Mitochondrial transcription, translation, and respiration require interactions between genes encoded in two distinct genomes, generating the potential for mutations in nuclear and mitochondrial genomes to interact epistatically and cause incompatibilities that decrease fitness. Mitochondrial-nuclear epistasis for fitness has been documented within and between populations and species of diverse taxa, but rarely has the genetic or mechanistic basis of these mitochondrial–nuclear interactions been elucidated, limiting our understanding of which genes harbor variants causing mitochondrial–nuclear disruption and of the pathways and processes that are impacted by mitochondrial–nuclear coevolution. Here we identify an amino acid polymorphism in the Drosophila melanogaster nuclear-encoded mitochondrial tyrosyl–tRNA synthetase that interacts epistatically with a polymorphism in the D. simulans mitochondrial-encoded tRNATyr to significantly delay development, compromise bristle formation, and decrease fecundity. The incompatible genotype specifically decreases the activities of oxidative phosphorylation complexes I, III, and IV that contain mitochondrial-encoded subunits. Combined with the identity of the interacting alleles, this pattern indicates that mitochondrial protein translation is affected by this interaction. Our findings suggest that interactions between mitochondrial tRNAs and their nuclear-encoded tRNA synthetases may be targets of compensatory molecular evolution. Human mitochondrial diseases are often genetically complex and variable in penetrance, and the mitochondrial–nuclear interaction we document provides a plausible mechanism to explain this complexity. PMID:23382693

  7. Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

    PubMed Central

    Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

    2007-01-01

    Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

  8. The TL region gene 37 encodes a Qa-1 antigen

    PubMed Central

    1990-01-01

    Of all the biochemically defined mouse MHC class I molecules, the Qa-1 antigens are the only ones for which a gene has not been identified. Recent evidence has suggested that Qa-1 antigens are functional class I molecules and can function as restriction elements for gamma/delta T cells. We have examined the relationship between Qa-1 and the product of gene 37, a presumed novel class I antigen encoded within the TL region. Immunoprecipitation and polyacrylamide gel electrophoresis analysis of the molecules reactive with anti-Qa-1 and anti-37 sera show that the Qa-1 molecule of Qa-1b (Qa-1.2) mouse strains is identical to the product of gene 37 on the basis of molecular weight, pI, and strain distribution. Immunodepletion, biosynthetic labeling, and tunicamycin treatment confirm that the protein encoded by gene 37 in Qa-1b mice is Qa-1.2. In contrast, the anti-37 serum was unable to recognize the Qa-1 molecule in Qa-1a strains. Given the fact that the only allele to gene 37 thus far identified in a Qa-1a strain (A/J) has a termination codon in the alpha 3 domain, our data lead us to conclude that the Qa-1 molecule expressed in Qa-1a mice is not a true allelic product of the gene 37 encoded antigen of Qa-1b mouse strains. PMID:2258708

  9. Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens

    PubMed Central

    Kirner, Sabine; Hammer, Philip E.; Hill, D. Steven; Altmann, Annett; Fischer, Ilona; Weislo, Laura J.; Lanahan, Mike; van Pée, Karl-Heinz; Ligon, James M.

    1998-01-01

    Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway. PMID:9537395

  10. Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens.

    PubMed

    Kirner, S; Hammer, P E; Hill, D S; Altmann, A; Fischer, I; Weislo, L J; Lanahan, M; van Pée, K H; Ligon, J M

    1998-04-01

    Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of L-tryptophan to form 7-chloro-L-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-L-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.

  11. Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus

    Treesearch

    David S. Bischoff; James M. Slavicek

    1997-01-01

    A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) gene has been identified that encodes a homolog to the granulovirus (GV) enhancin proteins that are capable of enhancing the infection of other baculoviruses. Enhancin genes have been identified and sequenced for three species of GVs but have not been found in any other nuclear...

  12. The exception proves the rule? Dual targeting of nuclear-encoded proteins into endosymbiotic organelles.

    PubMed

    Baudisch, Bianca; Langner, Uwe; Garz, Ingo; Klösgen, Ralf Bernd

    2014-01-01

    Plant cells harbor two types of endosymbiotic organelle: mitochondria and chloroplasts. As a consequence of endosymbiotic gene transfer, the majority of their proteins are encoded in the nucleus and post-translationally 're'-imported into the respective target organelle. The corresponding transport signals are usually selective for a single organelle, but several proteins are transported into both the mitochondria and chloroplasts. To estimate the number of proteins with such dual targeting properties in Arabidopsis, we classified the proteins encoded by nuclear genes of endosymbiotic origin according to the respective targeting specificity of their N-terminal transport signals as predicted by the TargetP software package. Selected examples of the resulting protein classes were subsequently analyzed by transient transformation assays as well as by in organello protein transport experiments. It was found that most proteins with high prediction values for both organelles show dual targeting with both experimental approaches. Unexpectedly, however, dual targeting was even found among those proteins that are predicted to be localized solely in one of the two endosymbiotic organelles. In total, among the 16 candidate proteins analyzed, we identified 10 proteins with dual targeting properties. This unexpectedly high proportion suggests that such transport properties are much more abundant than anticipated.

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

    SciTech Connect

    Beachy, R.N.

    1985-12-18

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

  14. The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase.

    PubMed Central

    Kanipes, M I; Hill, J E; Henry, S A

    1998-01-01

    The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms. PMID:9755189

  15. Identification and use of genes encoding amatoxin and phallotoxin

    DOEpatents

    Hallen, Heather E.; Walton, Jonathan D.; Luo, Hong; Scott-Craig, John S.

    2016-12-13

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In particular, the present invention relates to using genes and proteins from Amanita species encoding Amanita peptides, specifically relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and monitoring suspected cases of mushroom poisoning in patients.

  16. Clustered Genes Encoding the Methyltransferases of Methanogenesis from Monomethylamine

    PubMed Central

    Burke, Stephen A.; Lo, Sam L.; Krzycki, Joseph A.

    1998-01-01

    Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in Methanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region between mtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of mtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of mtmB in this methanogen. PMID:9642198

  17. Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine.

    PubMed

    Burke, S A; Lo, S L; Krzycki, J A

    1998-07-01

    Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in Methanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region between mtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of mtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of mtmB in this methanogen.

  18. Extranuclear gene expression in yeast: evidence for a plasmid-encoded RNA polymerase of unique structure.

    PubMed Central

    Wilson, D W; Meacock, P A

    1988-01-01

    Strains of the yeast Kluyveromyces lactis that produce killer-toxin have been found to contain two linear dsDNA plasmids, k1 (8.9 Kb) and k2 (13.4 Kb). The four transcribed open reading frames of plasmid k1 contain no recognisable yeast nuclear expression signals. Moreover, a toxin subunit gene fused with the lacZ gene of Escherichia coli is not detectably expressed when introduced to K.lactis or Saccharomyces cerevisiae on a nuclear vector, even when native k1 and k2 are present in the cell. This and other evidence is consistent with the hypothesis that k1 and k2 reside in an extranuclear location, and do not utilise the nuclear RNA polymerases I, II or III for transcription of their genes. Sequencing of plasmid k2, which is thought to encode factors necessary for the maintenance or expression of k1, reveals an open reading frame predicted to encode a 974 amino acid polypeptide with homology to several DNA-directed RNA polymerases. We suggest that this is a component of a novel plasmid-specific extranuclear gene expression system. PMID:3138657

  19. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus.

    PubMed

    Kim, Seongman; Ahn, Byung Chul; O'Callaghan, Dennis J; Kim, Seong Kee

    2012-10-25

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  20. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

    SciTech Connect

    Kim, Seongman; Chul Ahn, Byung; O'Callaghan, Dennis J.; Kim, Seong Kee

    2012-10-25

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  1. Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number.

    PubMed Central

    Huang, J; Struck, F; Matzinger, D F; Levings, C S

    1994-01-01

    The mitochondrial Rieske iron-sulfur protein is an obligatory component of the respiratory electron transport chain that is encoded by a single-copy gene in mammals and fungi. In contrast, this protein is encoded by a small gene family in dicotyledonous tobacco and monocotyledonous maize. We cloned four cDNAs from tobacco that encode the mitochondrial Rieske iron-sulfur protein. These clones, along with a previously isolated cDNA, represent five independent members of the gene family that can be divided into three subfamilies. All of these genes were derived from the two progenitor species and were expressed in amphidiploid tobacco. The proteins encoded by these five genes are probably functional because they all contain the universally conserved hexyl peptides necessary for the 2Fe-2S cluster formation. The expression of the Rieske protein gene family is differentially regulated; a 6- to 11-fold higher level of steady state transcripts was found in flowers than in leaves, stems, and roots. Members of at least two subfamilies were preferentially expressed in flowers, indicating that they share a common cis-regulatory element(s), which can respond to a flower-specific signal(s). Although approximately 10 times more transcripts occurred in flowers than in leaves, flower and leaf mitochondria contained a similar amount of the Rieske protein. Flowers, however, contained seven times more Rieske proteins than leaves. These results indicated an increase in mitochondrion number in flowers. High-energy demands during anther development might bring about an increase in mitochondrion numbers in flowers and the flower-enhanced expression of the Rieske protein gene family. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism and/or changes in mitochondrion numbers. PMID:8180500

  2. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA.

    PubMed

    Mueller, Edith E; Mayr, Johannes A; Zimmermann, Franz A; Feichtinger, René G; Stanger, Olaf; Sperl, Wolfgang; Kofler, Barbara

    2012-01-20

    Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 ρ(0) cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in ρ(0) cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  3. Nuclear gene dosage effects upon the expression of maize mitochondrial genes.

    PubMed Central

    Auger, D L; Newton, K J; Birchler, J A

    2001-01-01

    Each mitochondrion possesses a genome that encodes some of its own components. The nucleus encodes most of the mitochondrial proteins, including the polymerases and factors that regulate the expression of mitochondrial genes. Little is known about the number or location of these nuclear factors. B-A translocations were used to create dosage series for 14 different chromosome arms in maize plants with normal cytoplasm. The presence of one or more regulatory factors on a chromosome arm was indicated when variation of its dosage resulted in the alteration in the amount of a mitochondrial transcript. We used quantitative Northern analysis to assay the transcript levels of three mitochondrially encoded components of the cytochrome c oxidase complex (cox1, cox2, and cox3). Data for a nuclearly encoded component (cox5b) and for two mitochondrial genes that are unrelated to cytochrome c oxidase, ATP synthase alpha-subunit and 18S rRNA, were also determined. Two tissues, embryo and endosperm, were compared and most effects were found to be tissue specific. Significantly, the array of dosage effects upon mitochondrial genes was similar to what had been previously found for nuclear genes. These results support the concept that although mitochondrial genes are prokaryotic in origin, their regulation has been extensively integrated into the eukaryotic cell. PMID:11290725

  4. Identification of β-haemolysin-encoding genes in Streptococcus anginosus.

    PubMed

    Asam, D; Mauerer, S; Walheim, E; Spellerberg, B

    2013-08-01

    Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for β-haemolysis we performed genomic mutagenesis of the β-haemolytic S. anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. Integration site analysis of 15 non-haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S (SLS) encoding sag gene cluster of S. pyogenes. The gene cluster harbours 10 open reading frames displaying significant similarities to the S. pyogenes genes sagA-sagI, with the identities on protein level ranging from 38 to 87%. Complementation assays of S. anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for β-haemolysin production and suggest the presence of post-translational modifications in S. anginosus SLS similar to SLS of S. pyogenes. Characterization of the S. anginosus haemolysin in comparison to the S. pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S. pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S. anginosus in culture supernatants. Inhibition of β-haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore-forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of Streptococcus constellatus, a closely related streptococcal species that belongs to the S. anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S. anginosus and demonstrate its importance for β-haemolysin production in S. anginosus.

  5. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    PubMed

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  6. Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).

    PubMed

    Guo, Yong; Li, Hejuan; Wang, Ying; Yan, Xingrong; Sheng, Xihui; Chang, Di; Qi, Xiaolong; Wang, Xiangguo; Liu, Yunhai; Li, Junya; Ni, Hemin

    2017-02-01

    Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

  7. The EWS–Oct-4 fusion gene encodes a transforming gene

    PubMed Central

    Lee, Jungwoon; Kim, Ja Young; Kang, In Young; Kim, Hye Kyoung; Han, Yong-Mahn; Kim, Jungho

    2007-01-01

    The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS–Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS–Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS–Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS–Oct-4 are necessary for full transactivation potential. EWS–Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS–Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS–Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product. PMID:17564582

  8. Characterization and mapping of human genes encoding zinc finger proteins.

    PubMed Central

    Bray, P; Lichter, P; Thiesen, H J; Ward, D C; Dawid, I B

    1991-01-01

    The zinc finger motif, exemplified by a segment of the Drosophila gap gene Krüppel, is a nucleic acid-binding domain present in many transcription factors. To investigate the gene family encoding this motif in the human genome, a placental genomic library was screened at moderate stringency with a degenerate oligodeoxynucleotide probe designed to hybridize to the His/Cys (H/C) link region between adjoining zinc fingers. Over 200 phage clones were obtained and are being sorted into groups by partial sequencing, cross-hybridization with oligodeoxynucleotide probes, and PCR amplification. Further, the genomic clones were cross-hybridized with a set of 30 zinc finger-encoding cDNAs (Kox1-Kox30) isolated from a human T-cell cDNA library. Four cDNAs (Kox4, Kox7, Kox12, and Kox15) were identified that match one or more genomic clones; these matches were confirmed by nucleotide sequence analysis. One or more clones from each locus were mapped onto human metaphase chromosomes by chromosomal in situ suppression hybridization with fluorescent probe detection. We mapped ZNF7/Kox4 to chromosome 8qter, ZNF19/Kox12 to 16q22, ZNF22/Kox15 to 10q11, and ZNF44/Kox7 to 16p11. The results of these analyses support the conclusion that the human genome contains many, probably several hundred, zinc finger genes with consensus H/C link regions. Images PMID:1946370

  9. Rubisco in marine symbiotic dinoflagellates: form II enzymes in eukaryotic oxygenic phototrophs encoded by a nuclear multigene family.

    PubMed Central

    Rowan, R; Whitney, S M; Fowler, A; Yellowlees, D

    1996-01-01

    Genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were cloned from dinoflagellate symbionts (Symbiodinium spp) of the giant clam Tridacna gigas and characterized. Strikingly, Symbiodinium Rubisco is completely different from other eukaryotic (form I) Rubiscos: it is a form II enzyme that is approximately 65% identical to Rubisco from Rhodospirillum rubrum (Rubisco forms I and II are approximately 25 to 30% identical); it is nuclear encoded by a multigene family; and the predominantly expressed Rubisco is encoded as a precursor polyprotein. One clone appears to contain a predominantly expressed Rubisco locus (rbcA), as determined by RNA gel blot analysis of Symbiodinium RNA and sequencing of purified Rubisco protein. Another contains an enigmatic locus (rbcG) that exhibits an unprecedented pattern of amino acid replacement but does not appear to be a pseudogene. The expression of rbcG has not been analyzed; it was detected only in the minor of two taxa of Symbiodinium that occur together in T. gigas. This study confirms and describes a previously unrecognized branch of Rubisco's evolution: a eukaryotic form II enzyme that participates in oxygenic photosynthesis and is encoded by a diverse, nuclear multigene family. PMID:8721755

  10. A rhomboid gene controls speciation through regulation of nuclear-mitochondrial compatibility in Triticum

    USDA-ARS?s Scientific Manuscript database

    The nuclear encoded species cytoplasm specific (scs) genes control nuclear-cytoplasmic compatibility in Triticum. Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus....

  11. Gene family encoding the major toxins of lethal Amanita mushrooms.

    PubMed

    Hallen, Heather E; Luo, Hong; Scott-Craig, John S; Walton, Jonathan D

    2007-11-27

    Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode alpha-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. alpha-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable "toxin" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.

  12. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

    SciTech Connect

    Mueller, Edith E.; Mayr, Johannes A.; Zimmermann, Franz A.; Feichtinger, Rene G.; Stanger, Olaf; Sperl, Wolfgang; Kofler, Barbara

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  13. Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

    Treesearch

    David S. Bischoff; James M. Slavicek

    1995-01-01

    The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding G22 was cloned and sequenced. The G22 gene codes for a 191 amino acid protein with a predicted Mr of 22000. Expression of G22 in a rabbit reticulocyte system generated a protein with an M...

  14. The Pea Gene LH Encodes ent-Kaurene Oxidase1

    PubMed Central

    Davidson, Sandra E.; Smith, Jennifer J.; Helliwell, Chris A.; Poole, Andrew T.; Reid, James B.

    2004-01-01

    The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants. PMID:14988475

  15. The pea gene LH encodes ent-kaurene oxidase.

    PubMed

    Davidson, Sandra E; Smith, Jennifer J; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2004-03-01

    The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants.

  16. Genome-wide analysis of NBS-encoding disease resistance genes in Cucumis sativus and phylogenetic study of NBS-encoding genes in Cucurbitaceae crops

    PubMed Central

    2013-01-01

    Background Plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) proteins encoded by resistance genes play an important role in the responses of plants to various pathogens, including viruses, bacteria, fungi, and nematodes. In this study, a comprehensive analysis of NBS-encoding genes within the whole cucumber genome was performed, and the phylogenetic relationships of NBS-encoding resistance gene homologues (RGHs) belonging to six species in five genera of Cucurbitaceae crops were compared. Results Cucumber has relatively few NBS-encoding genes. Nevertheless, cucumber maintains genes belonging to both Toll/interleukine-1 receptor (TIR) and CC (coiled-coil) families. Eight commonly conserved motifs have been established in these two families which support the grouping into TIR and CC families. Moreover, three additional conserved motifs, namely, CNBS-1, CNBS-2 and TNBS-1, have been identified in sequences from CC and TIR families. Analyses of exon/intron configurations revealed that some intron loss or gain events occurred during the structural evolution between the two families. Phylogenetic analyses revealed that gene duplication, sequence divergence, and gene loss were proposed as the major modes of evolution of NBS-encoding genes in Cucurbitaceae species. Compared with NBS-encoding sequences from the Arabidopsis thaliana genome, the remaining seven TIR familes of NBS proteins and RGHs from Cucurbitaceae species have been shown to be phylogenetically distinct from the TIR family of NBS-encoding genes in Arabidopsis, except for two subfamilies (TIR4 and TIR9). On the other hand, in the CC-NBS family, they grouped closely with the CC family of NBS-encoding genes in Arabidopsis. Thus, the NBS-encoding genes in Cucurbitaceae crops are shown to be ancient, and NBS-encoding gene expansions (especially the TIR family) may have occurred before the divergence of Cucurbitaceae and Arabidopsis. Conclusion The results of this paper will provide a genomic framework

  17. Genome-wide analysis of NBS-encoding disease resistance genes in Cucumis sativus and phylogenetic study of NBS-encoding genes in Cucurbitaceae crops.

    PubMed

    Wan, Hongjian; Yuan, Wei; Bo, Kailiang; Shen, Jia; Pang, Xin; Chen, Jinfeng

    2013-02-19

    Plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) proteins encoded by resistance genes play an important role in the responses of plants to various pathogens, including viruses, bacteria, fungi, and nematodes. In this study, a comprehensive analysis of NBS-encoding genes within the whole cucumber genome was performed, and the phylogenetic relationships of NBS-encoding resistance gene homologues (RGHs) belonging to six species in five genera of Cucurbitaceae crops were compared. Cucumber has relatively few NBS-encoding genes. Nevertheless, cucumber maintains genes belonging to both Toll/interleukine-1 receptor (TIR) and CC (coiled-coil) families. Eight commonly conserved motifs have been established in these two families which support the grouping into TIR and CC families. Moreover, three additional conserved motifs, namely, CNBS-1, CNBS-2 and TNBS-1, have been identified in sequences from CC and TIR families. Analyses of exon/intron configurations revealed that some intron loss or gain events occurred during the structural evolution between the two families. Phylogenetic analyses revealed that gene duplication, sequence divergence, and gene loss were proposed as the major modes of evolution of NBS-encoding genes in Cucurbitaceae species. Compared with NBS-encoding sequences from the Arabidopsis thaliana genome, the remaining seven TIR familes of NBS proteins and RGHs from Cucurbitaceae species have been shown to be phylogenetically distinct from the TIR family of NBS-encoding genes in Arabidopsis, except for two subfamilies (TIR4 and TIR9). On the other hand, in the CC-NBS family, they grouped closely with the CC family of NBS-encoding genes in Arabidopsis. Thus, the NBS-encoding genes in Cucurbitaceae crops are shown to be ancient, and NBS-encoding gene expansions (especially the TIR family) may have occurred before the divergence of Cucurbitaceae and Arabidopsis. The results of this paper will provide a genomic framework for the further isolation of

  18. Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni

    PubMed Central

    Isokpehi, Raphael D.; Mahmud, Ousman; Mbah, Andreas N.; Simmons, Shaneka S.; Avelar, Lívia; Rajnarayanan, Rajendram V.; Udensi, Udensi K.; Ayensu, Wellington K.; Cohly, Hari H.; Brown, Shyretha D.; Dates, Centdrika R.; Hentz, Sonya D.; Hughes, Shawntae J.; Smith-McInnis, Dominique R.; Patterson, Carvey O.; Sims, Jennifer N.; Turner, Kelisha T.; Williams, Baraka S.; Johnson, Matilda O.; Adubi, Taiwo; Mbuh, Judith V.; Anumudu, Chiaka I.; Adeoye, Grace O.; Thomas, Bolaji N.; Nashiru, Oyekanmi; Oliveira, Guilherme

    2011-01-01

    The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the

  19. Nuclear-encoded factors involved in post-transcriptional processing and modification of mitochondrial tRNAs in human disease

    PubMed Central

    Powell, Christopher A.; Nicholls, Thomas J.; Minczuk, Michal

    2015-01-01

    The human mitochondrial genome (mtDNA) encodes 22 tRNAs (mt-tRNAs) that are necessary for the intraorganellar translation of the 13 mtDNA-encoded subunits of the mitochondrial respiratory chain complexes. Maturation of mt-tRNAs involves 5′ and 3′ nucleolytic excision from precursor RNAs, as well as extensive post-transcriptional modifications. Recent data suggest that over 7% of all mt-tRNA residues in mammals undergo post-transcriptional modification, with over 30 different modified mt-tRNA positions so far described. These processing and modification steps are necessary for proper mt-tRNA function, and are performed by dedicated, nuclear-encoded enzymes. Recent growing evidence suggests that mutations in these nuclear genes (nDNA), leading to incorrect maturation of mt-tRNAs, are a cause of human mitochondrial disease. Furthermore, mtDNA mutations in mt-tRNA genes, which may also affect mt-tRNA function, processing, and modification, are also frequently associated with human disease. In theory, all pathogenic mt-tRNA variants should be expected to affect only a single process, which is mitochondrial translation, albeit to various extents. However, the clinical manifestations of mitochondrial disorders linked to mutations in mt-tRNAs are extremely heterogeneous, ranging from defects of a single tissue to complex multisystem disorders. This review focuses on the current knowledge of nDNA coding for proteins involved in mt-tRNA maturation that have been linked to human mitochondrial pathologies. We further discuss the possibility that tissue specific regulation of mt-tRNA modifying enzymes could play an important role in the clinical heterogeneity observed for mitochondrial diseases caused by mutations in mt-tRNA genes. PMID:25806043

  20. lin-8, which antagonizes Caenorhabditis elegans Ras-mediated vulval induction, encodes a novel nuclear protein that interacts with the LIN-35 Rb protein.

    PubMed

    Davison, Ewa M; Harrison, Melissa M; Walhout, Albertha J M; Vidal, Marc; Horvitz, H Robert

    2005-11-01

    Ras-mediated vulval development in C. elegans is inhibited by the functionally redundant sets of class A, B, and C synthetic Multivulva (synMuv) genes. Three of the class B synMuv genes encode an Rb/DP/E2F complex that, by analogy with its mammalian and Drosophila counterparts, has been proposed to silence genes required for vulval specification through chromatin modification and remodeling. Two class A synMuv genes, lin-15A and lin-56, encode novel nuclear proteins that appear to function as a complex. We show that a third class A synMuv gene, lin-8, is the defining member of a novel C. elegans gene family. The LIN-8 protein is nuclear and can interact physically with the product of the class B synMuv gene lin-35, the C. elegans homolog of mammalian Rb. LIN-8 likely acts with the synMuv A proteins LIN-15A and LIN-56 in the nucleus, possibly in a protein complex with the synMuv B protein LIN-35 Rb. Other LIN-8 family members may function in similar complexes in different cells or at different stages. The nuclear localization of LIN-15A, LIN-56, and LIN-8, as well as our observation of a direct physical interaction between class A and class B synMuv proteins, supports the hypothesis that the class A synMuv genes control vulval induction through the transcriptional regulation of gene expression.

  1. RAG4 gene encodes a glucose sensor in Kluyveromyces lactis.

    PubMed Central

    Betina, S; Goffrini, P; Ferrero, I; Wésolowski-Louvel, M

    2001-01-01

    The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished. PMID:11404320

  2. The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

    PubMed

    Gogos, J A; Santha, M; Takacs, Z; Beck, K D; Luine, V; Lucas, L R; Nadler, J V; Karayiorgou, M

    1999-04-01

    Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

  3. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  4. Characterization of a plasmid-encoded urease gene cluster found in members of the family Enterobacteriaceae.

    PubMed

    D'Orazio, S E; Collins, C M

    1993-03-01

    Plasmid-encoded urease gene clusters found in uropathogenic isolates of Escherichia coli, Providencia stuartii, and Salmonella cubana demonstrated DNA homology, similar positions of restriction endonuclease cleavage sites, and manners of urease expression and therefore represent the same locus. DNA sequence analysis indicated that the plasmid-encoded urease genes are closely related to the Proteus mirabilis urease genes.

  5. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  6. Plastid-Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae.

    PubMed

    Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

    2016-06-27

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid-nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae

    PubMed Central

    Weng, Mao-Lun; Ruhlman, Tracey A.; Jansen, Robert K.

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid–nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  8. Inactivation of the Neurospora Crassa Gene Encoding the Mitochondrial Protein Import Receptor Mom19 by the Technique of ``sheltered Rip''

    PubMed Central

    Harkness, TAA.; Metzenberg, R. L.; Schneider, H.; Lill, R.; Neupert, W.; Nargang, F. E.

    1994-01-01

    We have used a technique referred to as ``sheltered RIP'' (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa. PMID:8138148

  9. Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

    PubMed

    Gale, Jenna R; Aschrafi, Armaz; Gioio, Anthony E; Kaplan, Barry B

    2017-06-01

    Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

  10. A mutation in At-nMat1a, which encodes a nuclear gene having high similarity to group II intron maturase, causes impaired splicing of mitochondrial NAD4 transcript and altered carbon metabolism in Arabidopsis thaliana.

    PubMed

    Nakagawa, Naoki; Sakurai, Naoki

    2006-06-01

    To elucidate the mechanism of cellulose synthesis, we isolated a mutant of Arabidopsis (changed sensitivity to cellulose synthesis inhibitors 1, css1) that showed changed sensitivity to cellulose biosynthesis inhibitor. The analysis of phenotypes indicated that the css1 mutation influenced various fundamental metabolic pathways including amino acid metabolism, triacylglycerol degradation and polysaccharide synthesis (cellulose and starch) during the early stage of plant growth. Unexpectedly, the map-based cloning of the gene responsible for the css1 mutation identified a protein (At-nMat1a) that was assumed to be a splicing factor of the mitochondrial group II intron. In accordance with this result, this mutant exhibited improper splicing of the mitochondrial NAD4 transcript. We noticed that the phenotypes of the css1 mutant are similar to the responses to anoxia that hinders mitochondrial aerobic respiration. It seems that the defect in the function of mitochondria influences various aspects of fundamental cellular metabolism including cellulose synthesis. Our results suggested that sucrose synthase (SuSy), an enzyme involved in the biosynthesis of cellulose, plays key roles in the connection between mitochondria and cellulose synthesis. The isolation of the css1 mutant also provides a useful resource in the study of post-transcriptional gene regulation in mitochondria.

  11. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    PubMed

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  12. Eukaryotic expression vectors bearing genes encoding cytotoxic proteins for cancer gene therapy.

    PubMed

    Glinka, Elena M

    2012-09-01

    Cancer gene therapy is a promising direction for the treatment of cancer patients. A primary goal of all cancer therapies is to selectively target and kill tumour cells. Such therapies are administered via different approaches, including both viral and non-viral delivery; however, both methods have advantages and disadvantages. Transcriptional targeting enables genes encoding toxic proteins to be expressed directly in cancer cells. Numerous vectors have been created with the purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Data concerning the function of vectors bearing genes that encode cytotoxic proteins under the control of different promoters, including tissue/tumour specific and constitutive promoters, is summarised here. This review focuses on vectors that bear genes encoding diphtheria toxin, Pseudomonas exotoxin A, caspases, gef, streptolysin, and melittin. Data describing the efficacy of such vectors have been summarised. Notably, there are vectors that killed cancer cell lines originating from the same type of cancer with differential efficiency. Thus, there is differential inhibition of cancer cell growth dependent on the cell line. In this review, the constructs employing genes whose expression induces cell death and the efficiency with which they suppress cancer cell growth will be summarised.

  13. Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

    PubMed Central

    Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

    2013-01-01

    Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns. PMID:24391881

  14. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    PubMed

    Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

    2013-01-01

    Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  15. The maize brittle 1 gene encodes amyloplast membrane polypeptides.

    PubMed

    Sullivan, T D; Kaneko, Y

    1995-01-01

    A chimeric protein, formed of 56 amino acids from the carboxy terminus of the maize (Zea mays L.) wild-type Brittle1 (Bt1) protein fused to the glutathione-S-transferase gene, was synthesized in Escherichia coli, and used to raise antibodies. Following affinity purification, the antibodies recognized a set of 38- to 42-kDa proteins in endosperm from wild-type Bt1 plants, as well as from brittle2, shrunken2 and sugary1 plants, but not in mutant bt1 endosperm. Bt1 proteins were not detected with the preimmune antibodies. A low level of Bt1-specific proteins was detected at 10 d after pollination (DAP) and increased to a plateau at 16 DAP. At the same time, the ratio of slow- to fast-migrating forms of the protein decreased. During endosperm fractionation by differential centrifugation and membrane sedimentation in sucrose gradients, the Bt1 proteins co-purified with the carotenoid-containing plastid membranes. They were localized to amyloplasts by electron-microscopic immunocytochemistry; most of the signal was detected at the plastid periphery. These results are consistent with predictions made from the deduced amino-acid sequence and previous in-vitro experiments that the bt1 locus encodes amyloplast membrane proteins.

  16. [Association of schizophrenia with variations in genes encoding transcription factors].

    PubMed

    Boyajyan, A S; Atshemyan, S A; Zakharyan, R V

    2015-01-01

    Alterations in neuronal plasticity and immune system play a key role in pathogenesis of schizophrenia. Identification of genetic factors contributing to these alterations will significantly encourage elucidation of molecular etiopathomechanisms of this disorder. Transcription factors c-Fos, c-Jun, and Ier5 are the important regulators of neuronal plasticity and immune response. In the present work we investigated a potential association of schizophrenia with a number of single nucleotide polymorphisms of c-Fos-,c-Jun and Ier5 encoding genes (FOS, JUN, and IER5 respectively). Genotyping of DNA samples of patients with schizophrenia and healthy individuals was performed using polymerase chain reaction with allele specific primers. The results obtained demonstrated association between schizophrenia and FOS rs1063169, FOS rs7101, JUN rs11688, and IER5 rs6425663 polymorphisms. Namely, it was found that the inheritance of FOS rs1063169*T, JUN rs11688*A, and IER5 rs6425663*T minor variants decreases risk for development of schizophrenia whereas the inheritance of FOS rs7101*T minor variant, especially its homozygous form, increases risk for development of this disorder.

  17. Cell type-specific transcriptional regulation of the gene encoding importin-{alpha}1

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro

    2011-08-15

    Importin-{alpha}1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position - 251 to - 179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krueppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells. -- Research Highlights: {yields} We showed the cell type-specific transcriptional regulation of Kpna2 encoding importin-al. {yields} NF-Y binds the CCAAT boxes to activate Kpna2 transcription in NIH3T3 cells. {yields} Klf2 and Klf4 redundantly activate the expression of Kpna2 in ES cells.

  18. Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

    PubMed Central

    DeHoratius, C; Silver, P A

    1996-01-01

    To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport. Images PMID:8930904

  19. SUPPRESSOR OF FRIGIDA3 Encodes a Nuclear ACTIN-RELATED PROTEIN6 Required for Floral Repression in ArabidopsisW⃞

    PubMed Central

    Choi, Kyuha; Kim, Sanghee; Kim, Sang Yeol; Kim, Minsoo; Hyun, Youbong; Lee, Horim; Choe, Sunghwa; Kim, Sang-Gu; Michaels, Scott; Lee, Ilha

    2005-01-01

    Flowering traits in winter annual Arabidopsis thaliana are conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC acts as a flowering repressor and is regulated by multiple flowering pathways. We isolated an early-flowering mutant, suppressor of FRIGIDA3 (suf3), which also shows leaf serration, weak apical dominance, and infrequent conversion of the inflorescence shoot to a terminal flower. The suf3 mutation caused a decrease in the transcript level of FLC in both a FRI-containing line and autonomous pathway mutants. However, suf3 showed only a partial reduction of FLC transcript level, although it largely suppressed the late-flowering phenotype. In addition, the suf3 mutation caused acceleration of flowering in both 35S-FLC and a flc null mutant, indicating that SUF3 regulates additional factor(s) for the repression of flowering. SUF3 is highly expressed in the shoot apex, but the expression is not regulated by FRI, autonomous pathway genes, or vernalization. SUF3 encodes the nuclear ACTIN-RELATED PROTEIN6 (ARP6), the homolog of which in yeast is a component of an ATP-dependent chromatin-remodeling SWR1 complex. Our analyses showed that SUF3 regulates FLC expression independent of vernalization, FRI, and an autonomous pathway gene, all of which affect the histone modification of FLC chromatin. Subcellular localization using a green fluorescent protein fusion showed that Arabidopsis ARP6 is located at distinct regions of the nuclear periphery. PMID:16155178

  20. (Genetic engineering with a gene encoding a soybean storage protein). Progress report

    SciTech Connect

    Beachy, R.N.

    1985-01-01

    Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the ..cap alpha..'-subunit of ..beta..-conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ACR)

  1. Tracing the origin and evolution of plant TIR-encoding genes.

    PubMed

    Sun, Xiaoqin; Pang, Hui; Li, Mimi; Chen, Jianqun; Hang, Yueyu

    2014-08-10

    Toll-interleukin-1 receptor (TIR)-encoding proteins represent one of the most important families of disease resistance genes in plants. Studies that have explored the functional details of these genes tended to focus on only a few limited groups; the origin and evolutionary history of these genes were therefore unclear. In this study, focusing on the four principal groups of TIR-encoding genes, we conducted an extensive genome-wide survey of 32 fully sequenced plant genomes and Expressed Sequence Tags (ESTs) from the gymnosperm Pinus taeda and explored the origins and evolution of these genes. Through the identification of the TIR-encoding genes, the analysis of chromosome positions, the identification and analysis of conserved motifs, and sequence alignment and phylogenetic reconstruction, our results showed that the genes of the TIR-X family (TXs) had an earlier origin and a wider distribution than the genes from the other three groups. TIR-encoding genes experienced large-scale gene duplications during evolution. A skeleton motif pattern of the TIR domain was present in all spermatophytes, and the genes with this skeleton pattern exhibited a conserved and independent evolutionary history in all spermatophytes, including monocots, that followed their gymnosperm origin. This study used comparative genomics to explore the origin and evolutionary history of the four main groups of TIR-encoding genes. Additionally, we unraveled the mechanism behind the uneven distribution of TIR-encoding genes in dicots and monocots. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. HMGN4, a newly discovered nucleosome-binding protein encoded by an intronless gene.

    PubMed

    Birger, Y; Ito, Y; West, K L; Landsman, D; Bustin, M

    2001-05-01

    We describe a newly discovered nuclear protein, HMGN4, that is closely related to the canonical HMGN2 nucleosome-binding protein. The protein is encoded by an intronless gene, which, in humans, is located in the hereditary hemochromatosis [correction of hemachromatosis] region at position 6p21.3. A single approximately 2-kb HMGN4 mRNA was found to be expressed, in variable amounts, in all human tissues tested; however, the HMGN4 transcript was significantly less abundant than that of HMGN2. The HMGN4 protein could be detected in HeLa cells by Western analysis with an antibody elicited against a unique region of the protein. Transfection of HeLa cells with a plasmid expressing HMGN4-GFP indicated that the protein localizes to the nucleus. Our results expand the multiplicity of the HMGN protein family and increase the known cellular repertoire of nucleosome-binding proteins.

  3. Concerted evolution of the tandemly repeated genes encoding primate U2 small nuclear RNA (the RNU2 locus) does not prevent rapid diversification of the (CT){sub n} {center_dot} (GA){sub n} microsatellite embedded within the U2 repeat unit

    SciTech Connect

    Liao, D.; Weiner, A.M.

    1995-12-10

    The RNU2 locus encoding human U2 small nuclear RNA (snRNA) is organized as a nearly perfect tandem array containing 5 to 22 copies of a 5.8-kb repeat unit. Just downstream of the U2 snRNA gene in each 5.8-kb repeat unit lies a large (CT){sub n}{center_dot}(GA){sub n} dinucleotide repeat (n {approx} 70). This form of genomic organization, in which one repeat is embedded within another, provides an unusual opportunity to study the balance of forces maintaining the homogeneity of both kinds of repeats. Using a combination of field inversion gel electrophoresis and polymerase chain reaction, we have been able to study the CT microsatellites within individual U2 tandem arrays. We find that the CT microsatellites within an RNU2 allele exhibit significant length polymorphism, despite the remarkable homogeneity of the surrounding U2 repeat units. Length polymorphism is due primarily to loss or gain of CT dinucleotide repeats, but other types of deletions, insertions, and substitutions are also frequent. Polymorphism is greatly reduced in regions where pure (CT){sub n} tracts are interrupted by occasional G residues, suggesting that irregularities stabilize both the length and the sequence of the dinucleotide repeat. We further show that the RNU2 loci of other catarrhine primates (gorilla, chimpanzee, ogangutan, and baboon) contain orthologous CT microsatellites; these also exhibit length polymorphism, but are highly divergent from each other. Thus, although the CT microsatellite is evolving far more rapidly than the rest of the U2 repeat unit, it has persisted through multiple speciation events spanning >35 Myr. The persistence of the CT microsatellite, despite polymorphism and rapid evolution, suggests that it might play a functional role in concerted evolution of the RNU2 loci, perhaps as an initiation site for recombination and/or gene conversion. 70 refs., 5 figs.

  4. Fibroblast growth factor-1-inducible gene FR-17 encodes a nonmuscle alpha-actinin isoform.

    PubMed

    Hsu, D K; Guo, Y; Alberts, G F; Peifley, K A; Winkles, J A

    1996-05-01

    Polypeptide growth factor binding to cell surface receptors activates a cytoplasmic signaling cascade that ultimately promotes the expression of specific nuclear genes. As an approach to investigate the molecular mechanism of fibroblast growth factor (FGF)-1 mitogenic signaling, we have begun to identify and characterize FGF-1-inducible genes in murine NIH 3T3 cells. Here we report that one of these genes, termed FGF-regulated (FR)-17, is predicted to encode a nonmuscle isoform of alpha-actinin, an actin cross-linking protein found along microfilaments and in focal adhesion plaques. FGF-1 induction of alpha-actinin mRNA expression is first detectable at 2 h after mitogen addition and is dependent on the novo RNA and protein synthesis. Maximal alpha-actinin mRNA expression, corresponding to an approximately nineteenfold level of induction, is present after 12 h of FGF-1 stimulation. Western blot analysis indicated that FGF-1-stimulated cells also produce an increased amount of alpha-actinin protein. The FGF-1-related mitogen FGF-2, calf serum, several of the polypeptide growth factors present in serum, and the tumor promoter phorbol myristate acetate can also induce alpha-actinin mRNA expression. Finally, nonmuscle alpha-actinin mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in adult mouse intestine and kidney. These results indicate that nonmuscle alpha-actinin is a serum-, polypeptide growth factor-, and tumor promoter-inducible gene in mouse fibroblasts.

  5. Dissecting signal and noise in diatom chloroplast protein encoding genes with phylogenetic information profiling.

    PubMed

    Theriot, Edward C; Ashworth, Matt P; Nakov, Teofil; Ruck, Elizabeth; Jansen, Robert K

    2015-08-01

    Previous analyses of single diatom chloroplast protein-encoded genes recovered results highly incongruent with both traditional phylogenies and phylogenies derived from the nuclear encoded small subunit (SSU) gene. Our analysis here of six individual chloroplast genes (atpB, psaA, psaB, psbA, psbC and rbcL) obtained similar anomalous results. However, phylogenetic noise in these genes did not appear to be correlated, and their concatenation appeared to effectively sum their collective signal. We empirically demonstrated the value of combining phylogenetic information profiling, partitioned Bremer support and entropy analysis in examining the utility of various partitions in phylogenetic analysis. Noise was low in the 1st and 2nd codon positions, but so was signal. Conversely, high noise levels in the 3rd codon position was accompanied by high signal. Perhaps counterintuitively, simple exclusion experiments demonstrated this was especially true at deeper nodes where the 3rd codon position contributed most to a result congruent with morphology and SSU (and the total evidence tree here). Correlated with our empirical findings, probability of correct signal (derived from information profiling) increased and the statistical significance of substitutional saturation decreased as data were aggregated. In this regard, the aggregated 3rd codon position performed as well or better than more slowly evolving sites. Simply put, direct methods of noise removal (elimination of fast-evolving sites) disproportionately removed signal. Information profiling and partitioned Bremer support suggest that addition of chloroplast data will rapidly improve our understanding of the diatom phylogeny, but conversely also illustrate that some parts of the diatom tree are likely to remain recalcitrant to addition of molecular data. The methods based on information profiling have been criticized for their numerous assumptions and parameter estimates and the fact that they are based on quartets of

  6. Biochemical evidence that secretor gene, Se, is a structural gene encoding a specific fucosyltransferase.

    PubMed Central

    Kumazaki, T; Yoshida, A

    1984-01-01

    Nonsecretors have no ABH blood group substances in their saliva and milk, but their erythrocytes contain the blood group substances. It has been generally believed that the secretor gene, Se, is a regulatory gene, not a structural gene, controlling the expression of (alpha 1----2)fucosyltransferase, which synthesizes the blood group H substance from its precursor, in secretions. To account for the existence of the blood type of "para Bombay" phenotype--i.e., H-negative in erythrocytes but H-positive in secretory fluids, another regulatory gene, Z, which would regulate the expression of the enzyme in the hematopoietic tissues, has been proposed. Contrary to this, a more simple model, in which the H gene and Se gene are both structural genes, encoding two separate fucosyltransferases in different tissues, was recently proposed. To settle the controversy, (alpha 1----2)fucosyltransferases were partially purified from human plasma and milk. The two enzymes differed from each other in the following respects: (i) the milk enzyme adsorbed to SP-Sephadex at pH 6.0, while the plasma enzyme did not; (ii) pH-activity profiles, with phenyl beta-D-galactoside as an acceptor, differed between the two enzymes; (iii) the milk enzyme exhibited lower thermal stability than the plasma enzyme; and (iv) Km values for several oligosaccharides with Gal(beta 1----3)GlcNAc and Gal(beta 1----4)GlcNAc as acceptors differed between the two enzymes. These results support the model that the Se gene is a structural gene encoding a distinctive (alpha 1----2)fucosyltransferase, refuting the classical regulatory gene model for the Se locus. The anomeric configuration of the fucosylated galactose residue produced by the action of enzyme was identified, thus establishing the specificity of the enzyme. Images PMID:6588382

  7. Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris.

    PubMed Central

    Broglie, K E; Gaynor, J J; Broglie, R M

    1986-01-01

    A full-length copy of bean leaf chitinase mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized. Images PMID:2428042

  8. Genome-wide comparative analysis of NBS-encoding genes in four Gossypium species.

    PubMed

    Xiang, Liuxin; Liu, Jinggao; Wu, Chaofeng; Deng, Yushan; Cai, Chaowei; Zhang, Xiao; Cai, Yingfan

    2017-04-12

    Nucleotide binding site (NBS) genes encode a large family of disease resistance (R) proteins in plants. The availability of genomic data of the two diploid cotton species, Gossypium arboreum and Gossypium raimondii, and the two allotetraploid cotton species, Gossypium hirsutum (TM-1) and Gossypium barbadense allow for a more comprehensive and systematic comparative study of NBS-encoding genes to elucidate the mechanisms of cotton disease resistance. Based on the genome assembly data, 246, 365, 588 and 682 NBS-encoding genes were identified in G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. The distribution of NBS-encoding genes among the chromosomes was nonrandom and uneven, and was tended to form clusters. Gene structure analysis showed that G. arboreum and G. hirsutum possessed a greater proportion of CN, CNL, and N genes and a lower proportion of NL, TN and TNL genes compared to that of G. raimondii and G. barbadense, while the percentages of RN and RNL genes remained relatively unchanged. The percentage changes among them were largest for TNL genes, about 7 times. Exon statistics showed that the average exon numbers per NBS gene in G. raimondii and G. barbadense were all greater than that in G. arboretum and G. hirsutum. Phylogenetic analysis revealed that the TIR-NBS genes of G. barbadense were closely related with that of G. raimondii. Sequence similarity analysis showed that diploid cotton G. arboreum possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. hirsutum, while diploid G. raimondii possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. barbadense. The synteny analysis showed that more NBS genes in G. raimondii and G. arboreum were syntenic with that in G. barbadense and G. hirsutum, respectively. The structural architectures, amino acid sequence similarities and synteny of NBS-encoding genes between G. arboreum and G. hirsutum, and between G

  9. NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization.

    PubMed

    Portereiko, Michael F; Sandaklie-Nikolova, Linda; Lloyd, Alan; Dever, Chad A; Otsuga, Denichiro; Drews, Gary N

    2006-07-01

    Karyogamy, or nuclear fusion, is essential for sexual reproduction. In angiosperms, karyogamy occurs three times: twice during double fertilization of the egg cell and the central cell and once during female gametophyte development when the two polar nuclei fuse to form the diploid central cell nucleus. The molecular mechanisms controlling karyogamy are poorly understood. We have identified nine female gametophyte mutants in Arabidopsis (Arabidopsis thaliana), nuclear fusion defective1 (nfd1) to nfd9, that are defective in fusion of the polar nuclei. In the nfd1 to nfd6 mutants, failure of fusion of the polar nuclei is the only defect detected during megagametogenesis. nfd1 is also affected in karyogamy during double fertilization. Using transmission electron microscopy, we showed that nfd1 nuclei fail to undergo fusion of the outer nuclear membranes. nfd1 contains a T-DNA insertion in RPL21M that is predicted to encode the mitochondrial 50S ribosomal subunit L21, and a wild-type copy of this gene rescues the mutant phenotype. Consistent with the predicted function of this gene, an NFD1-green fluorescent protein fusion protein localizes to mitochondria and the NFD1/RPL21M gene is expressed throughout the plant. The nfd3, nfd4, nfd5, and nfd6 mutants also contain T-DNA insertions in genes predicted to encode proteins that localize to mitochondria, suggesting a role for this organelle in nuclear fusion.

  10. Identification and characterization of a protein kinase gene in the Lymantria dispar nultinucleocapsid nuclear polyhedrosis virus

    Treesearch

    David S. Bischoff; James M. Slavicek

    1994-01-01

    The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding vPK has been cloned and sequenced. LdMNPV vPK shows a 24% amino acid identity to the catalytic domains of the eucaryotic protein kinases nPKC from rabbits, HSPKCE from humans, APLPKCB from Aplysia californica, and dPKC98F from ...

  11. Interference with nuclear factor kappaB signaling pathway by pathogen-encoded proteases: global and selective inhibition.

    PubMed

    Hodgson, Andrea; Wan, Fengyi

    2016-02-01

    Pathogens have evolved a myriad of ways to abrogate and manipulate the host response to infections. Of the various mechanisms involved, pathogen-encoded and sometimes host-encoded proteases are an important category of virulence factors that cause robust changes on the host response by targeting key proteins along signaling cascades. The nuclear factor kappaB (NF-κB) signaling pathway is a crucial regulatory mechanism for the cell, controlling the expression of survival, immune and proliferation genes. Proteases from pathogens of almost all types have been demonstrated to target and cleave members of the NF-κB signaling pathway at nearly every level. This review provides discussion of proteases targeting the most abundant NF-κB subunit, p65, and the impact of protease-mediated p65 cleavage on the immune responses and survival of the infected host cell. After examining various examples of protease interference, it becomes evident that the cleavage fragments produced by pathogen-driven proteolytic processing should be further characterized to determine whether they have novel and unique functions within the cell. The selective targeting of p65 and its effect on gene transcription reveals unique mechanisms by which pathogens acutely alter their microenvironment, and further research may open new opportunities for novel therapeutics to combat pathogens.

  12. Species-specific duplications of NBS-encoding genes in Chinese chestnut (Castanea mollissima)

    PubMed Central

    Zhong, Yan; Li, Yingjun; Huang, Kaihui; Cheng, Zong-Ming

    2015-01-01

    The disease resistance (R) genes play an important role in protecting plants from infection by diverse pathogens in the environment. The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of genes is one of the largest R gene families. Chinese chestnut (Castanea mollissima) is resistant to Chestnut Blight Disease, but relatively little is known about the resistance mechanism. We identified 519 NBS-encoding genes, including 374 NBS-LRR genes and 145 NBS-only genes. The majority of Ka/Ks were less than 1, suggesting the purifying selection operated during the evolutionary history of NBS-encoding genes. A minority (4/34) of Ka/Ks in non-TIR gene families were greater than 1, showing that some genes were under positive selection pressure. Furthermore, Ks peaked at a range of 0.4 to 0.5, indicating that ancient duplications arose during the evolution. The relationship between Ka/Ks and Ks indicated greater selective pressure on the newer and older genes with the critical value of Ks = 0.4–0.5. Notably, species-specific duplications were detected in NBS-encoding genes. In addition, the group of RPW8-NBS-encoding genes clustered together as an independent clade located at a relatively basal position in the phylogenetic tree. Many cis-acting elements related to plant defense responses were detected in promoters of NBS-encoding genes. PMID:26559332

  13. The Role of Nuclear Bodies in Gene Expression and Disease

    PubMed Central

    Morimoto, Marie; Boerkoel, Cornelius F.

    2013-01-01

    This review summarizes the current understanding of the role of nuclear bodies in regulating gene expression. The compartmentalization of cellular processes, such as ribosome biogenesis, RNA processing, cellular response to stress, transcription, modification and assembly of spliceosomal snRNPs, histone gene synthesis and nuclear RNA retention, has significant implications for gene regulation. These functional nuclear domains include the nucleolus, nuclear speckle, nuclear stress body, transcription factory, Cajal body, Gemini of Cajal body, histone locus body and paraspeckle. We herein review the roles of nuclear bodies in regulating gene expression and their relation to human health and disease. PMID:24040563

  14. Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function.

    PubMed

    Yumura, Mio; Yamamoto, Natsuko; Yokoyama, Katsushi; Mori, Hirotada; Yomo, Tetsuya; Ichihashi, Norikazu

    2017-01-01

    Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function.

  15. Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function

    PubMed Central

    Yumura, Mio; Yamamoto, Natsuko; Yokoyama, Katsushi; Mori, Hirotada; Yomo, Tetsuya

    2017-01-01

    Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function. PMID:28328998

  16. Isolation of a gene encoding a putative polyamine transporter from Candida albicans, GPT1.

    PubMed

    McNemar, M D; Gorman, J A; Buckley, H R

    2001-04-01

    A gene encoding a transport protein from the pathogenic yeast, Candida albicans, has been isolated during a complementation experiment utilizing an ornithine decarboxylase-negative (spe1 Delta) strain of Saccharomyces cerevisiae. This gene restores gamma-aminobutyric acid (GABA) transport to a GABA transport-negative mutant of S. cerevisiae and encodes a protein which putatively allows transport of one or more of the polyamines. We have assigned the name GPT1 (GABA/polyamine transporter) to this gene.

  17. TCA1, a single nuclear-encoded translational activator specific for petA mRNA in Chlamydomonas reinhardtii chloroplast.

    PubMed Central

    Wostrikoff, K; Choquet, Y; Wollman, F A; Girard-Bascou, J

    2001-01-01

    We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5' untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b(6)f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation--known as the CES process--in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process. PMID:11560891

  18. TIME FOR COFFEE Encodes a Nuclear Regulator in the Arabidopsis thaliana Circadian Clock[W

    PubMed Central

    Ding, Zhaojun; Millar, Andrew J.; Davis, Amanda M.; Davis, Seth J.

    2007-01-01

    The plant circadian clock is required for daily anticipation of the diurnal environment. Mutation in Arabidopsis thaliana TIME FOR COFFEE (TIC) affects free-running circadian rhythms. To investigate how TIC functions within the circadian system, we introduced markers for the evening and morning phases of the clock into tic and measured evident rhythms. The phases of evening clock genes in tic were all advanced under light/dark cycles without major expression level defects. With regard to morning-acting genes, we unexpectedly found that TIC has a closer relationship with LATE ELONGATED HYPOCOTYL (LHY) than with CIRCADIAN CLOCK ASSOCIATED1, as tic has a specific LHY expression level defect. Epistasis analysis demonstrated that there were no clear rhythms in double mutants of tic and evening-acting clock genes, although double mutants of tic and morning-acting genes exhibited a similar free-running period as tic. We isolated TIC and found that its mRNA expression is continuously present over the diurnal cycle, and the encoded protein appears to be strictly localized to the nucleus. Neither its abundance nor its cellular distribution was found to be clock regulated. We suggest that TIC encodes a nucleus-acting clock regulator working close to the central oscillator. PMID:17496120

  19. The naked endosperm Genes Encode Duplicate INDETERMINATE Domain Transcription Factors Required for Maize Endosperm Cell Patterning and Differentiation1[OPEN

    PubMed Central

    Yi, Gibum; Neelakandan, Anjanasree K.; Gontarek, Bryan C.; Vollbrecht, Erik; Becraft, Philip W.

    2015-01-01

    The aleurone is the outermost layer of cereal endosperm and functions to digest storage products accumulated in starchy endosperm cells as well as to confer important dietary health benefits. Whereas normal maize (Zea mays [Zm]) has a single aleurone layer, naked endosperm (nkd) mutants produce multiple outer cell layers of partially differentiated cells that show sporadic expression of aleurone identity markers such as a viviparous1 promoter-β-glucuronidase transgene. The 15:1 F2 segregation ratio suggested that two recessive genes were involved, and map-based cloning identified two homologous genes in duplicated regions of the genome. The nkd1 and nkd2 genes encode the INDETERMINATE1 domain (IDD) containing transcription factors ZmIDDveg9 and ZmIDD9 on chromosomes 2 and 10, respectively. Independent mutant alleles of nkd1 and nkd2, as well as nkd2-RNA interference lines in which both nkd genes were knocked down, also showed the nkd mutant phenotype, confirming the gene identities. In wild-type kernels, the nkd transcripts were most abundant around 11 to 16 d after pollination. The NKD proteins have putative nuclear localization signals, and green fluorescent protein fusion proteins showed nuclear localization. The mutant phenotype and gene identities suggest that NKD controls a gene regulatory network involved in aleurone cell fate specification and cell differentiation. PMID:25552497

  20. Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

    PubMed

    Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

    2013-02-01

    Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

  1. V-myc- and c-myc-encoded proteins are associated with the nuclear matrix.

    PubMed Central

    Eisenman, R N; Tachibana, C Y; Abrams, H D; Hann, S R

    1985-01-01

    A series of extraction procedures were applied to avian nuclei which allowed us to define three types of association of v-myc- and c-myc-encoded proteins with nuclei: (i) a major fraction (60 to 90%) which is retained in DNA- and RNA-depleted nuclei after low- and high-salt extraction, (ii) a small fraction (1%) released during nuclease digestion of DNA in intact nuclei in the presence of low-salt buffer, and (iii) a fraction of myc protein (less than 10%) extractable with salt or detergents and found to have affinity for both single- and double-stranded DNA. Immunofluorescence analysis with anti-myc peptide sera on cells extracted sequentially with nucleases and salts confirmed the idea that myc proteins were associated with a complex residual nuclear structure (matrix-lamin fraction) which also contained avian nuclear lamin protein. Dispersal of myc proteins into the cytoplasm was found to occur during mitosis. Both c-myc and v-myc proteins were associated with the matrix-lamin, suggesting that the function of myc may relate to nuclear structural organization. Images PMID:3872410

  2. Mutation in the novel nuclear-encoded mitochondrial protein CHCHD10 in a family with autosomal dominant mitochondrial myopathy.

    PubMed

    Ajroud-Driss, Senda; Fecto, Faisal; Ajroud, Kaouther; Lalani, Irfan; Calvo, Sarah E; Mootha, Vamsi K; Deng, Han-Xiang; Siddique, Nailah; Tahmoush, Albert J; Heiman-Patterson, Terry D; Siddique, Teepu

    2015-01-01

    Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5% of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

  3. SURVEY AND SUMMARY: A survey of small RNA-encoding genes in Escherichia coli

    PubMed Central

    Hershberg, Ruth; Altuvia, Shoshy; Margalit, Hanah

    2003-01-01

    Small RNA (sRNA) molecules have gained much interest lately, as recent genome-wide studies have shown that they are widespread in a variety of organisms. The relatively small family of 10 known sRNA-encoding genes in Escherichia coli has been significantly expanded during the past two years with the discovery of 45 novel genes. Most of these genes are still uncharacterized and their cellular roles are unknown. In this survey we examined the sequence and genomic features of the 55 currently known sRNA-encoding genes in E.coli, attempting to identify their common characteristics. Such characterization is important for both expanding our understanding of this unique gene family and for improving the methods to predict and identify sRNA-encoding genes based on genomic information. PMID:12654996

  4. Computational Characterization of Modes of Transcriptional Regulation of Nuclear Receptor Genes

    PubMed Central

    Sharma, Yogita; Chilamakuri, Chandra Sekhar Reddy; Bakke, Marit; Lenhard, Boris

    2014-01-01

    Background Nuclear receptors are a large structural class of transcription factors that act with their co-regulators and repressors to maintain a variety of biological and physiological processes such as metabolism, development and reproduction. They are activated through the binding of small ligands, which can be replaced by drug molecules, making nuclear receptors promising drug targets. Transcriptional regulation of the genes that encode them is central to gaining a deeper understanding of the diversity of their biochemical and biophysical roles and their role in disease and therapy. Even though they share evolutionary history, nuclear receptor genes have fundamentally different expression patterns, ranging from ubiquitously expressed to tissue-specific and spatiotemporally complex. However, current understanding of regulation in nuclear receptor gene family is still nascent. Methodology/Principal Findings In this study, we investigate the relationship between long-range regulation of nuclear receptor family and their known functionality. Towards this goal, we identify the nuclear receptor genes that are potential targets based on counts of highly conserved non-coding elements. We validate our results using publicly available expression (RNA-seq) and histone modification (ChIP-seq) data from the ENCODE project. We find that nuclear receptor genes involved in developmental roles show strong evidence of long-range mechanism of transcription regulation with distinct cis-regulatory content they feature clusters of highly conserved non-coding elements distributed in regions spanning several Megabases, long and multiple CpG islands, bivalent promoter marks and statistically significant higher enrichment of enhancer mark around their gene loci. On the other hand nuclear receptor genes that are involved in tissue-specific roles lack these features, having simple transcriptional controls and a greater variety of mechanisms for producing paralogs. We further examine the

  5. Computational characterization of modes of transcriptional regulation of nuclear receptor genes.

    PubMed

    Sharma, Yogita; Chilamakuri, Chandra Sekhar Reddy; Bakke, Marit; Lenhard, Boris

    2014-01-01

    Nuclear receptors are a large structural class of transcription factors that act with their co-regulators and repressors to maintain a variety of biological and physiological processes such as metabolism, development and reproduction. They are activated through the binding of small ligands, which can be replaced by drug molecules, making nuclear receptors promising drug targets. Transcriptional regulation of the genes that encode them is central to gaining a deeper understanding of the diversity of their biochemical and biophysical roles and their role in disease and therapy. Even though they share evolutionary history, nuclear receptor genes have fundamentally different expression patterns, ranging from ubiquitously expressed to tissue-specific and spatiotemporally complex. However, current understanding of regulation in nuclear receptor gene family is still nascent. In this study, we investigate the relationship between long-range regulation of nuclear receptor family and their known functionality. Towards this goal, we identify the nuclear receptor genes that are potential targets based on counts of highly conserved non-coding elements. We validate our results using publicly available expression (RNA-seq) and histone modification (ChIP-seq) data from the ENCODE project. We find that nuclear receptor genes involved in developmental roles show strong evidence of long-range mechanism of transcription regulation with distinct cis-regulatory content they feature clusters of highly conserved non-coding elements distributed in regions spanning several Megabases, long and multiple CpG islands, bivalent promoter marks and statistically significant higher enrichment of enhancer mark around their gene loci. On the other hand nuclear receptor genes that are involved in tissue-specific roles lack these features, having simple transcriptional controls and a greater variety of mechanisms for producing paralogs. We further examine the combinatorial patterns of histone maps

  6. Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

    PubMed Central

    Zusman, Tal; Speiser, Yariv

    2014-01-01

    Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes. PMID:25225276

  7. Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

    PubMed Central

    Suter, T M; Viswanathan, V K; Cianciotto, N P

    1997-01-01

    A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae. PMID:9174205

  8. Gene encoding human Ro-associated autoantigen Y5 RNA.

    PubMed Central

    Maraia, R; Sakulich, A L; Brinkmann, E; Green, E D

    1996-01-01

    Ro ribonucleoproteins are composed of Y RNAs and the Ro 60 kDa protein. While the Ro 60 kDa protein is implicated in an RNA discard pathway that recognizes 3'-extended 5S rRNAs, the function of Y RNAs remains unknown [O'Brien,C.A. and Wolin,S.L. (1995) Genes Dev. 8,2891-2903]. Y5 RNA occupies a large fraction of Ro 60 kDa protein in human Ro RNPs, contains an atypical 3'-extension not found on other Y RNAs, and constitutes an RNA antigen in certain autoimmune patients [Boulanger et al. (1995) Clin. Exp. Immunol. 99, 29-36]. An overabundance of Y RNA retroposed pseudogenes has previously complicated the isolation of mammalian Y RNA genes. The source gene for Y5 RNA was isolated from human DNA as well as from Galago senegalis DNA. Authenticity of the hY5 RNA gene was demonstrated in vivo and its activity was compared with the hY4 RNA gene that also uses a type 3 promoter for RNA polymerase III. The hY5 RNA gene was subsequently found to reside within a few hundred thousand base pairs of other Y RNA genes and the linear order of the four human Y RNA genes on chromosome 7q36 was determined. Phylogenetic comparative analyses of promoter and RNA structure indicate that the Y5 RNA gene has been subjected to positive selection during primate evolution. Consistent with the proposal of O'Brien and Harley [O'Brian,C.A. and Wolin,S.L. (1992) Gene 116, 285-289], analysis of flanking sequences suggest that the hY5 RNA gene may have originated as a retroposon. PMID:8836182

  9. Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase epsilon.

    PubMed Central

    Huang, D; Pospiech, H; Kesti, T; Syväoja, J E

    1999-01-01

    The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters. PMID:10215605

  10. Molecular and Genomic Analysis of Genes Encoding Surface-Anchored Proteins from Clostridium difficile

    PubMed Central

    Karjalainen, Tuomo; Waligora-Dupriet, Anne-Judith; Cerquetti, Marina; Spigaglia, Patrizia; Maggioni, Andrea; Mauri, Pierluigi; Mastrantonio, Paola

    2001-01-01

    The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins. PMID:11292772

  11. Molecular cloning and characterization of the gene encoding rat submandibular gland apomucin, Mucsmg.

    PubMed

    Albone, E F; Hagen, F K; Szpirer, C; Tabak, L A

    1996-10-01

    Mucin glycoproteins are a major constituent of salivary secretions and play a primary role in the protection of the oral cavity. Rat submandibular glands (RSMG) synthesize and secrete a low molecular weight (114 kDa) mucin glycoprotein. We have isolated, partially sequenced, and characterized the gene which encodes the RSMG apomucin. The gene is encoded by three exons of 106 nt, 69 nt, and 991 nt, separated by introns of 921 nt and 12.5 kb. CAAT and TATA elements are present, at -68 and -26, respectively, in the 5' flanking sequence of the RSMG apomucin gene. The tandem repeat domain present in exon III consists of ten tandem repeats of 39 nt encoding the consensus sequence PTTDSTTPAPTTK. Sequence comparison and organization of the nucleic acid sequence encoding the tandem repeats of two alleles for this gene suggests that the apomucin gene has undergone recombinational events during its evolution. No significant sequence similarity was found with other mucin genes, or with other known salivary gland-specific genes. The gene was localized to rat chromosome 14 using somatic cell hybrids that segregate rat chromosomes. Since this, to our knowledge, represents the first RSMG mucin gene cloned, we have designated this gene Mucsmg.

  12. Nuclear AXIN2 represses MYC gene expression

    SciTech Connect

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

  13. Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

    PubMed

    Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

    2016-12-01

    Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

  14. Human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment

    SciTech Connect

    Geraghty, D.E.; Koller, B.H.; Orr, H.T.

    1987-12-01

    The authors have cloned genomic DNA encoding a non-HLA-A, -B, -C class I gene located within a HindIII-generated restriction fragment of 6.0 kilobase pairs. This gene, designated HLA-6.0, is as homologous to HLA-A and HLA-B as they are to each other. The HLA class I protein encoded by HLA-6.0 is similar in organization to the HLA-A-, -B-, and -C-encoded proteins except that an in-frame termination codon prevents translation of a majority of the cytoplasmic region of the HLA-6.0 polypeptide. Moreover, the promoter region of HLA-6.0 resembles the promoter region of a Qa region gene. These structural features of HLA-6.0 suggest that this nonHLA-A, -B, -C gene is a structural homolog of a murine Qa region class I gene.

  15. Nuclear magnetic resonance diffusion pore imaging: Experimental phase detection by double diffusion encoding

    NASA Astrophysics Data System (ADS)

    Demberg, Kerstin; Laun, Frederik Bernd; Windschuh, Johannes; Umathum, Reiner; Bachert, Peter; Kuder, Tristan Anselm

    2017-02-01

    Diffusion pore imaging is an extension of diffusion-weighted nuclear magnetic resonance imaging enabling the direct measurement of the shape of arbitrarily formed, closed pores by probing diffusion restrictions using the motion of spin-bearing particles. Examples of such pores comprise cells in biological tissue or oil containing cavities in porous rocks. All pores contained in the measurement volume contribute to one reconstructed image, which reduces the problem of vanishing signal at increasing resolution present in conventional magnetic resonance imaging. It has been previously experimentally demonstrated that pore imaging using a combination of a long and a narrow magnetic field gradient pulse is feasible. In this work, an experimental verification is presented showing that pores can be imaged using short gradient pulses only. Experiments were carried out using hyperpolarized xenon gas in well-defined pores. The phase required for pore image reconstruction was retrieved from double diffusion encoded (DDE) measurements, while the magnitude could either be obtained from DDE signals or classical diffusion measurements with single encoding. The occurring image artifacts caused by restrictions of the gradient system, insufficient diffusion time, and by the phase reconstruction approach were investigated. Employing short gradient pulses only is advantageous compared to the initial long-narrow approach due to a more flexible sequence design when omitting the long gradient and due to faster convergence to the diffusion long-time limit, which may enable application to larger pores.

  16. Nuclear magnetic resonance diffusion pore imaging: Experimental phase detection by double diffusion encoding.

    PubMed

    Demberg, Kerstin; Laun, Frederik Bernd; Windschuh, Johannes; Umathum, Reiner; Bachert, Peter; Kuder, Tristan Anselm

    2017-02-01

    Diffusion pore imaging is an extension of diffusion-weighted nuclear magnetic resonance imaging enabling the direct measurement of the shape of arbitrarily formed, closed pores by probing diffusion restrictions using the motion of spin-bearing particles. Examples of such pores comprise cells in biological tissue or oil containing cavities in porous rocks. All pores contained in the measurement volume contribute to one reconstructed image, which reduces the problem of vanishing signal at increasing resolution present in conventional magnetic resonance imaging. It has been previously experimentally demonstrated that pore imaging using a combination of a long and a narrow magnetic field gradient pulse is feasible. In this work, an experimental verification is presented showing that pores can be imaged using short gradient pulses only. Experiments were carried out using hyperpolarized xenon gas in well-defined pores. The phase required for pore image reconstruction was retrieved from double diffusion encoded (DDE) measurements, while the magnitude could either be obtained from DDE signals or classical diffusion measurements with single encoding. The occurring image artifacts caused by restrictions of the gradient system, insufficient diffusion time, and by the phase reconstruction approach were investigated. Employing short gradient pulses only is advantageous compared to the initial long-narrow approach due to a more flexible sequence design when omitting the long gradient and due to faster convergence to the diffusion long-time limit, which may enable application to larger pores.

  17. A sex recognition glycoprotein is encoded by the plus mating-type gene fus1 of Chlamydomonas reinhardtii.

    PubMed Central

    Ferris, P J; Woessner, J P; Goodenough, U W

    1996-01-01

    Sexual fusion between plus and minus gametes of the unicellular green alga Chlamydomonas reinhardtii entails adhesion between plus-specific and minus-specific "fringe" proteins displayed on the plasma membrane of gametic mating structures. We report the identification of the gene (fus1) encoding the plus fringe glycoprotein, which resides in a unique domain of the mating-type plus (mt+) locus, and which was identified by transposon insertions in three fusion-defective mutant strains. Transformation with fus1+ restores fringe and fusion competence to these mutants and to the pseudo-plus mutant imp11 mt-, defective in minus differentiation. The fus1 gene is remarkable in lacking the codon bias found in all other nuclear genes of C. reinhardtii. Images PMID:8856667

  18. An intron-encoded protein assists RNA splicing of multiple similar introns of different bacterial genes.

    PubMed

    Meng, Qing; Wang, Yanfei; Liu, Xiang-Qin

    2005-10-21

    Four group II introns were found in an unusually intron-rich dnaN gene (encoding the beta subunit of DNA polymerase III) of the cyanobacterium Trichodesmium erythraeum, and they have strong similarities to two introns of the RIR gene (encoding ribonucleotide reductase) of the same organism. Of these six introns, only the RIR-3 intron encodes a maturase protein and showed efficient RNA splicing when expressed in Escherichia coli cells. The other five introns do not encode a maturase protein and did not show RNA splicing in E. coli. But these maturase-less introns showed efficient RNA splicing when the RIR-3 intron-encoded maturase protein was co-expressed from a freestanding gene in the same cell. These findings demonstrated that an intron-encoded protein could function as a general maturase for multiple introns of different genes. Major implications may include an intron-mediated co-regulation of the different genes and a resemblance of the evolutionary origin of spliceosomal introns.

  19. Concomitant loss of NDH complex-related genes within chloroplast and nuclear genomes in some orchids.

    PubMed

    Lin, Choun-Sea; Chen, Jeremy J W; Chiu, Chi-Chou; Hsiao, Han C W; Yang, Chen-Jui; Jin, Xiao-Hua; Leebens-Mack, James; dePamphilis, Claude W; Huang, Yao-Ting; Yang, Ling-Hung; Chang, Wan-Jung; Kui, Ling; Wong, Gane Ka-Shu; Hu, Jer-Ming; Wang, Wen; Shih, Ming-Che

    2017-03-04

    Chloroplast NAD(P)H dehydrogenase-like (NDH) complex consists of ~30 subunits from both the nuclear and chloroplast genomes and is ubiquitous across most land plants. In some orchids, such as Phalaenopsis equestris, Dendrobium officinale and D. catenatum, most of the 11 chloroplast genome encoded ndh genes (cp-ndh) have been lost. Here we investigated whether functional cp-ndh genes have been completely lost in these orchids or whether they have been transferred and retained in the nuclear genomes. Further, we assessed whether both cp-ndh genes and nucleus-encoded NDH-related genes can be lost resulting in the absence of the NDH complex. Comparative analyses of the genome of Apostasia odorata, an orchid species with a complete complement of cp-ndh genes which represents the sister lineage to all other orchids, and three published orchid genome sequences for Phalaenopsis equestris, D. officinale and D. catenatum which are all missing cp-ndh genes, indicated that copies of cp-ndh genes are not present in any of these four nuclear genomes. This observation suggests that the NDH complex is not necessary for some plants. Comparative genomic/transcriptomic analyses of currently available plastid genome sequences and nuclear transcriptome data showed that 47 out of 660 photoautotrophic plants and all the heterotrophic plants are missing plastid encoded cp-ndh genes and exhibit no evidence for maintenance of a functional NDH complex. Our data indicate that the NDH complex can be lost in photoautotrophic plant species. Further, the loss of the NDH complex may increase the probability of transition from a photoautotrophic to a heterotrophic life history. This article is protected by copyright. All rights reserved.

  20. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the [beta]-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate [beta]-carboxy-cis,cis-muconate. [beta]-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for [beta]-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to [beta]-carboxy-cis,cis-muconate.

  1. Eukaryotic Peptide Deformylases. Nuclear-Encoded and Chloroplast-Targeted Enzymes in Arabidopsis1

    PubMed Central

    Dirk, Lynnette M.A.; Williams, Mark A.; Houtz, Robert L.

    2001-01-01

    Arabidopsis (ecotype Columbia-0) genes, AtDEF1and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids. PMID:11553738

  2. A highly divergent gene cluster in honey bees encodes a novel silk family.

    PubMed

    Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

    2006-11-01

    The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

  3. Characterization of a novel gene encoding ankyrin repeat domain from Cotesia vestalis polydnavirus (CvBV)

    SciTech Connect

    Shi Min; Chen Yafeng; Huang Fang; Liu Pengcheng; Zhou Xueping; Chen Xuexin

    2008-06-05

    Cotesia vestalis (Haliday) is an endoparasitoid of Plutella xylostella (L.) larvae and injects a polydnavirus (CvBV) into its host during oviposition. In this report we describe the characterization of a gene (CvBV805) and its products. CvBV805 is located on the segment S8 of CvBV genome; it has a size of 909 bp and encodes a predicted protein of 125 amino acids. This protein contains an ankyrin repeat domain with a high degree of similarity with I{kappa}B-like genes. Gene transcripts were detected in extracts of the host as early as 2 h post-parasitization (p.p.) and continued to be detected through 24 h. Tissue-specific expression patterns showed that CvBV805 might be involved in early host immunosuppression. CvBV805 was detected in parasitized hosts at 12 h p.p. and in rBac-eGFP-CvBV805-infected Tn-5B1-4 cells at 72 h.p.i. by using western blots analysis. The size of the protein expressed in the host hemocytes and infected Tn-5B1-4 cells was 17 kDa and 56 kDa (including eGFP), respectively, which nearly corresponded with the predicted molecular weight (14.31 kDa) of CvBV805, suggesting that the protein did not undergo extensive post-translational modification. The protein was confirmed to be present within the nuclear region in hemocytes of the parasitized P. xylostella larvae at 48 h p.p. using confocal laser scanning microscopy.

  4. Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins.

    PubMed

    Satou, Masakazu; Enoki, Harumi; Oikawa, Akira; Ohta, Daisaku; Saito, Kazunori; Hachiya, Takushi; Sakakibara, Hitoshi; Kusano, Miyako; Fukushima, Atsushi; Saito, Kazuki; Kobayashi, Masatomo; Nagata, Noriko; Myouga, Fumiyoshi; Shinozaki, Kazuo; Motohashi, Reiko

    2014-07-01

    We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

  5. Identification of three related human GRO genes encoding cytokine functions

    SciTech Connect

    Haskill, S.; Peace, A.; Morris, J.; Sporn, S.A. ); Anisowicz, A.; Lee, S.W.; Sager, R. ); Smith, T. ); Martin, G.; Ralph, P. )

    1990-10-01

    The product of the human GRO gene is a cytokine with inflammatory and growth-regulatory properties; GRO is also called MGSA for melanoma growth-stimulatory activity. The authors have identified two additional genes, GRO{beta} and GRO{gamma}, that share 90{percent} and 86{percent} identity at the deduced amino acid level with the original GRO{alpha} isolate. One amino acid substitution of proline in GRO{alpha} by leucine in GRO{beta} and GRO{gamma} leads to a large predicted change in protein conformation. Significant differences also exist in the 3' untranslated region, including different numbers of ATTTA repeats associated with mRNA instability. A 122-base-pair region in the 3' region is conserved among the three GRO genes, and a part of it is also conserved in the Chinese hamster genome, suggesting a role in regulation. DNA hybridization with oligonucleotide probes and partial sequence analysis of the genomic clones confirm that the three forms are derived from related but different genes. Only one chromosomal locus has been identified, at 4q21, by using a GRO{alpha} cDNA clone that hybridized to all three genes. Expression studies reveal tissue-specific regulation as well as regulation by specific inducing agents, including interleukin 1, tumor necrosis factor, phorbol 12-myristate 13-acetate, and lipopolysaccharide.

  6. Mitochondrial electron transport regulation of nuclear gene expression. Studies with the alternative oxidase gene of tobacco.

    PubMed Central

    Vanlerberghe, G C; McIntosh, L

    1994-01-01

    We have isolated a cDNA representing the tobacco (Nicotiana tabacum L. cv Bright Yellow) nuclear gene Aox1, which encodes the alternative oxidase of plant mitochondria. The clone contains the complete coding region (1059 base pairs) of a precursor protein of 353 amino acids with a calculated molecular mass of 39.8 kD. A putative transit peptide contains common signals believed to be important for import and processing of mitochondrially localized proteins. We have studied changes in Aox1 gene expression in tobacco in response to changes in cytochrome pathway activity. Inhibition of the cytochrome pathway by antimycin A resulted in a rapid and dramatic accumulation of Aox1 mRNA, whereas the level of mRNAs encoding two proteins of the cytochrome pathway did not change appreciably. This was accompanied by a dramatic increase in alternative pathway capacity and engagement in whole cells. Respiration under these conditions was unaffected by the uncoupler p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, levels of Aox1 mRNA returned to control levels, alternative pathway capacity and engagement declined, and respiration could once again be stimulated by FCCP. The results show that a mechanism involving changes in Aox1 gene expression exists whereby the capacity of the alternative pathway can be adjusted in response to changes in the activity of the cytochrome pathway. PMID:8058837

  7. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

    PubMed Central

    Jones, Jonathan D. G.; Grady, Karen L.; Suslow, Trevor V.; Bedbrook, John R.

    1986-01-01

    Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen. ImagesFig. 2. PMID:16453672

  8. The transferome of metabolic genes explored: analysis of the horizontal transfer of enzyme encoding genes in unicellular eukaryotes.

    PubMed

    Whitaker, John W; McConkey, Glenn A; Westhead, David R

    2009-01-01

    Metabolic networks are responsible for many essential cellular processes, and exhibit a high level of evolutionary conservation from bacteria to eukaryotes. If genes encoding metabolic enzymes are horizontally transferred and are advantageous, they are likely to become fixed. Horizontal gene transfer (HGT) has played a key role in prokaryotic evolution and its importance in eukaryotes is increasingly evident. High levels of endosymbiotic gene transfer (EGT) accompanied the establishment of plastids and mitochondria, and more recent events have allowed further acquisition of bacterial genes. Here, we present the first comprehensive multi-species analysis of E/HGT of genes encoding metabolic enzymes from bacteria to unicellular eukaryotes. The phylogenetic trees of 2,257 metabolic enzymes were used to make E/HGT assertions in ten groups of unicellular eukaryotes, revealing the sources and metabolic processes of the transferred genes. Analyses revealed a preference for enzymes encoded by genes gained through horizontal and endosymbiotic transfers to be connected in the metabolic network. Enrichment in particular functional classes was particularly revealing: alongside plastid related processes and carbohydrate metabolism, this highlighted a number of pathways in eukaryotic parasites that are rich in enzymes encoded by transferred genes, and potentially key to pathogenicity. The plant parasites Phytophthora were discovered to have a potential pathway for lipopolysaccharide biosynthesis of E/HGT origin not seen before in eukaryotes outside the Plantae. The number of enzymes encoded by genes gained through E/HGT has been established, providing insight into functional gain during the evolution of unicellular eukaryotes. In eukaryotic parasites, genes encoding enzymes that have been gained through horizontal transfer may be attractive drug targets if they are part of processes not present in the host, or are significantly diverged from equivalent host enzymes.

  9. NBS-LRR-Encoding genes in sorghum and their role in plant defense

    USDA-ARS?s Scientific Manuscript database

    Nucleotide-binding site leucine-rich repeats (NBS-LRR) proteins are encoded by a large class of plant genes and many of them play an important role in plant defense against pest attack. Identification and characterization of the whole set of NBS-LRR genes in a plant genome will provide insights int...

  10. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    PubMed

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

  11. Escherichia coli yjjPB genes encode a succinate transporter important for succinate production.

    PubMed

    Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Yamakami, Suguru; Yahagi, Daiki; Chinen, Akito; Tokura, Mitsunori; Abe, Keietsu

    2017-09-01

    Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.

  12. Nuclear Compartmentalization Contributes to Stage-Specific Gene Expression Control in Trypanosoma cruzi

    PubMed Central

    Pastro, Lucía; Smircich, Pablo; Di Paolo, Andrés; Becco, Lorena; Duhagon, María A.; Sotelo-Silveira, José; Garat, Beatriz

    2017-01-01

    In the protozoan parasite Trypanosoma cruzi, as in other trypanosomatids, transcription of protein coding genes occurs in a constitutive fashion, producing large polycistronic transcription units. These units are composed of non-functionally related genes which are pervasively processed to yield each mRNA. Therefore, post-transcriptional processes are crucial to regulate gene expression. Considering that nuclear compartmentalization could contribute to gene expression regulation, we comparatively studied the nuclear, cytoplasmic and whole cell transcriptomes of the non-infective epimastigote stage of T. cruzi, using RNA-Seq. We found that the cytoplasmic transcriptome tightly correlates with the whole cell transcriptome and both equally correlate with the proteome. Nonetheless, 1,200 transcripts showed differential abundance between the nuclear and cytoplasmic fractions. For the genes with transcript content augmented in the nucleus, significant structural and compositional differences were found. The analysis of the reported epimastigote translatome and proteome, revealed scarce ribosome footprints and encoded proteins for them. Ontology analyses unveiled that many of these genes are distinctive of other parasite life-cycle stages. Finally, the relocalization of transcript abundance in the metacyclic trypomastigote infective stage was confirmed for specific genes. While gene expression is strongly dependent on transcript steady-state level, we here highlight the importance of the distribution of transcripts abundance between compartments in T. cruzi. Particularly, we show that nuclear compartmentation is playing an active role in the developmental stage determination preventing off-stage expression. PMID:28243589

  13. Nuclear Compartmentalization Contributes to Stage-Specific Gene Expression Control in Trypanosoma cruzi.

    PubMed

    Pastro, Lucía; Smircich, Pablo; Di Paolo, Andrés; Becco, Lorena; Duhagon, María A; Sotelo-Silveira, José; Garat, Beatriz

    2017-01-01

    In the protozoan parasite Trypanosoma cruzi, as in other trypanosomatids, transcription of protein coding genes occurs in a constitutive fashion, producing large polycistronic transcription units. These units are composed of non-functionally related genes which are pervasively processed to yield each mRNA. Therefore, post-transcriptional processes are crucial to regulate gene expression. Considering that nuclear compartmentalization could contribute to gene expression regulation, we comparatively studied the nuclear, cytoplasmic and whole cell transcriptomes of the non-infective epimastigote stage of T. cruzi, using RNA-Seq. We found that the cytoplasmic transcriptome tightly correlates with the whole cell transcriptome and both equally correlate with the proteome. Nonetheless, 1,200 transcripts showed differential abundance between the nuclear and cytoplasmic fractions. For the genes with transcript content augmented in the nucleus, significant structural and compositional differences were found. The analysis of the reported epimastigote translatome and proteome, revealed scarce ribosome footprints and encoded proteins for them. Ontology analyses unveiled that many of these genes are distinctive of other parasite life-cycle stages. Finally, the relocalization of transcript abundance in the metacyclic trypomastigote infective stage was confirmed for specific genes. While gene expression is strongly dependent on transcript steady-state level, we here highlight the importance of the distribution of transcripts abundance between compartments in T. cruzi. Particularly, we show that nuclear compartmentation is playing an active role in the developmental stage determination preventing off-stage expression.

  14. Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum.

    PubMed

    Warnecke, D; Erdmann, R; Fahl, A; Hube, B; Müller, F; Zank, T; Zähringer, U; Heinz, E

    1999-05-07

    Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.

  15. Cloning of a Serratia marcescens Gene Encoding Chitinase

    PubMed Central

    Fuchs, R. L.; McPherson, S. A.; Drahos, D. J.

    1986-01-01

    Serratia marcescens, a chitinase-producing microorganism, was shown to produce five unique chitinolytic proteins with subunit molecular masses of 21, 36, 48, 52, and 57 kilodaltons. A cosmid library of S. marcescens DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in Escherichia coli for clones capable of degrading chitin. A total of four independent clones (22- to 27-kilobase inserts) were isolated, characterized by restriction endonuclease digestion, and shown to share a common 9.5-kilobase EcoR1 fragment apparently encoding the same 57-kilodalton chitinase, the most abundant chitinase produced by S. marcescens. Chitinase expression from these constructs in both E. coli and Pseudomonas fluorescens 701E1 is apparently driven by an S. marcescens promoter. The significantly higher chitinase levels produced in E. coli relative to those in P. fluorescens 701E1 suggest that E. coli may recognize this promoter sequence more efficiently than P. fluorescens. Images PMID:16347012

  16. The gene encoding topoisomerase II from Plasmodium falciparum.

    PubMed

    Cheesman, S; McAleese, S; Goman, M; Johnson, D; Horrocks, P; Ridley, R G; Kilbey, B J

    1994-07-11

    The gene for topoisomerase II has been isolated from genomic libraries of strain K1 of the human malarial parasite, Plasmodium falciparum. The sequence reveals an open reading frame of 4194 nucleotides which predicts a polypeptide of 1398 amino acids. There are apparently no introns. The sequence is present as a single copy which has an identity of 47.4% and a similarity of 65.4% with its human homologue. Sequences conserved in topoisomerase II from other species are present in Pftopoisomerase II but in addition it has two adjacent asparagine-rich insertions which are unique to it. We have also detected asparagine-rich regions in the gene for PfDNA polymerase alpha. The gene for Pftopoisomerase II has been localised to chromosome 14 and northern analysis reveals a transcript of 5.8 kb. Two independent antisera raised in mice against glutathione-S-transferase fusion proteins containing the amino terminal portion of the malarial protein detect a weak band on western blots at about 160kDa, the expected size of the protein. Use of the same antisera for immunofluorescence analysis suggests that the protein is present at all stages of intraerythrocytic growth of the parasite.

  17. Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells.

    PubMed

    Glinka, Elena M

    2013-12-01

    Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Expression of hcp in freshwater Synechococcus spp., a gene encoding a hyperconserved protein in picocyanobacteria.

    PubMed

    Kutovaya, Olga A; McKay, R Michael L; Bullerjahn, George S

    2010-06-01

    Marine picoplankton of the genus Synechococcus and Prochlorococcus spp. are widely studied members of the picocyanobacterial clade, composed of unicellular cyanobacteria that dominate pelagic regions of the ocean. Less studied are the related freshwater Synechococcus spp. that similarly dominate the euphotic zone of oligotrophic lakes. Previous work has shown that marine picocyanobacteria harbor a small gene, hcp, that encodes a 62 amino acid protein 100% conserved among all strains examined. The gene is restricted exclusively to the picocyanobacterial lineage. The current study reveals that hcp is also 100% conserved in four freshwater Synechococcus spp. strains isolated from the Laurentian Great Lakes, and that the gene constitutively expressed with genes encoding a ribosomal protein and two tRNA genes. The synteny of the hcp region is also conserved between the marine and freshwater strains. Last, the hcp gene and the organization of the surrounding genetic region has been retained in the reduced genome of a picocyanobacterial endosymbiont of the amoeba Paulinella sp.

  19. The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis.

    PubMed

    Houtmeyers, Rob; Souopgui, Jacob; Tejpar, Sabine; Arkell, Ruth

    2013-10-01

    The zinc finger of the cerebellum gene (ZIC) discovered in Drosophila melanogaster (odd-paired) has five homologs in Xenopus, chicken, mice, and humans, and seven in zebrafish. This pattern of gene copy expansion is accompanied by a divergence in gene and protein structure, suggesting that Zic family members share some, but not all, functions. ZIC genes are implicated in neuroectodermal development and neural crest cell induction. All share conserved regions encoding zinc finger domains, however their heterogeneity and specification remain unexplained. In this review, the evolution, structure, and expression patterns of the ZIC homologs are described; specific functions attributable to individual family members are supported. A review of data from functional studies in Xenopus and murine models suggest that ZIC genes encode multifunctional proteins operating in a context-specific manner to drive critical events during embryogenesis. The identification of ZIC mutations in congenital syndromes highlights the relevance of these genes in human development.

  20. A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina.

    PubMed

    Wang, H; Jones, R W

    1995-05-01

    The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.

  1. A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina.

    PubMed Central

    Wang, H; Jones, R W

    1995-01-01

    The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific. PMID:7646037

  2. Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

    2015-09-01

    Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

  3. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  4. Cloning and sequencing of the gene encoding cytochrome c sub 553 from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    van Rooijen, G.J.H.; Voordouw, G. ); Bruschi, M. )

    1989-06-01

    The gene encoding cytochrome c{sub 553} from Desulfovibrio vulgaris Hildenborough was cloned by using two synthetic deoxyoligonucleotide probes. The amino acid sequence derived from the sequence of the gene differs from that reported by Bruschi and LeGall. Renewed protein sequencing confirmed the correctness of the DNA-derived sequence. The gene sequence indicates cytochrome c{sub 553} to be synthesized as a precursor protein with an NH{sub 2}-terminal signal sequence of 24 residues.

  5. Characterization of transcript processing of the gene encoding precerebellin-1.

    PubMed

    Kavety, B; Morgan, J I

    1998-12-10

    Precerebellin-1 (Cbln1) is a cerebellum-specific protein that shares significant sequence identity with the globular domains of the complement components C1qA, B and C, suggesting some common aspects of function and/or structure. As the C1q complex is composed of heterotrimers of C1qA, B and C it was hypothesized that multiple precerebellins may exist in a ternary complex. Northern blotting for cbln1 revealed multiple bands that could represent further family members or alternatively spliced variants. To discriminate these alternatives, probes derived from different regions of the cbln1 gene were used to identify and clone the transcripts detected on Northern blots. Four independent transcripts were repeatedly cloned from an adult mouse cerebellum cDNA library. Upon sequencing, all of these clones were found to be derived from the cbln1 gene and no additional precerebellin-related genes were isolated. Moreover, these clones accounted for the four cbln1-hybridizing bands (1.9, 2. 2, 3.2 and 5.5 kb) detected on Northern blots of adult cerebellum RNA. With one possible exception, these clones were all derived through alterations in the 3'-untranslated region (3'-UTR) of cbln1 that did not affect the coding sequence. This was achieved by the use of two polyadenylation sites and alternative (non-canonical) splicing in the 3'-UTR. Some additional variation in mRNA structure is provided by the use of alternative transcription start sites in cbln1. The possible significance of this level of diversity in the 3'-UTR is discussed.

  6. The Drosophila Hrb87F gene encodes a new member of the A and B hnRNP protein group.

    PubMed Central

    Haynes, S R; Johnson, D; Raychaudhuri, G; Beyer, A L

    1991-01-01

    Nascent premessenger RNA transcripts are packaged into heterogeneous nuclear ribonucleoprotein (hnRNP) complexes containing specific nuclear proteins, the hnRNP proteins. The A and B group proteins constitute a major class of small basic proteins found in mammalian hnRNP complexes. We have previously characterized the Drosophila melanogaster Hrb98DE gene, which is alternatively spliced to encode four protein isoforms closely related to the A and B proteins. We report here that the Drosophila genome contains a family of genes related to the Hrb98DE gene. One member of the family, Hrb87F, is very homologous to Hrb98DE in both sequence and structure. The Hrb87F transcripts (1.7 and 2.2 kb) utilize two alternative polyadenylation sites, are abundant in ovaries and early embryos, and are present in lesser amounts throughout development. In one wildtype strain of Drosophila there is a naturally-occurring polymorphism in this gene due to the insertion of a 412 transposable element in the 3' untranslated region. The larger transcript is not produced in these files and thus is not required for viability. Sequence identities among the Drosophila Hrb proteins and the vertebrate A and B hnRNP proteins suggest that these proteins may form a distinct subfamily within the larger family of related RNA binding proteins. Images PMID:1849257

  7. A neurotransmitter transporter encoded by the Drosophila inebriated gene

    PubMed Central

    Soehnge, Holly; Huang, Xi; Becker, Marie; Whitley, Penn; Conover, Diana; Stern, Michael

    1996-01-01

    Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron. PMID:8917579

  8. Phylogeny of the bears (Ursidae) based on nuclear and mitochondrial genes.

    PubMed

    Yu, Li; Li, Qing-wei; Ryder, O A; Zhang, Ya-ping

    2004-08-01

    The taxomic classification and phylogenetic relationships within the bear family remain argumentative subjects in recent years. Prior investigation has been concentrated on the application of different mitochondrial (mt) sequence data, herein we employ two nuclear single-copy gene segments, the partial exon 1 from gene encoding interphotoreceptor retinoid binding protein (IRBP) and the complete intron 1 from transthyretin (TTR) gene, in conjunction with previously published mt data, to clarify these enigmatic problems. The combined analyses of nuclear IRBP and TTR datasets not only corroborated prior hypotheses, positioning the spectacled bear most basally and grouping the brown and polar bear together but also provided new insights into the bear phylogeny, suggesting the sister-taxa association of sloth bear and sun bear with strong support. Analyses based on combination of nuclear and mt genes differed from nuclear analysis in recognizing the sloth bears as the earliest diverging species among the subfamily ursine representatives while the exact placement of the sun bear did not resolved. Asiatic and American black bears clustered as sister group in all analyses with moderate levels of bootstrap support and high posterior probabilities. Comparisons between the nuclear and mtDNA findings suggested that our combined nuclear dataset have the resolving power comparable to mtDNA dataset for the phylogenetic interpretation of the bear family. As can be seen from present study, the unanimous phylogeny for this recently derived family was still not produced and additional independent genetic markers were in need.

  9. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    PubMed Central

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-01-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians. PMID:27311567

  10. Characterization of two genes, Impa1 and Impa2 encoding mouse myo-inositol monophosphatases.

    PubMed

    Shamir, A; Sjøholt, G; Ebstein, R P; Agam, G; Steen, V M

    2001-06-27

    The enzyme myo-inositol monophosphatase (Impa) catalyzes the synthesis of free myo-inositol from various myo-inositol monophosphates in the phosphatidylinositol signaling system. Impa is a lithium-blockable enzyme that has been hypothesized to be the biological target for lithium-salts used as mood-stabilizing drugs in the treatment of manic-depressive (bipolar) illness. As an initial step to explore the functional consequences of reduced or absent Impa activity in an animal model we here report the isolation of two Impa-encoding mouse genes, Impa1 and Impa2. Impa1 spans approximately 17.5 kb and contains nine exons of 46--1354 bp encoding a protein of 277 amino acids. Impa2 spans at least 19.5 kb and contains eight exons of 46--444 bp size encoding a protein of 290 amino acids. The genomic structure including the positions of the exon-intron splice sites seems to be conserved among myo-inositol monophosphatase genes in mammalian species. One or more Impa-like genes do also exist in evolutionary more distant species like invertebrates, plants and bacteria. The proteins encoded by the non-vertebrate genes seem to be equally related to Impa1 and Impa2. We therefore suggest that the Impa1 and Impa2 genes duplicated from a common ancestral gene after the evolutionary divergence of vertebrates.

  11. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    NASA Astrophysics Data System (ADS)

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-06-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

  12. Unusual conservation among genes encoding small secreted salivary gland proteins from a gall midge.

    PubMed

    Chen, Ming-Shun; Liu, Xuming; Yang, Ziheng; Zhao, Huixian; Shukle, Richard H; Stuart, Jeffrey J; Hulbert, Scot

    2010-09-28

    In most protein-coding genes, greater sequence variation is observed in noncoding regions (introns and untranslated regions) than in coding regions due to selective constraints. During characterization of genes and transcripts encoding small secreted salivary gland proteins (SSSGPs) from the Hessian fly, we found exactly the opposite pattern of conservation in several families of genes: the non-coding regions were highly conserved, but the coding regions were highly variable. Seven genes from the SSSGP-1 family are clustered as one inverted and six tandem repeats within a 15 kb region of the genome. Except for SSSGP-1A2, a gene that encodes a protein identical to that encoded by SSSGP-1A1, the other six genes consist of a highly diversified, mature protein-coding region as well as highly conserved regions including the promoter, 5'- and 3'-UTRs, a signal peptide coding region, and an intron. This unusual pattern of highly diversified coding regions coupled with highly conserved regions in the rest of the gene was also observed in several other groups of SSSGP-encoding genes or cDNAs. The unusual conservation pattern was also found in some of the SSSGP cDNAs from the Asian rice gall midge, but not from the orange wheat blossom midge. Strong positive selection was one of the forces driving for diversification whereas concerted homogenization was likely a mechanism for sequence conservation. Rapid diversification in mature SSSGPs suggests that the genes are under selection pressure for functional adaptation. The conservation in the noncoding regions of these genes including introns also suggested potential mechanisms for sequence homogenization that are not yet fully understood. This report should be useful for future studies on genetic mechanisms involved in evolution and functional adaptation of parasite genes.

  13. Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein.

    PubMed

    Lan, Ke; Verma, Subhash C; Murakami, Masanao; Bajaj, Bharat; Kaul, Rajeev; Robertson, Erle S

    2007-10-09

    Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    PubMed

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

    2002-03-22

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

  17. Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

    PubMed

    Peoples, R J; Cisco, M J; Kaplan, P; Francke, U

    1998-01-01

    We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

  18. A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.

    PubMed Central

    Reeve, J N; Beckler, G S; Cram, D S; Hamilton, P T; Brown, J W; Krzycki, J A; Kolodziej, A F; Alex, L; Orme-Johnson, W H; Walsh, C T

    1989-01-01

    The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters. PMID:2654933

  19. Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein

    PubMed Central

    Lagos, Rosalba; Villanueva, Jorge E.; Monasterio, Octavio

    1999-01-01

    The gene coding for the immunity protein (mceB) and the structural gene of microcin E492 (mceA), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (mceB) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed. PMID:9864332

  20. The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

    PubMed

    Segal-Kischinevzky, Claudia; Rodarte-Murguía, Beatriz; Valdés-López, Victor; Mendoza-Hernández, Guillermo; González, Alicia; Alba-Lois, Luisa

    2011-03-01

    Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

  1. Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

    PubMed Central

    Mizumoto, Shuji; Ikegawa, Shiro; Sugahara, Kazuyuki

    2013-01-01

    A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs. PMID:23457301

  2. Common origin of plasmid encoded alpha-hemolysin genes in Escherichia coli

    PubMed Central

    2010-01-01

    Background Alpha (α)-hemolysin is a pore forming cytolysin and serves as a virulence factor in intestinal and extraintestinal pathogenic strains of E. coli. It was suggested that the genes encoding α-hemolysin (hlyCABD) which can be found on the chromosome and plasmid, were acquired through horizontal gene transfer. Plasmid-encoded α-hly is associated with certain enterotoxigenic (ETEC), shigatoxigenic (STEC) and enteropathogenic E. coli (EPEC) strains. In uropathogenic E. coli (UPEC), the α-hly genes are located on chromosomal pathogenicity islands. Previous work suggested that plasmid and chromosomally encoded α-hly may have evolved independently. This was explored in our study. Results We have investigated 11 α-hly plasmids from animal and human ETEC, STEC and EPEC strains. The size of α-hly plasmids ranges from 48-157 kb and eight plasmids are conjugative. The regulatory gene (hlyR) located upstream of the hlyCABD gene operon and an IS911 element located downstream of hlyD are conserved. Chromosomally-encoded α-hly operons lack the hlyR and IS911 elements. The DNA sequence of hlyC and hlyA divided the plasmid- and chromosomally-encoded α-hemolysins into two clusters. The plasmid-encoded α-hly genes could be further divided into three groups based on the insertion of IS1 and IS2 in the regulatory region upstream of the α-hly operon. Transcription of the hlyA gene was higher than the housekeeping icdA gene in all strains (rq 4.8 to 143.2). Nucleotide sequence analysis of a chromosomally located α-hly determinant in Enterobacter cloacae strain indicates that it originates from an E. coli α-hly plasmid. Conclusion Our data indicate that plasmids encoding α-hly in E. coli descended from a common ancestor independent of the plasmid size and the origin of the strains. Conjugative plasmids could contribute to the spread of the α-hly determinant to Enterobacter cloacae. The presence of IS-elements flanking the plasmid-encoded α-hly indicate that they

  3. Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus

    PubMed Central

    Richards, Amber D.; Vargas-Muñiz, José M.; Renshaw, Hilary; Steinbach, William J.

    2015-01-01

    Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A

  4. Association of the gene encoding neurogranin with schizophrenia in males.

    PubMed

    Ruano, Dina; Aulchenko, Yurii S; Macedo, António; Soares, Maria J; Valente, José; Azevedo, Maria H; Hutz, Mara H; Gama, Clarissa S; Lobato, Maria I; Belmonte-de-Abreu, Paulo; Goodman, Ann B; Pato, Carlos; Heutink, Peter; Palha, Joana A

    2008-01-01

    The neurogranin (NRGN) gene produces a postsynaptic brain-specific protein that regulates calmodulin-Ca(2+) availability in neurons. Acting downstream of the NMDA receptor and upstream of calcineurin and other proteins implicated in schizophrenia, NRGN is a good candidate for association studies in schizophrenia. NRGN expression is regulated during development and is modulated by thyroid hormones and retinoids, molecules essential for the proper development of the central nervous system. Given the genetic complexity of schizophrenia and the potential genetic heterogeneity in different populations, we studied a possible association of NRGN with schizophrenia in 73 Azorean proband-parent triads and in two independent case-control samples from the Portuguese-mainland (244 schizophrenic and 210 controls) and Brazil (69 schizophrenic and 85 mentally healthy individuals). Genotype distribution showed association of the rs7113041 SNP with schizophrenia in males of Portuguese origin, which was confirmed by the analysis of the proband-parent triads. This evidence, implicating NRGN in schizophrenia, introduces another player into the glutamatergic hypothesis of schizophrenia.

  5. Cloning, characterization and subcellular localization of a gene encoding a human Ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product.

    PubMed

    Yin, Gang; Ji, Chaoneng; Wu, Tong; Shen, Zhouliang; Xu, Xin; Xie, Yi; Mao, Yumin

    2006-05-01

    Ubiquitin charging and activation of class III E2 enzymes has been directly linked to their nuclear import. It has not been published whether other classes E2s also abide by this mechanism. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone that is 2252 base pair in length, encoding a putative 162 amino acid protein, which shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16 (Accession number NP_565110, 51% identity and 71% similarity) at protein level. Bioinformatics analysis revealed that the gene is composed of 7 exons, located on human chromosome 8q13-8q21.1, and that the predicted protein of the gene is a class I E2, for only composed of a conserved approximately 150-amino acid catalytic core, ubiquitin-conjugating enzyme E2 domain (UBC domain). In the C-terminal of the UBC domain sequence, there are two nuclear localization signals (NLSs). RT-PCR showed that this gene is ubiquitously expressed in 16 kinds of normal human tissues, but expression level is very low, unless in human heart, brain, liver, and pancreas. The subcellular localizations of the new human Ubiquitin conjugating enzyme E2 and its mutation were also examined, which showed that the nuclear localization of hUBC16 depended on two conditions: It has NLS, and at the same time, has enzyme active site, too, at least in HEK293 cells.

  6. [Differential expression of genes that encode glycolysis enzymes in kidney and lung cancer in humans].

    PubMed

    Oparina, N Yu; Snezhkina, A V; Sadritdinova, A F; Veselovskii, V A; Dmitriev, A A; Senchenko, V N; Mel'nikova, N V; Speranskaya, A S; Darii, M V; Stepanov, O A; Barkhatov, I M; Kudryavtseva, A V

    2013-07-01

    Glycolysis is a main catabolic pathway of glucose metabolism, accompanied by ATP synthesis. More than 30 enzymes are involved in glycolysis, and genes that encode them can be considered housekeeping genes due to the high conservatism and evolutionary antiquity of the process. We studied the expression of these genes in kidney papillary cancer and planocellular lung cancer via the bioinformatic analysis of transcriptome database and method of quantitative real time PCR. Quantitative analysis of mRNA level demonstrated that only a part ofgenes that encode glycolysis enzymes maintain relatively stable mRNA level, including the HK1, ADPGK, GPI, PGK1, and PKM2 genes in kidney papillary cancer and the ADPGK, ALDOA, GAPDH, PGK1, BPGM, ENO1, and PKM2 genes in planocellular lung cancer. The frequent increase in the mRNA expression of PFKP, ALDOA, and GAPDH genes in kidney cancer, as well as the GPI gene in lung cancer, were detected for the first time by real time PCR. For other genes, their differential expression was demonstrated; the cases of both a decrease and increase in the mRNA level were detected. Thus, several genes that can be used as control genes in transcriptome analysis by real time PCR in kidney and lung cancer, as well as a number of differentially expressed genes that can be potential oncomarkers, were identified.

  7. Cloning and expression of genes encoding Haemophilus somnus antigens.

    PubMed Central

    Corbeil, L B; Chikami, G; Yarnall, M; Smith, J; Guiney, D G

    1988-01-01

    A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease. Images PMID:2843469

  8. Identification of the VH genes encoding xenoantibodies in non-immunosuppressed rhesus monkeys

    PubMed Central

    Kleihauer, Annette; Gregory, Clare R; Borie, Dominic C; Kyles, Andrew E; Shulkin, Irina; Patanwala, Insiyyah; Zahorsky-Reeves, Joanne; Starnes, Vaughn A; Mullen, Yoko; Todorov, Ivan T; Kearns-Jonker, Mary

    2005-01-01

    The major immunological barrier that prevents the use of wild-type pig xenografts as an alternative source of organs for human xenotransplantation is antibody-mediated rejection. In this study, we identify the immunoglobulin variable region heavy (IgVH) chain genes encoding xenoantibodies to porcine heart and fetal porcine islet xenografts in non-immunosuppressed rhesus monkeys. We sought to compare the IgVH genes encoding xenoantibodies to porcine islets and solid organ xenografts. The immunoglobulin M (IgM) and IgG xenoantibody response was analysed by enzyme-linked immunosorbent assay and cDNA libraries from peripheral blood lymphocytes were prepared and sequenced. The relative frequency of IgVH gene usage was established by colony filter hybridization. Induced xenoantibodies were encoded by the IGHV3-11 germline progenitor, the same germline gene that encodes xenoantibodies in humans mounting active xenoantibody responses. The immune response to pig xenografts presented as solid organs or isolated cells is mediated by identical IgVH genes in rhesus monkeys. These animals represent a clinically relevant model to identify the immunological basis of pig-to-human xenograft rejection. PMID:16108821

  9. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters

    PubMed Central

    Zeevi, Danny; Sharon, Eilon; Lotan-Pompan, Maya; Lubling, Yaniv; Shipony, Zohar; Raveh-Sadka, Tali; Keren, Leeat; Levo, Michal; Weinberger, Adina; Segal, Eran

    2011-01-01

    Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ∼10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded. PMID:22009988

  10. Nuclear and mitochondrial genes for inferring Trichuris phylogeny.

    PubMed

    Callejón, Rocío; Cutillas, Cristina; Nadler, Steven A

    2015-12-01

    Nucleotide sequences of the triose phosphate isomerase (TPI) gene (624 bp) and mitochondrial cytochrome b (cob) gene (520 bp) were obtained by PCR and evaluated for utility in inferring the phylogenetic relationships among Trichuris species. Published sequences of one other nuclear gene (18S or SSU rRNA, 1816-1846 bp) and one additional mitochondrial (mtDNA) gene (cytochrome oxidase 1, cox1, 342 bp) were also analyzed. Maximum likelihood and Bayesian inference methods were used to infer phylogenies for each gene separately but also for the combined mitochondrial data (two genes), the combined nuclear data (two genes), and the total evidence (four gene) dataset. Few Trichuris clades were uniformly resolved across separate analyses of individual genes. For the mtDNA, the cob gene trees had greater phylogenetic resolution and tended to have higher support values than the cox1 analyses. For nuclear genes, the SSU gene trees had slightly greater resolution and support values than the TPI analyses, but TPI was the only gene with reliable support for the deepest nodes in the tree. Combined analyses of genes yielded strongly supported clades in most cases, with the exception of the relationship among Trichuris clades 1, 2, and 3, which showed conflicting results between nuclear and mitochondrial genes. Both the TPI and cob genes proved valuable for inferring Trichuris relationships, with greatest resolution and support values achieved through combined analysis of multiple genes. Based on the phylogeny of the combined analysis of nuclear and mitochondrial genes, parsimony mapping of definitive host utilization depicts artiodactyls as the ancestral hosts for these Trichuris, with host-shifts into primates, rodents, and Carnivora.

  11. Characteristics analysis of the luzA gene encoding chaperone from Photobacterium leiognathi related to bioluminescence.

    PubMed

    Lin, J W; Lin, B J; Chen, H Y; Weng, S F

    1998-03-27

    Nucleotide sequence of the luzA gene (GenBank accession No. AF039303) from Photobacterium leiognathi ATCC 25521 (NCIMB 2193) has been determined, and the chaperone encoded by the luzA gene was deduced. The LuzA chaperone has a calculated M(r) 26,295 and comprises 230 amino acid residues; the hydrophobic alpha-helix N-terminal 21 amino acid residues MKKTIFALLFMSVFI SYPSFA is the leader peptide, therefore the matured LuzA chaperone has a calculated M(r) 23,871 and comprises 209 amino acid residues only. The periplasmic LuzA chaperone is the protein concerned with the protein folding, assembly and stability. The luzA gene and the related genes are closely linked to the sod gene, that encoding Cu/Zn superoxide dismutase enables to enhance bioluminescence of the lux operon; the gene order of the luzA gene and related genes is -ufo'-luzA-ufoI-ufoII-ter->-R&R'-sod-ufo-- >. In trans complementation bioluminoassays in vivo elicit that the LuzA chaperone might be not directly concerned with bioluminescence of the lux operon from P. leiognathi in E. coli, but might enable to stabilize the proteins related to bioluminescence. The unidentified ufoII gene closely linked to the luzA gene is able to enhance bioluminescence.

  12. Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

    PubMed Central

    Gregerson, R. G.; Cameron, L.; McLean, M.; Dennis, P.; Strommer, J.

    1993-01-01

    In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes. PMID:8096485

  13. Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

    PubMed

    Chen, John; Carpena, Nuria; Quiles-Puchalt, Nuria; Ram, Geeta; Novick, Richard P; Penadés, José R

    2015-05-01

    Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

  14. Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila.

    PubMed Central

    Singh-Wissmann, K; Ferry, J G

    1995-01-01

    Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M.T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript. PMID:7896690

  15. Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

    PubMed Central

    Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

    1994-01-01

    Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years. PMID:7937917

  16. Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

    PubMed

    Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

    1994-10-11

    Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years.

  17. Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei.

    PubMed

    Krügel, H; Fiedler, G; Smith, C; Baumberg, S

    1993-05-15

    We have determined the nucleotide (nt) sequence of nat1, a gene encoding nourseothricin (Nc) acetyltransferase (AT) from Streptomyces noursei, and its transcriptional start point (tsp). The nt sequence upstream from the coding region is completely different from that of the stat gene (encoding streptothricin AT) from Streptomyces lavendulae [S. Horinouchi, K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu, J. Bacteriol. 169 (1987) 1929-1937], even though the nt sequences of the two genes and the deduced amino acid (aa) sequences of the two enzymes show a high degree of similarity. Another stat gene, derived from a Gram-negative plasmid, showed only deduced aa similarity, but not nt sequence similarity, to the above two. A database search for related aa sequences did not reveal any clear-cut homologies to other types of protein. A multiple aa sequence alignment of several ATs is presented.

  18. A Gene Selection Method for Microarray Data Based on Binary PSO Encoding Gene-to-Class Sensitivity Information.

    PubMed

    Han, Fei; Yang, Chun; Wu, Ya-Qi; Zhu, Jian-Sheng; Ling, Qing-Hua; Song, Yu-Qing; Huang, De-Shuang

    2017-01-01

    Traditional gene selection methods for microarray data mainly considered the features' relevance by evaluating their utility for achieving accurate predication or exploiting data variance and distribution, and the selected genes were usually poorly explicable. To improve the interpretability of the selected genes as well as prediction accuracy, an improved gene selection method based on binary particle swarm optimization (BPSO) and prior information is proposed in this paper. In the proposed method, BPSO encoding gene-to-class sensitivity (GCS) information is used to perform gene selection. The gene-to-class sensitivity information, extracted from the samples by extreme learning machine (ELM), is encoded into the selection process in four aspects: initializing particles, updating the particles, modifying maximum velocity, and adopting mutation operation adaptively. Constrained by the gene-to-class sensitivity information, the new method can select functional gene subsets which are significantly sensitive to the samples' classes. With the few discriminative genes selected by the proposed method, ELM, K-nearest neighbor and support vector machine classifiers achieve much high prediction accuracy on five public microarray data, which in turn verifies the efficiency and effectiveness of the proposed gene selection method.

  19. Genes Encoding Cher-TPR Fusion Proteins Are Predominantly Found in Gene Clusters Encoding Chemosensory Pathways with Alternative Cellular Functions

    PubMed Central

    Rico-Jiménez, Miriam; Alfonso, Carlos; Krell, Tino

    2012-01-01

    Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be

  20. Genes encoding Cher-TPR fusion proteins are predominantly found in gene clusters encoding chemosensory pathways with alternative cellular functions.

    PubMed

    Muñoz-Martínez, Francisco; García-Fontana, Cristina; Rico-Jiménez, Miriam; Alfonso, Carlos; Krell, Tino

    2012-01-01

    Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be

  1. Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

  2. Constitutive expression of Epstein-Barr virus-encoded RNAs and nuclear antigen during latency and after induction of Epstein-Barr virus replication.

    PubMed Central

    Weigel, R; Fischer, D K; Heston, L; Miller, G

    1985-01-01

    We examined the fate of two major products of latency as Epstein-Barr virus was induced to replicate. We studied a superinducible clone of HR-1 cells in the presence and absence of induction by phorbol ester, and we analyzed the X50-7 line with and without superinfection by an HR-1 viral variant which disrupts latency. The two methods of induction yielded qualitatively similar results. After induction, there was abundant synthesis of viral transcripts, amplification of viral DNA, and the appearance of many new viral polypeptides. Nonetheless, there were no changes in the cytoplasmic abundance of Epstein-Barr virus-encoded RNAs and no alteration in the level of Epstein-Barr virus nuclear antigen mRNA or polypeptide. Thus, under conditions in which numerous other Epstein-Barr virus gene products are activated, the two major latent gene products are expressed at a constitutive level. Expression of Epstein-Barr virus-encoded RNAs and nuclear antigen must therefore be regulated in a manner completely different from expression of replicative functions. Images PMID:2981344

  3. Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge.

    PubMed

    Shrestha, Sony; Park, Yoonseong; Stanley, David; Kim, Yonggyun

    2010-03-01

    We propose that expression of four genes encoding secretory phospholipases A(2) (sPLA(2)) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA(2)-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA(2) activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA(2) and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA(2)s. The recombinant sPLA(2)s were inhibited by sPLA(2) inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA(2) genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA(2)s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA(2)s showed the presence of the sPLA(2) enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA(2) genes that mediate nodulation reactions strongly supports our hypothesis that sPLA(2)s are central enzymes in insect cellular immune reactions. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  4. Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    PubMed Central

    Charlebois, Audrey; Jalbert, Louis-Alexandre; Harel, Josée; Masson, Luke; Archambault, Marie

    2012-01-01

    Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC90 (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC50 was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens. PMID:22970221

  5. Genome-wide comparative analysis of NBS-encoding genes in four Gossypium species

    USDA-ARS?s Scientific Manuscript database

    Nucleotide binding site (NBS) genes encode a large family of disease resistance (R) proteins in plants. The availability of genomic data of the two diploid cotton species, Gossypium arboreum and Gossypium raimondii, and the two allotetraploid cotton species, Gossypium hirsutum (TM-1) and Gossypium ...

  6. Genes Encoding Phospholipases A2 Mediate Insect Nodulation Reactions to Bacterial Challenge

    USDA-ARS?s Scientific Manuscript database

    We propose that expression of four genes encoding secretory phospholipases A2 (sPLA2) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis...

  7. Characterization of Genes Encoding Key Enzymes Involved in Anthocyanin Metabolism of Kiwifruit during Storage Period

    PubMed Central

    Li, Boqiang; Xia, Yongxiu; Wang, Yuying; Qin, Guozheng; Tian, Shiping

    2017-01-01

    ‘Hongyang’ is a red fleshed kiwifruit with high anthocyanin content. In this study, we mainly investigated effects of different temperatures (25 and 0°C) on anthocyanin biosynthesis in harvested kiwifruit, and characterized the genes encoding key enzymes involved in anthocyanin metabolism, as well as evaluated the mode of the action, by which low temperature regulates anthocyanin accumulation in ‘Hongyang’ kiwifruit during storage period. The results showed that low temperature could effectively enhance the anthocyanin accumulation of kiwifruit in the end of storage period (90 days), which related to the increase in mRNA levels of ANS1, ANS2, DRF1, DRF2, and UGFT2. Moreover, the transcript abundance of MYBA1-1 and MYB5-1, the genes encoding an important component of MYB–bHLH–WD40 (MBW) complex, was up-regulated, possibly contributing to the induction of specific anthocyanin biosynthesis genes under the low temperature. To further investigate the roles of AcMYB5-1/5-2/A1-1 in regulation of anthocyanin biosynthesis, genes encoding the three transcription factors were transiently transformed in Nicotiana benthamiana leaves. Overexpression of AcMYB5-1/5-2/A1-1 activated the gene expression of NtANS and NtDFR in tobacco. Our results suggested that low temperature storage could stimulate the anthocyanin accumulation in harvested kiwifruit via regulating several structural and regulatory genes involved in anthocyanin biosynthesis. PMID:28344589

  8. Cloning of human genes encoding novel G protein-coupled receptors

    SciTech Connect

    Marchese, A.; Docherty, J.M.; Heiber, M.

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  9. Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

    PubMed Central

    Morbidoni, H R; de Mendoza, D; Cronan, J E

    1996-01-01

    A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome. PMID:8759840

  10. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL

    PubMed Central

    Brickner, Anthony G.; Evans, Anne M.; Mito, Jeffrey K.; Xuereb, Suzanne M.; Feng, Xin; Nishida, Tetsuya; Fairfull, Liane; Ferrell, Robert E.; Foon, Kenneth A.; Hunt, Donald F.; Shabanowitz, Jeffrey; Engelhard, Victor H.; Riddell, Stanley R.; Warren, Edus H.

    2006-01-01

    Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A*0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19+ B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19+ cells, and identify PANE1 as a potential therapeutic target in B-CLL. PMID:16391015

  11. Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection.

    PubMed

    Beard, Cheryl E; Court, Leon; Boets, Annemie; Mourant, Roslyn; Van Rie, Jeroen; Akhurst, Raymond J

    2008-09-01

    Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.

  12. A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution.

    PubMed Central

    Henze, K; Badr, A; Wettern, M; Cerff, R; Martin, W

    1995-01-01

    Genes for glycolytic and Calvin-cycle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher eukaryotes derive from ancient gene duplications which occurred in eubacterial genomes; both were transferred to the nucleus during the course of endosymbiosis. We have cloned cDNAs encoding chloroplast and cytosolic GAPDH from the early-branching photosynthetic protist Euglena gracilis and have determined the structure of its nuclear gene for cytosolic GAPDH. The gene contains four introns which possess unusual secondary structures, do not obey the GT-AG rule, and are flanked by 2- to 3-bp direct repeats. A gene phylogeny for these sequences in the context of eubacterial homologues indicates that euglenozoa, like higher eukaryotes, have obtained their GAPDH genes from eubacteria via endosymbiotic (organelle-to-nucleus) gene transfer. The data further suggest that the early-branching protists Giardia lamblia and Entamoeba histolytica--which lack mitochondria--and portions of the trypanosome lineage have acquired GAPDH genes from eubacterial donors which did not ultimately give rise to contemporary membrane-bound organelles. Evidence that "cryptic" (possibly ephemeral) endosymbioses during evolution may have entailed successful gene transfer is preserved in protist nuclear gene sequences. PMID:7568085

  13. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

    PubMed

    Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

    2015-12-01

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon.

  14. Local synthesis of nuclear-encoded mitochondrial proteins in the presynaptic nerve terminal.

    PubMed

    Gioio, A E; Eyman, M; Zhang, H; Lavina, Z S; Giuditta, A; Kaplan, B B

    2001-06-01

    One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function. In contrast to this postulate, we have established previously that a heterogeneous population of mRNAs and biologically active polyribosomes exist in the giant axon and presynaptic nerve terminals of the photoreceptor neurons in squid. We report that these mRNA populations contain mRNAs for nuclear-encoded mitochondrial proteins to include: cytochrome oxidase subunit 17, propionyl-CoA carboxylase (EC 6.4.1.3), dihydrolipoamide dehydrogenase (EC 1.8.1.4), and coenzyme Q subunit 7. The mRNA for heat shock protein 70, a chaperone protein known to be involved in the import of proteins into mitochondria, has also been identified. Electrophoretic gel analysis of newly synthesized proteins in the synaptosomal fraction isolated from the squid optic lobe revealed that the large presynaptic terminals of the photoreceptor neuron contain a cytoplasmic protein synthetic system. Importantly, a significant amount of the cycloheximide resistant proteins locally synthesized in the terminal becomes associated with mitochondria. PCR analysis of RNA from synaptosomal polysomes establishes that COX17 and CoQ7 mRNAs are being actively translated. Taken together, these findings indicate that proteins required for the maintenance of mitochondrial function are synthesized locally in the presynaptic nerve terminal, and call attention to the intimacy of the relationship between the terminal and its energy generating system. J. Neurosci. Res. 64:447-453, 2001. Published 2001 Wiley-Liss, Inc.

  15. Screening of the Enterocin-Encoding Genes and Antimicrobial Activity in Enterococcus Species.

    PubMed

    Ogaki, Mayara Baptistucci; Rocha, Katia Real; Terra, MÁrcia Regina; Furlaneto, MÁrcia Cristina; Maia, Luciana Furlaneto

    2016-06-28

    In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.

  16. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  17. Molecular anatomy of tunicate senescence: reversible function of mitochondrial and nuclear genes associated with budding cycles.

    PubMed

    Kawamura, Kaz; Kitamura, Seigo; Sekida, Satoko; Tsuda, Masayuki; Sunanaga, Takeshi

    2012-11-01

    Zooids of the asexual strain of Polyandrocarpa misakiensis have a lifespan of 4-5 months; before dying, they produce many buds, enabling continuation of the strain. This study was designed to investigate the nature of gene inactivation and reactivation during this continuous process of senescence and budding. During senescence, the zooidal epidermis showed acid β-galactosidase activity, lost proliferating cell nuclear antigen immunoreactivity and became ultrastructurally worn, indicating that the epidermis is a major tissue affected by the ageing process. Semi-quantitative PCR analysis showed that the genes encoding mitochondrial respiratory chains (MRCs) engaged in decreased transcriptional activity in senescent adults compared with younger adults. The results of in situ hybridization showed that the epidermis dramatically attenuates MRC expression during ageing but restores gene activity when budding commences. During budding and ageing, the nuclear gene Eed (a polycomb group component) was activated and inactivated in a pattern similar to that observed in MRCs. In buds, RNA interference (RNAi) of Eed attenuated Eed transcripts but did not affect the gene expression of pre-activated MRCs. A tunicate humoral factor, TC14-3, could induce Eed, accompanying the reactivation of MRC in adult zooids. When RNAi of Eed and Eed induction were performed simultaneously, zooidal cells and tissues failed to engage in MRC reactivation, indicating the involvement of Eed in MRC activation. Results of this study provide evidence that the mitochondrial gene activities of Polyandrocarpa can be reversed during senescence and budding, suggesting that they are regulated by nuclear polycomb group genes.

  18. Expression of a synthetic gene encoding P2 ribonuclease from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus in mesophylic hosts.

    PubMed

    Fusi, P; Grisa, M; Mombelli, E; Consonni, R; Tortora, P; Vanoni, M

    1995-02-27

    This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus [Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in S. cerevisiae using the galactose-inducible GAL1 promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E. coli as judged by immunological and biochemical criteria. E. coli-produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3. S. cerevisiae-produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern. Furthermore, monodimensional nuclear magnetic resonance showed that the E. coli- and Su. solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys4 and Lys6 were not methylated in the re-enzyme, thus showing that lysine methylation does not play a role in P2 thermostabilization.

  19. Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes.

    PubMed

    Rees, Matthew G; Ng, David; Ruppert, Sarah; Turner, Clesson; Beer, Nicola L; Swift, Amy J; Morken, Mario A; Below, Jennifer E; Blech, Ilana; Mullikin, James C; McCarthy, Mark I; Biesecker, Leslie G; Gloyn, Anna L; Collins, Francis S

    2012-01-01

    Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

  20. A Single Gene May Encode Differentially Localized Ca2+-ATPases in Tomato.

    PubMed Central

    Ferrol, N; Bennett, AB

    1996-01-01

    Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon Ca2+-ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATP-dependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots. PMID:12239413

  1. Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello.

    PubMed Central

    Voordouw, G; Strang, J D; Wilson, F R

    1989-01-01

    The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of alpha and beta subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) alpha subunit precedes that of the 13.5-kDa beta subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both alpha subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed beta subunits (124 and 123 amino acid residues, respectively) share 71% sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was found approximately 90 base pairs upstream from the translational start of the hydA gene. The recently discovered hydC gene, which may code for a 65.8-kDa fusion protein (gamma) of the alpha and beta subunits and is present immediately downstream from the hydAB genes in the Hildenborough strain, was found to be absent from the Monticello strain. The implication of this result for the possible function of the hydC gene product in Desulfovibrio species is discussed. Images PMID:2661538

  2. The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase

    PubMed Central

    Li, Li; Hill-Skinner, Sarah; Liu, Sanzhen; Beuchle, Danielle; Tang, Ho Man; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

    2015-01-01

    Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine-mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one-carbon (C1) metabolism to polyglutamylate substrates of folate-dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS-deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild-type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis. PMID:25495051

  3. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  4. Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

    PubMed Central

    Cardoso, Mariana S.; Junqueira, Caroline; Trigueiro, Ricardo C.; Shams-Eldin, Hosam; Macedo, Cristiana S.; Araújo, Patrícia R.; Gomes, Dawidson A.; Martinelli, Patrícia M.; Kimmel, Jürgen; Stahl, Philipp; Niehus, Sebastian; Schwarz, Ralph T.; Previato, José O.; Mendonça-Previato, Lucia; Gazzinelli, Ricardo T.; Teixeira, Santuza M. R.

    2013-01-01

    Background Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. Methodology/Principal Findings In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. Conclusions/Significance Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this

  5. Filamentous-haemagglutinin-like protein genes encoded on a plasmid of Moraxella bovis.

    PubMed

    Kakuda, Tsutomu; Sarataphan, Nopporn; Tanaka, Tetsuya; Takai, Shinji

    2006-11-26

    The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

  6. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    PubMed

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber.

  7. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    NASA Astrophysics Data System (ADS)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  8. Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354.

    PubMed

    Kimura, T; Takahashi, M; Yoshihara, K; Furuichi, T; Suzuki, K; Imai, K; Karita, S; Sakka, K; Ohmiya, K

    1998-11-08

    We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.

  9. Isolation of the gene encoding the Hin recombinational enhancer binding protein.

    PubMed Central

    Johnson, R C; Ball, C A; Pfeffer, D; Simon, M I

    1988-01-01

    In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer. We have cloned and determined the primary sequence of the gene encoding Fis. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus. The gene encoding Fis maps at 72 min on the Escherichia coli chromosome. The construction of mutant strains of E. coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo. PMID:2835774

  10. Dynamics of nuclear receptor gene expression during Pacific oyster development.

    PubMed

    Vogeler, Susanne; Bean, Tim P; Lyons, Brett P; Galloway, Tamara S

    2016-09-29

    Nuclear receptors are a highly conserved set of ligand binding transcription factors, with essential roles regulating aspects of vertebrate and invertebrate biology alike. Current understanding of nuclear receptor regulated gene expression in invertebrates remains sparse, limiting our ability to elucidate gene function and the conservation of developmental processes across phyla. Here, we studied nuclear receptor expression in the early life stages of the Pacific oyster, Crassostrea gigas, to identify at which specific key stages nuclear receptors are expressed RESULTS: We used quantitative RT-PCR to determine the expression profiles of 34 nuclear receptors, revealing three developmental key stages, during which nuclear receptor expression is dynamically regulated: embryogenesis, mid development from gastrulation to trochophore larva, and late larval development prior to metamorphosis. Clustering of nuclear receptor expression patterns demonstrated that transcriptional regulation was not directly related to gene phylogeny, suggesting closely related genes may have distinct functions. Expression of gene homologs of vertebrate retinoid receptors suggests participation in organogenesis and shell-formation, as they are highly expressed at the gastrulation and trochophore larval initial shell formation stages. The ecdysone receptor homolog showed high expression just before larval settlement, suggesting a potential role in metamorphosis. Throughout early oyster development nuclear receptors exhibited highly dynamic expression profiles, which were not confined by gene phylogeny. These results provide fundamental information on the presence of nuclear receptors during key developmental stages, which aids elucidation of their function in the developmental process. This understanding is essential as ligand sensing nuclear receptors can be disrupted by xenobiotics, a mode of action through which anthropogenic environmental pollutants have been found to mediate effects.

  11. Horse cDNA clones encoding two MHC class I genes

    SciTech Connect

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

  12. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    PubMed Central

    Karlyshev, A.V.; Thacker, G.; Jones, M.A.; Clements, M.O.; Wren, B.W.

    2014-01-01

    According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection. PMID:24918062

  13. A mouse speciation gene encodes a meiotic histone H3 methyltransferase.

    PubMed

    Mihola, Ondrej; Trachtulec, Zdenek; Vlcek, Cestmir; Schimenti, John C; Forejt, Jiri

    2009-01-16

    Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the "fertility" Hst1(f) allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize gammaH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

  14. [Phylogenetic relationships among Asiatic salamanders of the genus Salamandrella based on variability of nuclear genes].

    PubMed

    Maliarchuk, B A; Derenko, M V; Denisova, G A

    2015-01-01

    Based on sequence variation of three nuclear genome genes (BDNF, POMC, and RAG1), the phylogenetic relationships among Asiatic salamanders of the genus Salamandrella, Siberian salamander (S. keyserlingii) and Schrenk salamander (S. schrenkii), were examined. Both species demonstrated high levels of heterozygosity determined by intraspecific polymorphism. Fixed interspecific differences were revealed at one nucleotide position of the RAG1 gene, and thus the level of interspecific divergence over the three genes constituted only 0.04%. Analysis of the RAG1 polymorphism across the whole range of S. keyserlingii showed that only one gene variant, encoding for modified RAG1 recombinase, had the highest distribution to the north of the Amur region (west and northeast of Siberia). It is possible that the changes in the RAG1 gene in Siberian salamander are of an adaptive nature. However, cases of interspecific hybridization were identified in Jewish autonomous oblast (JAO), which contains one of the range borders between the two Salamandrella species.

  15. [Identification of genes encoding ligninolytic enzymes in naturally occurring basidiomycete isolates].

    PubMed

    Shevchenko, E A; Bessolitsyna, E A; Darmov, I V

    2013-01-01

    The presence of genes encoding lignin peroxidase, laccase, and manganese peroxidase was assessed in more than 20 types of polypore fungi collected in the woods of Kirov oblast; the fungi studied had not been previously characterized with regard to ligninolytic enzyme production. Fifteen isolates of eleven basidiomycete species were shown to contain genes coding for all three ligninolytic enzymes. Genes coding for these enzymes were detected in D. mollis, D. quercina, F. pinicola, G. trabeum, G. lucidum, H. fasciculare, L. betulina, P. betulinus, P. igniarus, P. pomaceus, P. pini, and P. cinnabarinus for the first time.

  16. [Expression of the genes encoding RhtB family proteins depends on global regulator Lrp].

    PubMed

    Kutukova, E A; Zakataeva, N P; Livshits, V A

    2005-01-01

    In the present work, further study of the genes encoding RhtB family proteins is presented. In our previous work the involvement of two family members, RhtB and RhtC, in efflux of amino acids was demonstrated. Now we investigated regulation of expression of the rhtB, rhtC, yeaS and yahN genes. It is shown that expression of these genes is under control of the global regulator Lrp, depends on the presence of some amino acids in growth medium, and increases during different physiological stresses.

  17. Characterization of a ubiquitous expressed gene family encoding polygalacturonase in Arabidopsis thaliana.

    PubMed

    Torki, M; Mandaron, P; Mache, R; Falconet, D

    2000-01-25

    Pectin, as one of the major components of plant cell wall, has been implicated in many developmental processes occurring during plant growth. Among the different enzymes known to participate in the pectin structure modifications, polygalacturonase (PG) activity has been shown to be associated with fruit ripening, organ abscission and pollen grain development. Until now, sequence analyses of the deduced polypeptides of the plant PG genes allowed their grouping into three clades corresponding to genes involved in one of these three activities. In this study, we report the sequence of three genomic clones encoding PG in Arabidopsis thaliana. These genes, together with 16 other genes present in the databases form a large gene family, ubiquitously expressed, present on the five chromosomes with at least two gene clusters on chromosomes II and V, respectively. Phylogenetic analyses suggest that the A. thaliana gene family contains five classes of genes, with three of them corresponding to the previously defined clades. Comparison of positions and numbers of introns among the A. thaliana genes reveals structural conservation between genes belonging to the same class. The pattern of intron losses that could have given rise to the PG gene family is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a spliced mRNA. Following this event of intron loss, the acquisition of introns in novel positions is consistent with a mechanism of intron gain at proto-splice sites.

  18. A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response

    PubMed Central

    2014-01-01

    Background Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. PMID:24739417

  19. A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

    PubMed

    Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

    2014-04-16

    Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

  20. Encoded physics knowledge in checking codes for nuclear cross section libraries at Los Alamos

    NASA Astrophysics Data System (ADS)

    Parsons, D. Kent

    2017-09-01

    Checking procedures for processed nuclear data at Los Alamos are described. Both continuous energy and multi-group nuclear data are verified by locally developed checking codes which use basic physics knowledge and common-sense rules. A list of nuclear data problems which have been identified with help of these checking codes is also given.

  1. Analysis of genes encoding penicillin-binding proteins in clinical isolates of Acinetobacter baumannii.

    PubMed

    Cayô, Rodrigo; Rodríguez, María-Cruz; Espinal, Paula; Fernández-Cuenca, Felipe; Ocampo-Sosa, Alain A; Pascual, Alvaro; Ayala, Juan A; Vila, Jordi; Martínez-Martínez, Luis

    2011-12-01

    There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.

  2. Analysis of Genes Encoding Penicillin-Binding Proteins in Clinical Isolates of Acinetobacter baumannii ▿ †

    PubMed Central

    Cayô, Rodrigo; Rodríguez, María-Cruz; Espinal, Paula; Fernández-Cuenca, Felipe; Ocampo-Sosa, Alain A.; Pascual, Álvaro; Ayala, Juan A.; Vila, Jordi; Martínez-Martínez, Luis

    2011-01-01

    There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals. PMID:21947403

  3. Brassica rapa Has Three Genes That Encode Proteins Associated with Different Neutral Lipids in Plastids of Specific Tissues1

    PubMed Central

    Kim, Hyun Uk; Wu, Sherry S.H.; Ratnayake, Chandra; Huang, Anthony H.C.

    2001-01-01

    Plastid lipid-associated protein (PAP), a predominant structural protein associated with carotenoids and other non-green neutral lipids in plastids, was shown to be encoded by a single nuclear gene in several species. Here we report three PAP genes in the diploid Brassica rapa; the three PAPs are associated with different lipids in specific tissues. Pap1 and Pap2 are more similar to each other (84% amino acid sequence identity) than to Pap3 (46% and 44%, respectively) in the encoded mature proteins. Pap1 transcript was most abundant in the maturing anthers (tapetum) and in lesser amounts in leaves, fruit coats, seeds, and sepals; Pap2 transcript was abundant only in the petals; and Pap3 transcript had a wide distribution, but at minimal levels in numerous organs. Immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most organs had several nanograms of PAP1 or PAP2 per milligram of total protein, the highest amounts being in the anthers (10.9 μg mg−1 PAP1) and petals (6.6 μg mg−1 PAP2), and that they had much less PAP3 (<0.02 μg mg−1). In these organs PAP was localized in isolated plastid fractions. Plants were subjected to abiotic stresses; drought and ozone reduced the levels of the three Pap transcripts, whereas mechanical wounding and altering the light intensity enhanced their levels. We conclude that the PAP gene family consists of several members whose proteins are associated with different lipids and whose expressions are controlled by distinct mechanisms. Earlier reports of the expression of one Pap gene in various organs in a species need to be re-examined. PMID:11351096

  4. MYB98 positively regulates a battery of synergid-expressed genes encoding filiform apparatus localized proteins.

    PubMed

    Punwani, Jayson A; Rabiger, David S; Drews, Gary N

    2007-08-01

    The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98-green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation.

  5. Expression of neuropeptide- and hormone-encoding genes in the Ciona intestinalis larval brain.

    PubMed

    Hamada, Mayuko; Shimozono, Naoki; Ohta, Naoyuki; Satou, Yutaka; Horie, Takeo; Kawada, Tsuyoshi; Satake, Honoo; Sasakura, Yasunori; Satoh, Nori

    2011-04-15

    Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-β-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain.

  6. A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum.

    PubMed

    Mohammed, Suja; Te'o, Junior; Nevalainen, Helena

    2013-08-01

    Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

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

    PubMed Central

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

    1995-01-01

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

  8. Housekeeping genes essential for pantothenate biosynthesis are plasmid-encoded in Rhizobium etli and Rhizobium leguminosarum.

    PubMed

    Villaseñor, Tomás; Brom, Susana; Dávalos, Araceli; Lozano, Luis; Romero, David; Los Santos, Alejandro García-de

    2011-04-05

    A traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer. Analysis of mutants confirmed that the panC and panB genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of panCB genes among members of the Rhizobiales showed that only R. etli and R. leguminosarum strains carry panCB genes in plasmids. The panCB phylogeny attested a common origin for chromosomal and plasmid-borne panCB sequences, suggesting that the R. etli and R. leguminosarum panCB genes are orthologs rather than xenologs. The panCB genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium. This study shows experimental evidence that core panCB genes located in plasmids of R. etli and R. leguminosarum are indispensable for the synthesis of pantothenate. The

  9. Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica.

    PubMed

    Rumpho, Mary E; Worful, Jared M; Lee, Jungho; Kannan, Krishna; Tyler, Mary S; Bhattacharya, Debashish; Moustafa, Ahmed; Manhart, James R

    2008-11-18

    The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3' flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty ("green animals") and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

  10. Isolation and expression of two aquaporin-encoding genes from the marine phanerogam Posidonia oceanica.

    PubMed

    Maestrini, Pierluigi; Giordani, Tommaso; Lunardi, Andrea; Cavallini, Andrea; Natali, Lucia

    2004-12-01

    Seagrasses such as Posidonia oceanica (L.) Delile are marine phanerogams, widespread in various seas, where they form large prairies representing dynamic substrates exceeding the area of the sediment surface several times over and allowing settlement of epiphyte organisms. Studying mechanisms involved in water transport in marine plants, we isolated two aquaporin-encoding genes, PoPIP1;1 and PoTIP1;1, showing high similarity to plasma membrane- and tonoplast-intrinsic protein-encoding genes, respectively. PoPIP1;1 is unique in the genome of P. oceanica, while PoTIP1;1 belongs to an aquaporin subfamily of at least four members. PoPIP1;1 and PoTIP1;1 encode functional proteins, as indicated by expression experiments in Xenopus oocytes. Both genes are constitutively expressed in the leaves, with higher levels of transcripts in young than in differentiated leaf tissues. Variations of salt concentration in aquarium determined different PoPIP1;1 and PoTIP1;1 transcript accumulation, indicating the existence of adaptation mechanisms related to gene expression also in marine plants, i.e. adapted to very high salt concentrations. Hyposalinity induced lower levels of PIP1 transcripts, while hypersalinity determined more PIP1 transcripts than normal salinity. TIP1 transcripts increased in response to both hypo- and hypersalinity after 2 days of treatment and went back to control levels after 5 d.

  11. Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

    NASA Astrophysics Data System (ADS)

    Li, Shengjie; Bai, Junjie; Wang, Lin

    2008-08-01

    Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

  12. Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance

    PubMed Central

    Zhang, Lei; Xiao, Shanshan; Feng, Wei; Wu, Zhidan; Gao, Xuewen; Liu, Fengquan; Shao, Min

    2011-01-01

    Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops. PMID:21527628

  13. Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins▿

    PubMed Central

    Donato, Justin J.; Moe, Luke A.; Converse, Brandon J.; Smart, Keith D.; Berklein, Flora C.; McManus, Patricia S.; Handelsman, Jo

    2010-01-01

    To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a β-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional β-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a β-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance. PMID:20453147

  14. Identification and transcriptional control of Caulobacter crescentus genes encoding proteins containing a cold shock domain.

    PubMed

    Lang, Elza A S; Marques, Marilis V

    2004-09-01

    The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.

  15. Maize Cytolines Unmask Key Nuclear Genes That Are under the Control of Retrograde Signaling Pathways in Plants

    PubMed Central

    Miclaus, Mihai; Balacescu, Ovidiu; Has, Ioan; Balacescu, Loredana; Has, Voichita; Suteu, Dana; Neuenschwander, Samuel; Keller, Irene; Bruggmann, Rémy

    2016-01-01

    The genomes of the two plant organelles encode for a relatively small number of proteins. Thus, nuclear genes encode the vast majority of their proteome. Organelle-to-nucleus communication takes place through retrograde signaling (RS) pathways. Signals relayed through RS pathways have an impact on nuclear gene expression but their target-genes remain elusive in a normal state of the cell (considering that only mutants and stress have been used so far). Here, we use maize cytolines as an alternative. The nucleus of a donor line was transferred into two other cytoplasmic environments through at least nine back-crosses, in a time-span of > 10 years. The transcriptomes of the resulting cytolines were sequenced and compared. There are 96 differentially regulated nuclear genes in two cytoplasm-donor lines when compared with their nucleus-donor. They are expressed throughout plant development, in various tissues and organs. One-third of the 96 proteins have a human homolog, stressing their potential role in mitochondrial RS. We also identified syntenic orthologous genes in four other grasses and homologous genes in Arabidopsis thaliana. These findings contribute to the paradigm we use to describe the RS in plants. The 96 nuclear genes identified here are not differentially regulated as a result of mutation, or any kind of stress. They are rather key players of the organelle-to-nucleus communication in a normal state of the cell. PMID:27702813

  16. Dissection of the erythroid-specific transcriptional promoter used by the gene encoding aminolevulinic acid dehydratase (ALAD)

    SciTech Connect

    Bishop, T.R.; Schaffer, T.; Pien, B.

    1994-09-01

    The gene encoding delta-aminolevulinate dehydratase (ALAD), the second enzyme of the heme biosynthetic pathway, exists as a single gene in most mammalian genomes and we have sequenced over 12 kb from overlapping lambda clones containing the murine ALAD gene. The gene has a dual promoter driving expression of two different first exons; exon1A is expressed in all tissues and exon1B only in erythroid cells, where heme production is induced to exceptionally high levels for hemoglobin synthesis. Erythroid-specific expression of the ALAD gene is presumably accomplished by using the exon1B promoter which we hypothesize is responsive to erythroid-specific transcriptional activators. In order to test this, we have used gel mobility shift assays and DNase footprint analyses to dissect and identify the critical upstream regulatory elements. Nuclear extracts, prepared from murine erythroleukemia cells (MELC), human chronic myelogenous leukemia cell line (K562) and human fibroblast cell line (HeLa), were used as sources of proteins to analyze DNA binding sites in the ALAD erythroid-specific promoter from -307 to +1. In this region, there are three potential GATA1 sites, two CACCC boxes, a CCAAT box and a GGTGG box. NF-E2 sites were explored by using in vitro translation products of cloned p18 and p45, the two heterologous components of NF-E2, and successfully gel-shifted a 29 bp double-stranded oligo found at 2.6 kb in front of the ALAD gene. Thus, the ALAD gene utilizes both a housekeeping and a tissue-specific promoter.

  17. Isolation and expression analysis of FTZ-F1 encoding gene of black rock fish ( Sebastes schlegelii)

    NASA Astrophysics Data System (ADS)

    Shafi, Muhammad; Wang, Yanan; Zhou, Xiaosu; Ma, Liman; Muhammad, Faiz; Qi, Jie; Zhang, Quanqi

    2013-03-01

    Sex related FTZ-F1 is a transcriptional factor regulating the expression of fushi tarazu (a member of the orphan nuclear receptors) gene. In this study, FTZ-F1 gene ( FTZ-F1) was isolated from the testis of black rockfish ( Sebastes schlegeli) by homology cloning. The full-length cDNA of S. schlegeli FTZ-F1 ( ssFTZ-F1) contained a 232bp 5' UTR, a 1449bp ORF encoding FTZ-F1 (482 amino acid residules in length) with an estimated molecular weight of 5.4kD and a 105bp 3' UTR. Sequence, tissue distribution and phylogenic analysis showed that ssFTZ-F1 belonged to FTZ group, holding highly conserved regions including I, II and III FTZ-F1 boxes and an AF-2 hexamer. Relatively high expression was observed at different larva stages. In juveniles (105 days old), the transcript of ssFTZ-F1 can be detected in all tissues and the abuncance of the gene transcript in testis, ovary, spleen and brain was higher than that in other tissues. In mature fish, the abundance of gene transcript was higher in testis, ovary, spleen and brain than that in liver (trace amount), and the gene was not transcribed in other tissues. The highest abundance of gene transcript was always observed in gonads of both juvenile and mature fish. In addition, the abundance of gene transcript in male tissues were higher than that in female tissue counterparts ( P<0.05).

  18. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins.

    PubMed

    Hayward, B E; Moran, V; Strain, L; Bonthron, D T

    1998-12-22

    The GNAS1 gene encodes the alpha subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsalpha is biallelically encoded. In contrast, the large G protein XLalphas, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5' of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5' to the paternally expressed XLalphas exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLalphas), and biallelically (Gsalpha) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions.

  19. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins

    PubMed Central

    Hayward, Bruce E.; Moran, Veronica; Strain, Lisa; Bonthron, David T.

    1998-01-01

    The GNAS1 gene encodes the α subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsα is biallelically encoded. In contrast, the large G protein XLαs, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′ of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′ to the paternally expressed XLαs exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLαs), and biallelically (Gsα) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions. PMID:9860993

  20. Variants in genes that encode muscle contractile proteins influence risk for isolated clubfoot

    PubMed Central

    Weymouth, Katelyn S.; Blanton, Susan H.; Bamshad, Michael J.; Beck, Anita E.; Alvarez, Christine; Richards, Steve; Gurnett, Christina A.; Dobbs, Matthew B.; Barnes, Douglas; Mitchell, Laura E.; Hecht, Jacqueline T.

    2011-01-01

    Isolated clubfoot is a relatively common birth defect that affects approximately 4,000 newborns in the US each year. Calf muscles in the affected leg(s) are underdeveloped and remain small even after corrective treatment. This observation suggests that variants in genes that influence muscle development are priority candidate risk factors for clubfoot. This contention is further supported by the discovery that mutations in genes that encode components of the muscle contractile complex (MYH3, TPM2, TNNT3, TNNI2, and MYH8) cause congenital contractures, including clubfoot, in distal arthrogryposis (DA) syndromes. Interrogation of fifteen genes encoding proteins that control myofiber contractility in a cohort of both nonHispanic white (NHW) and Hispanic families, identified positive associations (p<0.05) with SNPs in twelve genes; only one was identified in a family-based validation dataset. Six SNPs in TNNC2 deviated from Hardy Weinberg Equilibrium (HWE) in mothers in our NHW discovery dataset. Relative risk and likelihood ratio tests showed evidence for a maternal genotypic effect with TNNC2/rs383112 and an inherited/child genotypic effect with two SNPs, TNNC2/rs4629 and rs383112. Associations with multiple SNPs in TPM1 were identified in the NHW discovery (rs4075583, p=0.01), family-based validation (rs1972041, p=0.000074) and case-control validation (rs12148828, p=0.04) datasets. Gene interactions were identified between multiple muscle contraction genes with many of the interactions involving at least one potential regulatory SNP. Collectively, our results suggest that variation in genes that encode contractile proteins of skeletal myofibers may play a role in the etiology of clubfoot. PMID:21834041

  1. Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci.

    PubMed

    Perumal, N; Murugesan, S; Krishnan, P

    2016-01-01

    The objective of this study was to determine the distribution of genes encoding aminoglycoside-modifying enzymes (AMEs) and staphylococcal cassette chromosome mec (SCCmec) elements among clinical isolates of methicillin-resistant staphylococci (MRS). Antibiotic susceptibility test was done using Kirby-Bauer disk diffusion method. The presence of SCCmec types and AME genes, namely, aac (6')-Ie-aph (2''), aph (3')-IIIa and ant (4')-Ia was determined using two different multiplex polymerase chain reaction. The most encountered AME genes were aac (6')-Ie-aph (2'') (55.4%) followed by aph (3')-IIIa (32.3%) and ant (4')-Ia gene (9%). SCCmec type I (34%) was predominant in this study. In conclusion, the aac (6')-Ie-aph (2'') was the most common AME gene and SCCmec type I was most predominant among the MRS isolates.

  2. A negative element involved in Kaposi's sarcoma-associated herpesvirus-encoded ORF11 gene expression

    SciTech Connect

    Chen, Lei

    2009-01-01

    The ORF11 of the Kaposi's sarcoma-associated herpesvirus (KSHV) is a lytic viral gene with delayed-early expression kinetics. How the ORF11 gene expression is regulated in the KSHV lytic cascade is largely unknown. Here we report that the deletion of the KSHV viral IL-6 gene from the viral genome leads to deregulated ORF11 gene expression. The KSHV-encoded viral IL-6 protein was found not to be essentially involved in the regulation of ORF11, suggesting a potential transcriptional cis-regulation. A negative element was identified downstream of the ORF11 gene, which suppresses the ORF11 basal promoter activity in a position-independent manner.

  3. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.

    PubMed

    Morris, R O; Bilyeu, K D; Laskey, J G; Cheikh, N N

    1999-02-16

    The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.

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

    PubMed Central

    Niskanen, Einari A; Hytönen, Vesa P; Grapputo, Alessandro; Nordlund, Henri R; Kulomaa, Markku S; Laitinen, Olli H

    2005-01-01

    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. PMID:15777476

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

    PubMed

    Niskanen, Einari A; Hytönen, Vesa P; Grapputo, Alessandro; Nordlund, Henri R; Kulomaa, Markku S; Laitinen, Olli H

    2005-03-18

    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. 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. 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.

  6. Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

    PubMed Central

    Watson, D K; McWilliams, M J; Lapis, P; Lautenberger, J A; Schweinfest, C W; Papas, T S

    1988-01-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains. PMID:2847145

  7. The ctnG gene encodes carbonic anhydrase involved in mycotoxin citrinin biosynthesis from Monascus aurantiacus.

    PubMed

    Li, Yan-Ping; Tang, Xiao; Wu, Wei; Xu, Yang; Huang, Zhi-Bing; He, Qing-Hua

    2015-01-01

    Citrinin, a fungal secondary metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. Citrinin is synthesised by condensation of acetyl-CoA and malonyl-CoA. Six genes involved in the citrinin biosynthesis, including pksCT, ctnA and ctnB, have been cloned in Monascus purpureus. The pksCT gene encodes a polyketide synthase; ctnA is a regulatory factor; and ctnB encodes an oxidoreductase. When the three genes were respectively disrupted, the disruption strains drastically decreased citrinin production or barely produced citrinin. Ten new genes have been discovered in Monascus aurantiacus besides the above six genes. One of these gene displayed the highest similarity to the β-carbonic anhydrase gene from Aspergillus oryzae (74% similarity) and was designated ctnG. To learn more about the citrinin biosynthetic pathway, a ctnG-replacement vector was constructed to disrupt ctnG with the hygromycin resistance gene as the selection marker, then transformed into M. aurantiacus Li AS3.4384 by a protoplast-PEG method. The citrinin content of three disruptants was reduced to about 50%, meanwhile pigment production decreased by 23%, respectively, over those of the wild-type strains. ctnG was deduced to be involved in the formation of malonyl-CoA as a common precursor of red pigments and citrinin. Therefore, the disruption of the ctnG gene decreased citrinin and pigment production. M. aurantiacus Li AS3.4384 can produce higher concentrations of citrinin than other strains such as M. purpureus and M. ruber. Establishing the function of citrinin biosynthetic genes in M. aurantiacus is helpful in understanding the citrinin synthetic pathway and adopting some strategies to control contamination.

  8. High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

    PubMed

    Kamenetzky, L; Muzulin, P M; Gutierrez, A M; Angel, S O; Zaha, A; Guarnera, E A; Rosenzvit, M C

    2005-12-01

    Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

  9. The Evolutionary Fate of the Genes Encoding the Purine Catabolic Enzymes in Hominoids, Birds, and Reptiles

    PubMed Central

    Keebaugh, Alaine C.; Thomas, James W.

    2010-01-01

    Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes. PMID:20106906

  10. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

  11. Characterization of emb, a Gene Encoding the Major Adhesin of Streptococcus defectivus

    PubMed Central

    Manganelli, Riccardo; van de Rijn, Ivo

    1999-01-01

    Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins. PMID:9864195

  12. Bacteriophage T5 gene D10 encodes a branch-migration protein

    PubMed Central

    Wong, Io Nam; Sayers, Jon R.; Sanders, Cyril M.

    2016-01-01

    Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms. PMID:28009009

  13. Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli.

    PubMed Central

    Gilbert, J V; Plaut, A G; Fishman, Y; Wright, A

    1988-01-01

    We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region. Images PMID:3294181

  14. Sulfotransferase genes: Regulation by nuclear receptors in response to xeno/endo-biotics

    PubMed Central

    Kodama, Susumu; Negishi, Masahiko

    2014-01-01

    Pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR), members of the nuclear receptor superfamily, are two major xeno-sensing transcription factors. They can be activated by a broad range of lipophilic xenobiotics including therapeutics drugs. In addition to xenobiotics, endogenous compounds such as steroid hormones and bile acids can also activate PXR and/or CAR. These nuclear receptors regulate genes that encode enzymes and transporters that metabolize and excrete both xenobiotics and endobiotics. Sulfotransferases (SULTs) are a group of these enzymes and sulfate xenobiotics for detoxification. In general, inactivation by sulfation constitutes the mechanism to maintain homeostasis of endobiotics. Thus, deciphering the molecular mechanism by which PXR and CAR regulate SULT genes is critical for understanding the roles of SULTs in the alterations of physiological and pathophysiological processes caused by drug treatment or environmental exposures. PMID:24025090

  15. The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

    PubMed Central

    Boonstra, Birgitte; French, Christopher E.; Wainwright, Ian; Bruce, Neil C.

    1999-01-01

    The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase. PMID:9922271

  16. Expression of the gene encoding secreted placental alkaline phosphatase (SEAP) by a nondefective adenovirus vector.

    PubMed

    Doronin, K K; Zakharchuk, A N; Grinenko, N F; Yurov, G K; Krougliak, V A; Naroditsky, B S

    1993-04-30

    A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.

  17. Strategies on the nuclear-targeted delivery of genes

    PubMed Central

    Yao, Jing; Fan, Ying; Li, Yuanke; Huang, Leaf

    2016-01-01

    To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. “Smart” non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus. PMID:23964565

  18. Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

    PubMed

    Ernst, Antonia M; Jekat, Stephan B; Zielonka, Sascia; Müller, Boje; Neumann, Ulla; Rüping, Boris; Twyman, Richard M; Krzyzanek, Vladislav; Prüfer, Dirk; Noll, Gundula A

    2012-07-10

    The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

  19. Enterotoxin-Encoding Genes in Staphylococcus spp. from Food Handlers in a University Restaurant.

    PubMed

    da Silva, Sabina Dos Santos Paulino; Cidral, Thiago André; Soares, Maria José dos Santos; de Melo, Maria Celeste Nunes

    2015-11-01

    Food handlers carrying enterotoxin-producing Staphylococcus are a potential source of food poisoning. The aim of this study was to analyze genes encoding enterotoxins in coagulase-positive Staphylococcus (CoPS) and coagulase-negative Staphylococcus (CoNS) isolated from the anterior nostrils and hands of food handlers at a university restaurant in the city of Natal, Northeast Brazil. Thirty food handlers were screened for the study. The isolates were subjected to Gram staining, a bacitracin sensitivity test, mannitol fermentation, and catalase and coagulase tests. CoNS and CoPS strains were subsequently identified by a Vitek 2 System (BioMerieux, France) and various biochemical tests. Polymerase chain reaction was used to detect genes for enterotoxins A, B, C, D, E, G, H, and I (sea, seb, sec, sed, see, seg, seh, and sei) and a disc-diffusion method was used to determine susceptibility to several classes of antimicrobials. All food handlers presented staphylococci on their hands and/or noses. The study found 58 Staphylococcus spp., of which 20.7% were CoPS and 79.3% were CoNS. S. epidermidis was the most prevalent species. Twenty-nine staphylococci (50%) were positive for one or more enterotoxin genes, and the most prevalent genes were seg and sei, each with a frequency of 29.3%. Indeed, CoNS encoded a high percentage of enterotoxin genes (43.5%). However, S. aureus encoded even more enterotoxin genes (75%). Most isolates showed sensitivity to the antibiotics used for testing, except for penicillin (only 35% sensitive). The results from this study reinforce that coagulase-negative as well as coagulase-positive staphylococci isolated from food handlers are capable of genotypic enterotoxigenicity.

  20. Mutations in the gene encoding epsilon-sarcoglycan cause myoclonus-dystonia syndrome.

    PubMed

    Zimprich, A; Grabowski, M; Asmus, F; Naumann, M; Berg, D; Bertram, M; Scheidtmann, K; Kern, P; Winkelmann, J; Müller-Myhsok, B; Riedel, L; Bauer, M; Müller, T; Castro, M; Meitinger, T; Strom, T M; Gasser, T

    2001-09-01

    The dystonias are a common clinically and genetically heterogeneous group of movement disorders. More than ten loci for inherited forms of dystonia have been mapped, but only three mutated genes have been identified so far. These are DYT1, encoding torsin A and mutant in the early-onset generalized form, GCH1 (formerly known as DYT5), encoding GTP-cyclohydrolase I and mutant in dominant dopa-responsive dystonia, and TH, encoding tyrosine hydroxylase and mutant in the recessive form of the disease. Myoclonus-dystonia syndrome (MDS; DYT11) is an autosomal dominant disorder characterized by bilateral, alcohol-sensitive myoclonic jerks involving mainly the arms and axial muscles. Dystonia, usually torticollis and/or writer's cramp, occurs in most but not all affected patients and may occasionally be the only symptom of the disease. In addition, patients often show prominent psychiatric abnormalities, including panic attacks and obsessive-compulsive behavior. In most MDS families, the disease is linked to a locus on chromosome 7q21 (refs. 11-13). Using a positional cloning approach, we have identified five different heterozygous loss-of-function mutations in the gene for epsilon-sarcoglycan (SGCE), which we mapped to a refined critical region of about 3.2 Mb. SGCE is expressed in all brain regions examined. Pedigree analysis shows a marked difference in penetrance depending on the parental origin of the disease allele. This is indicative of a maternal imprinting mechanism, which has been demonstrated in the mouse epsilon-sarcoglycan gene.

  1. Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

    PubMed Central

    Lewin, A S; Hines, V; Small, G M

    1990-01-01

    The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle. Images PMID:2181273

  2. Temporal regulation of genes encoding the flagellar proximal rod in Caulobacter crescentus.

    PubMed

    Boyd, C H; Gober, J W

    2001-01-01

    The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. The flgBC-fliE genes were identified as a result of the C. crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with sigma(70) is responsible for regulating transcription of other early flagellar genes in C. crescentus. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and flgB deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus.

  3. Differential transcription of multiple copies of a silk worm gene encoding tRNA(Gly1).

    PubMed

    Fournier, A; Taneja, R; Gopalkrishnan, R; Prudhomme, J C; Gopinathan, K P

    1993-12-08

    Ten different tRNA(Gly1) genes from the silk worm, Bombyx mori, have been cloned and characterized. These genes were transcribed in vitro in homologous nuclear extracts from the posterior silk gland (PSG) or nuclear extracts derived from the middle silk gland or ovarian tissues. Although the transcription levels were much higher in the PSG nuclear extracts, the transcriptional efficiency of the individual genes followed a similar pattern in all the extracts. Based on the levels of in vitro transcription, the ten tRNA(Gly1) genes could be divided into three groups, viz., those which were transcribed at very high levels (e.g., clone pR8), high to medium levels (e.g., pBmi1, pBmp1, pBmh1, pBmt1) and low to barely detectable levels (e.g., pBms1, pBmj1 and pBmk1). The coding sequences of all these tRNA genes being identical, the differential transcription suggested that the flanking sequences modulate their transcriptional efficiency. The presence of positive and negative regulatory elements in the 5' flanking regions of these genes was confirmed by transcription competition experiments. A positive element was present in the immediate upstream A+T-rich sequences in all the genes, but no consensus sequences correlating to the transcriptional status could be generated. The presence of negative elements on the other hand was indicated only in some of the genes and therefore may have a role in the differential transcription of these tRNA(Gly1) genes in vivo.

  4. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis.

    PubMed

    Town, M; Jean, G; Cherqui, S; Attard, M; Forestier, L; Whitmore, S A; Callen, D F; Gribouval, O; Broyer, M; Bates, G P; van't Hoff, W; Antignac, C

    1998-04-01

    Nephropathic cystinosis, an autosomal recessive disorder resulting from defective lysosomal transport of cystine, is the most common inherited cause of renal Fanconi syndrome. The cystinosis gene has been mapped to chromosome 17p13. We found that the locus D17S829 was homozygously deleted in 23 out of 70 patients, and identified a novel gene, CTNS, which mapped to the deletion interval. CTNS encodes an integral membrane protein, cystinosin, with features of a lysosomal membrane protein. Eleven different mutations, all predicted to cause loss of function of the protein, were found to segregate with the disorder.

  5. Localization of the human genes encoding the two subunits of general transcription factor TFIIE.

    PubMed

    Purrello, M; Di Pietro, C; Rapisarda, A; Motta, S; Pavone, L; Grzeschik, K H; Sichel, G

    1994-09-01

    TFIIE is a general transcription factor for class II genes composed of two types of subunits, a large one of 56 kDa and a small of 34 kDa. By Southern analysis at high and at low stringency of a panel of mouse/human hybrid cell lines and by in situ chromosomal hybridization, we have demonstrated that both polypeptides are encoded by genes that are single copy in the human genome and are localized at 3q13-q21 and at 8p12, respectively. A TaqI RFLP (heterozygosity index of 0.07) was detected at the locus for the 56-kDa subunit.

  6. Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase.

    PubMed

    Colombo, S; Grisa, M; Tortora, P; Vanoni, M

    1994-01-03

    Fumarase catalyzes the interconversion of L-malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C-terminal deletion.

  7. Chromosomal localization of the gene encoding the human DNA helicase RECQL and its mouse homologue

    SciTech Connect

    Puranam, K.L.; Kennington, E.; Blackshear, P.J.

    1995-04-10

    We have determined the chromosomal location of the human and mouse genes encoding the RECQL protein, a putative DNA helicase homologous to the bacterial DNA helicase, RecQ. RECQL was localized to human chromosome 12 by analysis of human-rodent somatic cell hybrid DNA, fine mapping of RECQL by fluorescence in situ hybridization revealed its chromosomal location to be 12p11-p12. The corresponding mouse gene, Recql, was mapped to the telomeric end of mouse chromosome 6 by analysis of DNA from an interspecific cross. 19 refs., 2 figs.

  8. The respiratory gene OXA1 has two fission yeast orthologues which together encode a function essential for cellular viability.

    PubMed

    Bonnefoy, N; Kermorgant, M; Groudinsky, O; Dujardin, G

    2000-03-01

    The Saccharomyces cerevisiae nuclear gene OXA1, which is conserved from prokaryotes to human, was shown to be essential for cytochrome c oxidase and F1F0-ATP synthase biogenesis. We have searched for an orthologue of OXA1 in Schizosaccharomyces pombe, another yeast that is highly diverged from S. cerevisiae and which could more closely model higher eukaryotes. In particular, S. pombe exhibits a limited growth under anaerobic conditions and is petite negative, that is it does not tolerate large deletions of its mitochondrial DNA. Surprisingly, two S. pombe cDNAs able to complement an S. cerevisiae oxa1 mutation were isolated. The corresponding genes have different chromosomal locations and intron contents. They encode distinct proteins, both sharing a weak sequence identity one with the other and with Oxa1p. A phenotypic analysis of both single inactivations demonstrates that only one gene is essential for respiration in S. pombe. However, the double inactivation is lethal. This work gives new insight into the dependence of S. pombe viability upon oxa1 function, providing evidence of a connection between petite negativity, a functional respiratory chain and F1F0-ATP synthase complex in S. pombe.

  9. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    PubMed Central

    Liao, Liao; Vimolmangkang, Sornkanok; Wei, Guochao; Zhou, Hui; Korban, Schuyler S.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis. PMID:25914714

  10. Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin.

    PubMed

    Hammer, P E; Hill, D S; Lam, S T; Van Pée, K H; Ligon, J M

    1997-06-01

    Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis.

  11. Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin.

    PubMed Central

    Hammer, P E; Hill, D S; Lam, S T; Van Pée, K H; Ligon, J M

    1997-01-01

    Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis. PMID:9172332

  12. Three hormone receptor-like Drosophila genes encode an identical DNA-binding finger.

    PubMed

    Rothe, M; Nauber, U; Jäckle, H

    1989-10-01

    The putative finger domain of knirps (kni), a member of the gap class of segmentation genes, was used to isolate two sequence-related genes of Drosophila melanogaster under reduced stringency hybridization conditions. The two kni homologous genes map close to kni in the proximal portion of the third chromosome. One of them is the previously identified gene knirps-related (knrl), kni and knrl are spatially co-regulated in both early and late stages of embryogenesis. Their posterior domains of expression at blastoderm stage are under the control of the maternal pattern organizer gene nanos. In contrast, the expression of the second kni homologous gene is restricted to the late embryonic gonads. Due to its site of expression, we termed this gene 'embryonic gonad' (egon). In addition to the conserved DNA-binding domain, these three genes share an additional sequence of 19 amino acids, the kni-box, adjacent to the finger region. The identical N-terminal Cys/Cys finger encoded by each of the three genes suggests that they code for DNA-binding proteins which might bind to similar (or even identical) target sequences.

  13. Three hormone receptor-like Drosophila genes encode an identical DNA-binding finger.

    PubMed Central

    Rothe, M; Nauber, U; Jäckle, H

    1989-01-01

    The putative finger domain of knirps (kni), a member of the gap class of segmentation genes, was used to isolate two sequence-related genes of Drosophila melanogaster under reduced stringency hybridization conditions. The two kni homologous genes map close to kni in the proximal portion of the third chromosome. One of them is the previously identified gene knirps-related (knrl), kni and knrl are spatially co-regulated in both early and late stages of embryogenesis. Their posterior domains of expression at blastoderm stage are under the control of the maternal pattern organizer gene nanos. In contrast, the expression of the second kni homologous gene is restricted to the late embryonic gonads. Due to its site of expression, we termed this gene 'embryonic gonad' (egon). In addition to the conserved DNA-binding domain, these three genes share an additional sequence of 19 amino acids, the kni-box, adjacent to the finger region. The identical N-terminal Cys/Cys finger encoded by each of the three genes suggests that they code for DNA-binding proteins which might bind to similar (or even identical) target sequences. Images PMID:2555153

  14. Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

    PubMed Central

    Capel, J; Jarillo, J A; Salinas, J; Martínez-Zapater, J M

    1997-01-01

    We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes. PMID:9342870

  15. Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats.

    PubMed

    Rajneesh; Pathak, Jainendra; Kannaujiya, Vinod K; Singh, Shailendra P; Sinha, Rajeshwar P

    2017-07-01

    Nucleotide and amino acid compositions were studied to determine the genomic and structural relationship of photolyase gene in freshwater, marine and hot spring cyanobacteria. Among three habitats, photolyase encoding genes from hot spring cyanobacteria were found to have highest GC content. The genomic GC content was found to influence the codon usage and amino acid variability in photolyases. The third position of codon was found to have more effect on amino acid variability in photolyases than the first and second positions of codon. The variation of amino acids Ala, Asp, Glu, Gly, His, Leu, Pro, Gln, Arg and Val in photolyases of three different habitats was found to be controlled by first position of codon (G1C1). However, second position (G2C2) of codon regulates variation of Ala, Cys, Gly, Pro, Arg, Ser, Thr and Tyr contents in photolyases. Third position (G3C3) of codon controls incorporation of amino acids such as Ala, Phe, Gly, Leu, Gln, Pro, Arg, Ser, Thr and Tyr in photolyases from three habitats. Photolyase encoding genes of hot spring cyanobacteria have 85% codons with G or C at third position, whereas marine and freshwater cyanobacteria showed 82 and 60% codons, respectively, with G or C at third position. Principal component analysis (PCA) showed that GC content has a profound effect in separating the genes along the first major axis according to their RSCU (relative synonymous codon usage) values, and neutrality analysis indicated that mutational pressure has resulted in codon bias in photolyase genes of cyanobacteria.

  16. A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

    PubMed

    Waldron, Ashley L; Cahan, Sara Helms; Franklyn, Christopher S; Ebert, Alicia M

    2017-01-01

    Histidyl tRNA Synthetase (HARS) is a member of the aminoacyl tRNA synthetase (ARS) family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.

  17. Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein.

    PubMed Central

    Taylor, M E; Brickell, P M; Craig, R K; Summerfield, J A

    1989-01-01

    The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver. PMID:2590164

  18. The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase.

    PubMed Central

    Thomson, G J; Howlett, G J; Ashcroft, A E; Berry, A

    1998-01-01

    The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies. PMID:9531482

  19. Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase.

    PubMed

    Casqueiro, J; Gutiérrez, S; Bañuelos, O; Fierro, F; Velasco, J; Martín, J F

    1998-09-01

    A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

  20. Genome analysis and identification of gelatinase encoded gene in Enterobacter aerogenes

    NASA Astrophysics Data System (ADS)

    Shahimi, Safiyyah; Mutalib, Sahilah Abdul; Khalid, Rozida Abdul; Repin, Rul Aisyah Mat; Lamri, Mohd Fadly; Bakar, Mohd Faizal Abu; Isa, Mohd Noor Mat

    2016-11-01

    In this study, bioinformatic analysis towards genome sequence of E. aerogenes was done to determine gene encoded for gelatinase. Enterobacter aerogenes was isolated from hot spring water and gelatinase species-specific bacterium to porcine and fish gelatin. This bacterium offers the possibility of enzymes production which is specific to both species gelatine, respectively. Enterobacter aerogenes was partially genome sequenced resulting in 5.0 mega basepair (Mbp) total size of sequence. From pre-process pipeline, 87.6 Mbp of total reads, 68.8 Mbp of total high quality reads and 78.58 percent of high quality percentage was determined. Genome assembly produced 120 contigs with 67.5% of contigs over 1 kilo base pair (kbp), 124856 bp of N50 contig length and 55.17 % of GC base content percentage. About 4705 protein gene was identified from protein prediction analysis. Two candidate genes selected have highest similarity identity percentage against gelatinase enzyme available in Swiss-Prot and NCBI online database. They were NODE_9_length_26866_cov_148.013245_12 containing 1029 base pair (bp) sequence with 342 amino acid sequence and NODE_24_length_155103_cov_177.082458_62 which containing 717 bp sequence with 238 amino acid sequence, respectively. Thus, two paired of primers (forward and reverse) were designed, based on the open reading frame (ORF) of selected genes. Genome analysis of E. aerogenes resulting genes encoded gelatinase were identified.

  1. The Embryonically Active Gene, Unkempt, of Drosophila Encodes a Cys(3)his Finger Protein

    PubMed Central

    Mohler, J.; Weiss, N.; Murli, S.; Mohammadi, S.; Vani, K.; Vasilakis, G.; Song, C. H.; Epstein, A.; Kuang, T.; English, J.; Cherdak, D.

    1992-01-01

    The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys(3)His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development. PMID:1339381

  2. Somatic instability of the DNA sequences encoding the polymorphic polyglutamine tract of the AIB1 gene

    PubMed Central

    Dai, P; Wong, L

    2003-01-01

    Background: AIB1 contains a polymorphic polyglutamine tract (poly Q) that is encoded by a trinucleotide CAG repeat. Previously there have been conflicting results regarding the effect of the poly Q tract length on breast cancer. Since poly Q is not encoded by a perfect CAG repeat, the heterozygous polymorphic alleles need to be resolved, to understand the exact DNA sequences encoding poly Q. Methods: Poly Q encoding sequences of AIB1 from 107 DNA samples, including breast cancer cell lines, sporadic primary breast tumours, and blood samples from BRCA1/BRCA2 mutation carriers and the general population, were resolved by PCR/cloning followed by sequencing of each individual clone. Results: 25 distinct poly Q encoding sequence patterns were found. More than two distinct sequence patterns were found in a significantly higher proportion of tumours and cell lines than that of the general population, suggesting somatic instability. A significantly higher proportion of cancer cell lines or primary breast tumours than that of the general population contained rare sequence patterns. The proportion of sporadic breast tumours having at least one allele ⩽27 repeats is significantly higher than that in the blood of BRCA1/BRCA2 mutation carrier breast cancer patients or the general population. Conclusion: The poly Q encoding DNA sequences are somatically unstable in tumour tissues and cell lines. A missense mutation and a very short glutamine repeat in primary tumours suggests that AIB1 activity may be modulated through poly Q, which in turn plays a role in the cotransactivation of gene expressions in breast cancers. PMID:14684685

  3. Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization

    PubMed Central

    Wang, Yong; Tao, Xia-Fang; Su, Zhi-Xi; Liu, A-Ke; Liu, Tian-Lei; Sun, Ling; Yao, Qin; Chen, Ke-Ping; Gu, Xun

    2016-01-01

    Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5′-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5′-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories. PMID:27980385

  4. Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

    PubMed Central

    van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

    2016-01-01

    Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol

  5. Identification of the gene encoding vaccinia virus immunodominant protein p35.

    PubMed

    Zinoviev, V V; Tchikaev, N A; Chertov OYu; Malygin, E G

    1994-09-30

    The major envelope protein, p35, of vaccinia virus (VV; strain LIVP) was purified by extraction from virions with the non-ionic detergent Nonidet P-40. The protein was cleaved with CNBr. Four homogeneous peptides were isolated and their N-terminal amino-acid (aa) sequences determined. A computer search of a protein sequence databank revealed complete identity of the determined sequences with aa 44-63, 144-149, 154-165 and 224-238 of ORF H3 of the HindIII-H fragment of the VV genome [Rosel et al., J. Virol. 60 (1989) 436-446]. Earlier, Gordon et al. [Virology 167 (1988) 361-369] determined that the p35 surface protein of VV strain IHD-W is encoded by the H6 gene. Muravlev et al. [Biopolymery i kletka 6 (1990) 83-89 (Russian)] deduced from their data that gene A2 encodes this prominent antigen. Taking into account this ambiguity, we cloned the genes H3, H6 and A2 in expression vectors, prepared the specific antisera against the expression products and conducted the immunochemical analysis of the recombinant and native VV-specific proteins. It has been established that the H6 codes for an early protein that is found only in the infected cell extracts, but is absent in mature virions. The immunodominant protein p35 of VV strain LIVP is encoded by the gene H3. The gene A2 protein product is present mainly in the infected cell extract, but the antiserum against the A2 product shows a rather weak interaction with the 35-kDa fraction of structural VV proteins resolved by electrophoresis.

  6. Identification and differential expression dynamics of peach small GTPases encoding genes during fruit development and ripening

    PubMed Central

    Falchi, Rachele; Cipriani, Guido; Marrazzo, Teresa; Nonis, Alberto; Vizzotto, Giannina; Ruperti, Benedetto

    2010-01-01

    The function of monomeric GTPases of the RAS superfamily in fruit development and ripening has been partially characterized. Here the identification of peach (Prunus persica) small GTPases of the RAS superfamily expressed in fruit and the characterization of their expression profiles during fruit development are described. Extensive searches on expressed sequence tag (EST) databases led to the selection of a total of 24 genes from peach encoding proteins with significant similarity to Arabidopsis small GTPases. Sequence similarity analyses and identification of conserved motifs, diagnostic of specific RAS families and subfamilies, enabled bona fide assignment of fourteen PpRAB, seven PpARF/ARL/SAR, two PpROP and one PpRAN GTPases. Transcriptional expression profiles of peach monomeric GTPases, analysed by real-time quantitative reverse transcription-PCR, were obtained for mesocarp samples, collected in two consecutive years. Reproducible patterns of expression could be identified for five peach RAB-encoding genes (PpRABA1-1, PpRABA2, PpRABD2-1, PpRABD2-2, and PpRABC2), two ARFs (PpARFA1-1 and PpARLB1), and two ROPs (PpROP3 and PpROP4). Interestingly, the transient transcriptional up-regulation of PpARF genes and of PpRAB genes of the A and D clades, putatively controlling the exocytic delivery of cell wall components and modifying enzymes, appeared to coincide with peaks of growth speed and sugar accumulation and with the final phases of ripening. To our knowledge, this is the first description of the co-ordinated differential expression of a set of genes encoding small GTPases of the ARF and RAB families which takes place during key moments of fruit development and maturation. PMID:20501747

  7. funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi

    PubMed Central

    2014-01-01

    Background RNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP. Description To identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities. Conclusions funRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi. PMID:25522231

  8. Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius).

    PubMed

    Mamidala, Praveen; Mittapelly, Priyanka; Jones, Susan C; Piermarini, Peter M; Mittapalli, Omprakash

    2013-04-01

    The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect 'Kir3' clade, which we designate as 'ClKir3'. The other was a partial cDNA that encodes a protein with similarity to both the insect 'Kir1' and 'Kir2' clades, which we designate as 'ClKir1/2'. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology.

  9. Entamoeba histolytica: a unicellular organism containing two active genes encoding for members of the TBP family.

    PubMed

    Castañon-Sanchez, Carlos Alberto; Luna-Arias, Juan Pedro; de Dios-Bravo, Ma Guadalupe; Herrera-Aguirre, Maria Esther; Olivares-Trejo, Jose J; Orozco, Esther; Hernandez, Jose Manuel

    2010-03-01

    Entamoeba histolytica is the protozoan parasite which causes human amoebiasis. In this parasite, few encoding genes for transcription factors have been cloned and characterized. The E. histolytica TATA-box binding protein (EhTBP) is the first basal transcription factor that has been studied. To continue with the identification of other members of the basal transcription machinery, we performed an in silico analysis of the E. histolytica genome and found three loci encoding for polypeptides with similarity to EhTBP. One locus has a 100% identity to the previously Ehtbp gene reported by our group. The second locus encodes for a 212 aa polypeptide that is 100% identical to residues 23-234 from EhTBP. The third one encodes for a 216 aa polypeptide of 24kDa that showed 42.6% identity and 73.7% similarity to EhTBP. This protein was named E. histolytica TBP-related factor 1 (EhTRF1). Ehtrf1 gene was expressed in bacteria and the purified 28kDa recombinant polypeptide showed the capacity to bind to TATTTAAA-box by electrophoretic mobility shift assays. K(D) values for rEhTBP and rEhTRF1 were (1.71+/-2.90)x10(-12)M and (1.12+/-0.160)x10(-11)M, respectively. Homology modeling of EhTRF1 and EhTBP revealed that, although they were very similar, they showed some differences on their surfaces. Thus, E. histolytica is a unicellular organism having two members of the TBP family.

  10. High evolutionary rates in nuclear genes of squamates.

    PubMed

    Hughes, S; Mouchiroud, D

    2001-07-01

    We compared nonsynonymous substitution rates (Ka) of nuclear coding genes between four major groups of living sauropsids (reptiles): birds, squamates, crocodiles, and turtles. Since only 9 orthologous genes are known in all the four taxonomic groups, we searched for orthologous genes known in chicken and at least one of any representative of poikilotherm sauropsids. Thus, we analyzed three additional data sets: 28 genes identified in chicken and various squamates, 24 genes identified in chicken and crocodilians, and 20 genes identified in chicken and turtles. To compare nonsynonymous substitution rates between all lineages of sauropsids, we used the relative-rate test with human genes as the outgroup. We show that 22/28 nuclear coding genes of squamates, especially snakes (15/16), have an higher evolutionary rate than those in chicken (in mean, 30-40% faster). However, no such difference is detected between crocodiles, turtles and chicken. Higher substitution rate in squamates nuclear coding genes than in chicken, and probably than in other sauropsids, could explain some of the difficulties in resolving the molecular phylogeny of reptiles.

  11. The PCBP1 gene encoding poly(rC) binding protein I is recurrently mutated in Burkitt lymphoma.

    PubMed

    Wagener, Rabea; Aukema, Sietse M; Schlesner, Matthias; Haake, Andrea; Burkhardt, Birgit; Claviez, Alexander; Drexler, Hans G; Hummel, Michael; Kreuz, Markus; Loeffler, Markus; Rosolowski, Maciej; López, Cristina; Möller, Peter; Richter, Julia; Rohde, Marius; Betts, Matthew J; Russell, Robert B; Bernhart, Stephan H; Hoffmann, Steve; Rosenstiel, Philip; Schilhabel, Markus; Szczepanowski, Monika; Trümper, Lorenz; Klapper, Wolfram; Siebert, Reiner

    2015-09-01

    The genetic hallmark of Burkitt lymphoma is the translocation t(8;14)(q24;q32), or one of its light chain variants, resulting in IG-MYC juxtaposition. However, these translocations alone are insufficient to drive lymphomagenesis, which requires additional genetic changes for malignant transformation. Recent studies of Burkitt lymphoma using next generation sequencing approaches have identified various recurrently mutated genes including ID3, TCF3, CCND3, and TP53. Here, by using similar approaches, we show that PCBP1 is a recurrently mutated gene in Burkitt lymphoma. By whole-genome sequencing, we identified somatic mutations in PCBP1 in 3/17 (18%) Burkitt lymphomas. We confirmed the recurrence of PCBP1 mutations by Sanger sequencing in an independent validation cohort, finding mutations in 3/28 (11%) Burkitt lymphomas and in 6/16 (38%) Burkitt lymphoma cell lines. PCBP1 is an intron-less gene encoding the 356 amino acid poly(rC) binding protein 1, which contains three K-Homology (KH) domains and two nuclear localization signals. The mutations predominantly (10/12, 83%) affect the KH III domain, either by complete domain loss or amino acid changes. Thus, these changes are predicted to alter the various functions of PCBP1, including nuclear trafficking and pre-mRNA splicing. Remarkably, all six primary Burkitt lymphomas with a PCBP1 mutation expressed MUM1/IRF4, which is otherwise detected in around 20-40% of Burkitt lymphomas. We conclude that PCBP1 mutations are recurrent in Burkitt lymphomas and might contribute, in cooperation with other mutations, to its pathogenesis.

  12. Involvement of the Serotonin Transporter Gene in Accurate Subcortical Speech Encoding.

    PubMed

    Selinger, Lenka; Zarnowiec, Katarzyna; Via, Marc; Clemente, Immaculada C; Escera, Carles

    2016-10-19

    A flourishing line of evidence has highlighted the encoding of speech sounds in the subcortical auditory system as being shaped by acoustic, linguistic, and musical experience and training. And while the heritability of auditory speech as well as nonspeech processing has been suggested, the genetic determinants of subcortical speech processing have not yet been uncovered. Here, we postulated that the serotonin transporter-linked polymorphic region (5-HTTLPR), a common functional polymorphism located in the promoter region of the serotonin transporter gene (SLC6A4), is implicated in speech encoding in the human subcortical auditory pathway. Serotonin has been shown as essential for modulating the brain response to sound both cortically and subcortically, yet the genetic factors regulating this modulation regarding speech sounds have not been disclosed. We recorded the frequency following response, a biomarker of the neural tracking of speech sounds in the subcortical auditory pathway, and cortical evoked potentials in 58 participants elicited to the syllable /ba/, which was presented >2000 times. Participants with low serotonin transporter expression had higher signal-to-noise ratios as well as a higher pitch strength representation of the periodic part of the syllable than participants with medium to high expression, possibly by tuning synaptic activity to the stimulus features and hence a more efficient suppression of noise. These results imply the 5-HTTLPR in subcortical auditory speech encoding and add an important, genetically determined layer to the factors shaping the human subcortical response to speech sounds. The accurate encoding of speech sounds in the subcortical auditory nervous system is of paramount relevance for human communication, and it has been shown to be altered in different disorders of speech and auditory processing. Importantly, this encoding is plastic and can therefore be enhanced by language and music experience. Whether genetic factors

  13. Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

    PubMed Central

    Daley, M D; Misener, V; Olee, T; Chen, P P; Siminovitch, K A

    1993-01-01

    Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection. PMID:8324896

  14. Identification of candidate genes encoding an LDL-C QTL in baboons[S

    PubMed Central

    Karere, Genesio M.; Glenn, Jeremy P.; Birnbaum, Shifra; Hafizi, Sussan; Rainwater, David L.; Mahaney, Michael C.; VandeBerg, John L.; Cox, Laura A.

    2013-01-01

    Cardiovascular disease (CVD) is the leading cause of death in developed countries, and dyslipidemia is a major risk factor for CVD. We previously identified a cluster of quantitative trait loci (QTL) on baboon chromosome 11 for multiple, related quantitative traits for serum LDL-cholesterol (LDL-C). Here we report differentially regulated hepatic genes encoding an LDL-C QTL that influences LDL-C levels in baboons. We performed hepatic whole-genome expression profiling for LDL-C-discordant baboons fed a high-cholesterol, high-fat (HCHF) diet for seven weeks. We detected expression of 117 genes within the QTL 2-LOD support interval. Three genes were differentially expressed in low LDL-C responders and 8 in high LDL-C responders in response to a HCHF diet. Seven genes (ACVR1B, CALCOCO1, DGKA, ERBB3, KRT73, MYL6B, TENC1) showed discordant expression between low and high LDL-C responders. To prioritize candidate genes, we integrated miRNA and mRNA expression profiles using network tools and found that four candidates (ACVR1B, DGKA, ERBB3, TENC1) were miRNA targets and that the miRNAs were inversely expressed to the target genes. Candidate gene expression was validated using QRT-PCR and Western blotting. This study reveals candidate genes that influence variation in LDL-C in baboons and potential genetic mechanisms for further investigation. PMID:23596326

  15. Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

    PubMed

    Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B

    2011-01-01

    Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity.

  16. Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.

    PubMed

    Gaines, W A; Marcotte, W R

    2008-09-01

    Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSps) that consist of a large repeat array flanked by nonrepetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species.

  17. Clusters of genes encoding fructan biosynthesizing enzymes in wheat and barley.

    PubMed

    Huynh, Bao-Lam; Mather, Diane E; Schreiber, Andreas W; Toubia, John; Baumann, Ute; Shoaei, Zahra; Stein, Nils; Ariyadasa, Ruvini; Stangoulis, James C R; Edwards, James; Shirley, Neil; Langridge, Peter; Fleury, Delphine

    2012-10-01

    Fructans are soluble carbohydrates with health benefits and possible roles in plant adaptation. Fructan biosynthetic genes were isolated using comparative genomics and physical mapping followed by BAC sequencing in barley. Genes encoding sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT) and sucrose:fructan 6-fructosyltransferase (6-SFT) were clustered together with multiple copies of vacuolar invertase genes and a transposable element on two barley BAC. Intron-exon structures of the genes were similar. Phylogenetic analysis of the fructosyltransferases and invertases in the Poaceae showed that the fructan biosynthetic genes may have evolved from vacuolar invertases. Quantitative real-time PCR was performed using leaf RNA extracted from three wheat cultivars grown under different conditions. The 1-SST, 1-FFT and 6-SFT genes had correlated expression patterns in our wheat experiment and in existing barley transcriptome database. Single nucleotide polymorphism (SNP) markers were developed and successfully mapped to a major QTL region affecting wheat grain fructan accumulation in two independent wheat populations. The alleles controlling high- and low- fructan in parental lines were also found to be associated in fructan production in a diverse set of 128 wheat lines. To the authors' knowledge, this is the first report on the mapping and sequencing of a fructan biosynthetic gene cluster and in particular, the isolation of a novel 1-FFT gene from barley.

  18. A highly conserved NB-LRR encoding gene cluster effective against Setosphaeria turcica in sorghum

    PubMed Central

    2011-01-01

    Background The fungal pathogen Setosphaeria turcica causes turcicum or northern leaf blight disease on maize, sorghum and related grasses. A prevalent foliar disease found worldwide where the two host crops, maize and sorghum are grown. The aim of the present study was to find genes controlling the host defense response to this devastating plant pathogen. A cDNA-AFLP approach was taken to identify candidate sequences, which functions were further validated via virus induced gene silencing (VIGS), and real-time PCR analysis. Phylogenetic analysis was performed to address evolutionary events. Results cDNA-AFLP analysis was run on susceptible and resistant sorghum and maize genotypes to identify resistance-related sequences. One CC-NB-LRR encoding gene GRMZM2G005347 was found among the up-regulated maize transcripts after fungal challenge. The new plant resistance gene was designated as St referring to S. turcica. Genome sequence comparison revealed that the CC-NB-LRR encoding St genes are located on chromosome 2 in maize, and on chromosome 5 in sorghum. The six St sorghum genes reside in three pairs in one locus. When the sorghum St genes were silenced via VIGS, the resistance was clearly compromised, an observation that was supported by real-time PCR. Database searches and phylogenetic analysis suggest that the St genes have a common ancestor present before the grass subfamily split 50-70 million years ago. Today, 6 genes are present in sorghum, 9 in rice and foxtail millet, respectively, 3 in maize and 4 in Brachypodium distachyon. The St gene homologs have all highly conserved sequences, and commonly reside as gene pairs in the grass genomes. Conclusions Resistance genes to S. turcica, with a CC-NB-LRR protein domain architecture, have been found in maize and sorghum. VIGS analysis revealed their importance in the surveillance to S. turcica in sorghum. The St genes are highly conserved in sorghum, rice, foxtail millet, maize and Brachypodium, suggesting an

  19. Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin.

    PubMed

    Jia, Sujuan; Omelchenko, Marina; Garland, Donita; Vasiliou, Vasilis; Kanungo, Jyotshnabala; Spencer, Michael; Wolf, Yuri; Koonin, Eugene; Piatigorsky, Joram

    2007-10-01

    We have previously identified a gelsolin-like protein (C/L-gelsolin) as a corneal crystallin in zebrafish. Here we show by phylogenetic analysis that there are at least six genes encoding gelsolin-like proteins based on their gelsolin domains in zebrafish: gsna and gsnb group with the vertebrate gelsolin gene, scina and scinb group with the scinderin (adseverin) gene, and scinla (C/L-gelsolin) and scinlb are novel scinderin-like genes. RT-PCR showed that scinla, scinlb, and gsnb are preferentially expressed in the adult cornea whereas gsna is expressed to a similar extent in cornea, lens, brain, and heart; scina and scinb expression were detectable only in whole zebrafish and not in these adult tissues. Quantitative RT-PCR and 2-dimensional polyacrylamide gel electrophoresis followed by MALDI/TOF mass spectroscopy confirmed high expression of beta-actin and scinla, moderate expression of scinlb, and very low expression of gsna and gsnb in the cornea. Finally, transgenic zebrafish carrying a green fluorescent protein reporter transgene driven by a 4 kb scinla promoter fragment showed expression in the cornea, snout, dorsal fin, and tail fin of 3-day-old zebrafish larvae. Our data suggest that scinla and scinlb are diverged paralogs of the vertebrate scinderin gene and show that scinla encodes the zebrafish corneal crystallin previously called C/L-gelsolin.

  20. Hypoxia: adapting to high altitude by mutating EPAS-1, the gene encoding HIF-2α.

    PubMed

    van Patot, Martha C Tissot; Gassmann, Max

    2011-01-01

    Living at high altitude is demanding and thus drives adaptational mechanisms. The Tibetan population has had a longer evolutionary period to adapt to high altitude than other mountain populations such as Andeans. As a result, some Tibetans living at high altitudes do not show markedly elevated red blood cell production as compared to South American high altitude natives such as Quechuas or Aymaras, thereby avoiding high blood viscosity creating cardiovascular risk. Unexpectedly, the responsible mutation(s) reducing red blood cell production do not involve either the gene encoding the blood hormone erythropoietin (Epo), or the corresponding regulatory sequences flanking the Epo gene. Similarly, functional mutations in the hypoxia-inducible transcription factor 1α (HIF-1α) gene that represents the oxygen-dependent subunit of the HIF-1 heterodimer, the latter being the main regulator of over 100 hypoxia-inducible genes, have not been described so far. It was not until very recently that three independent groups showed that the gene encoding HIF-2α, EPAS-1 (Wenger et al. 1997), represents a key gene mutated in Tibetan populations adapted to living at high altitudes (Beall et al. 2010 , Yi et al. 2010 , Simonson et al. 2010). Hypoxia-inducible transcription factors were first identified by the description of HIF-1 (Semenza et al. 1991 , 1992), which was subsequently found to enhance transcription of multiple genes that encode proteins necessary for rescuing from hypoxic exposure, including erythropoietic, angiogenic and glycolytic proteins. Then HIF-2 was identified (Ema et al. 1997 ; Flamme et al. 1997 ; Hogenesch et al. 1997 ; and Tian et al. 1997) and although it is highly similar to HIF-1 and has the potential to bind (Camenisch et al. 2001) and mediate (Mole et al. 2009) many of the same genes as HIF-1, its biological actions in response to hypoxia are distinct from those of HIF-1 (reviewed by Loboda et al. 2010). By now, several of these HIF-2 mediated

  1. Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

    PubMed

    Cabello-Díaz, Juan Miguel; Gálvez-Valdivieso, Gregorio; Caballo, Cristina; Lambert, Rocío; Quiles, Francisco Antonio; Pineda, Manuel; Piedras, Pedro

    2015-08-01

    Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  2. Characterization of the gene encoding the human LW blood group protein in LW+ and LW- phenotypes.

    PubMed

    Hermand, P; Le Pennec, P Y; Rouger, P; Cartron, J P; Bailly, P

    1996-04-01

    The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig-like domain are encoded by the first exon. The 5'UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5'UT region, has been detected by sequencing polymerase chain reaction-amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes.

  3. A corm-specific gene encodes tarin, a major globulin of taro (Colocasia esculenta L. Schott).

    PubMed

    Bezerra, I C; Castro, L A; Neshich, G; de Almeida, E R; de Sá, M F; Mello, L V; Monte-Neshich, D C

    1995-04-01

    A gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (G1, ca. 28 kDa) was isolated from a lambda Charon 35 library, using a cDNA derived from a highly abundant corm-specific mRNA, as probe. The gene, named tar1, and the corresponding cDNA were characterized and compared. No introns were found. The major transcription start site was determined by primer extension analysis. The gene has an open reading frame (ORF) of 765 bp, and the deduced amino acid sequence indicated a precursor polypeptide of 255 residues that is post-translationally processed into two subunits of about 12.5 kDa each. The deduced protein is 45% homologous to curculin, a sweet-tasting protein found in the fruit pulp of Curculigo latifolia and 40% homologous to a mannose-binding lectin from Galanthus nivalis. Significant similarity was also found at the nucleic acid sequence level with genes encoding lectins from plant species of the Amaryllidaceae and Lilliaceae families.

  4. Identification of the gene encoding scHelI, a DNA helicase from Saccharomyces cerevisiae.

    PubMed

    Bean, D W; Matson, S W

    1997-12-01

    The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a lambda gt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable for mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability.

  5. An Arabidopsis gene encoding an α-xylosyltransferase involved in xyloglucan biosynthesis

    PubMed Central

    Faik, Ahmed; Price, Nicholas J.; Raikhel, Natasha V.; Keegstra, Kenneth

    2002-01-01

    Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of α-xylosyltransferase and β-glucan synthase activities. Here we report that etiolated pea microsomes contain an α-xylosyltransferase that catalyzes the transfer of xylose from UDP-[14C]xylose onto β(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode α-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the 14C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an α(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an α-xylosyltransferase activity involved in xyloglucan biosynthesis. PMID:12032363

  6. Transcriptional analysis of genes encoding β-glucosidase of Schizophyllum commune KUC9397 under optimal conditions.

    PubMed

    Lee, Young Min; Lee, Hanbyul; Heo, Young Mok; Lee, Hwanhwi; Hong, Joo-Hyun; Kim, Jae-Jin

    2017-05-01

    The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

  7. K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase.

    PubMed Central

    Katoh, M; Hattori, Y; Sasaki, H; Tanaka, M; Sugano, K; Yazaki, Y; Sugimura, T; Terada, M

    1992-01-01

    K-sam was first identified as a gene amplified in the stomach cancer cell line KATO-III. The size of the major transcript of the K-sam gene was 3.5 kilobases in KATO-III cells, and we have previously shown that K-sam encodes a receptor tyrosine kinase that belongs to the heparin-binding growth factor receptor, or fibroblast growth factor receptor, gene family. The K-sam gene expresses multiple sizes of mRNAs in brain tissue, the immature teratoma cell line NCC-IT, and KATO-III. RNA blot analyses with a variety of K-sam probes indicate that there are at least four classes of K-sam mRNAs. Three types of K-sam cDNAs in addition to the previously reported type of K-sam cDNA were isolated, and their nucleotide sequences encode a full-length transmembrane receptor, a secreted receptor with a tyrosine kinase domain, and a secreted receptor without a tyrosine kinase domain. Images PMID:1313574

  8. Identification of Cellular Genes Targeted by KSHV-Encoded MicroRNAs

    PubMed Central

    Samols, Mark A; Skalsky, Rebecca L; Maldonado, Ann M; Riva, Alberto; Lopez, M. Cecilia; Baker, Henry V; Renne, Rolf

    2007-01-01

    MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis. PMID:17500590

  9. [Cloning and structure of gene encoded alpha-latrocrustoxin from the Black widow spider venom].

    PubMed

    Danilevich, V N; Luk'ianov, S A; Grishin, E V

    1999-07-01

    The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).

  10. A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

    PubMed

    Hayashi, Y; Yamagishi, M; Nishimoto, Y; Taguchi, O; Matsukage, A; Yamaguchi, M

    1999-12-03

    The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

  11. A horizontally transferred nuclear gene is associated with microhabitat variation in a natural plant population

    PubMed Central

    Tunlid, Anders; Ghatnekar, Lena

    2015-01-01

    Horizontal gene transfer involves the non-sexual interspecific transmission of genetic material. Even if they are initially functional, horizontally transferred genes are expected to deteriorate into non-expressed pseudogenes, unless they become adaptively relevant in the recipient organism. However, little is known about the distributions of natural transgenes within wild species or the adaptive significance of natural transgenes within wild populations. Here, we examine the distribution of a natural plant-to-plant nuclear transgene in relation to environmental variation within a wild population. Festuca ovina is polymorphic for an extra (second) expressed copy of the nuclear gene (PgiC) encoding cytosolic phosphoglucose isomerase, with the extra PgiC locus having been acquired horizontally from the distantly related grass genus Poa. We investigated variation at PgiC in samples of F. ovina from a fine-scale, repeating patchwork of grassland microhabitats, replicated within spatially separated sites. Even after accounting for spatial effects, the distributions of F. ovina individuals carrying the additional PgiC locus, and one of the enzyme products encoded by the locus, are significantly associated with fine-scale habitat variation. Our results suggest that the PgiC transgene contributes, together with the unlinked ‘native’ PgiC locus, to local adaptation to a fine-scale mosaic of edaphic and biotic grassland microhabitats. PMID:26674953

  12. A horizontally transferred nuclear gene is associated with microhabitat variation in a natural plant population.

    PubMed

    Prentice, Honor C; Li, Yuan; Lönn, Mikael; Tunlid, Anders; Ghatnekar, Lena

    2015-12-22

    Horizontal gene transfer involves the non-sexual interspecific transmission of genetic material. Even if they are initially functional, horizontally transferred genes are expected to deteriorate into non-expressed pseudogenes, unless they become adaptively relevant in the recipient organism. However, little is known about the distributions of natural transgenes within wild species or the adaptive significance of natural transgenes within wild populations. Here, we examine the distribution of a natural plant-to-plant nuclear transgene in relation to environmental variation within a wild population. Festuca ovina is polymorphic for an extra (second) expressed copy of the nuclear gene (PgiC) encoding cytosolic phosphoglucose isomerase, with the extra PgiC locus having been acquired horizontally from the distantly related grass genus Poa. We investigated variation at PgiC in samples of F. ovina from a fine-scale, repeating patchwork of grassland microhabitats, replicated within spatially separated sites. Even after accounting for spatial effects, the distributions of F. ovina individuals carrying the additional PgiC locus, and one of the enzyme products encoded by the locus, are significantly associated with fine-scale habitat variation. Our results suggest that the PgiC transgene contributes, together with the unlinked 'native' PgiC locus, to local adaptation to a fine-scale mosaic of edaphic and biotic grassland microhabitats.

  13. Two histone H1-encoding genes of the green alga Volvox carteri with features intermediate between plant and animal genes.

    PubMed

    Lindauer, A; Müller, K; Schmitt, R

    1993-07-15

    Southern hybridization indicated the presence of at least two and possibly four histone H1-encoding genes occurring as singlets in the Volvox carteri genome. Two of these genes, H1-I and H1-II, have been cloned and characterized. Their coding sequences are each interrupted by three introns, but only the position of the second intron is identically conserved in both H1-I and H1-II. The encoded 260-amino-acid (aa) (H1-I) and 240-aa (H1-II) polypeptides possess the typical tripartite organization of animal H1 histones, with variable N- and C-terminal domains flanking a conserved 'globular' DNA-binding domain. Extensive differences in their variable regions suggest that H1-I and H1-II (62% identity) represent two isotypes with different functions. A prominent KAPKAP-KAA motif in the H1-I N-terminal region, similarly seen in single H1 variants of a mosquito and a nematode, has a putative function in packing condensed subtypes of chromatin. Different from higher plants, but like animals, the H1 genes of V. carteri possess a typical 3' palindrome for mRNA processing, resulting in non-polyadenylated mRNAs. Transcription initiates 33 nucleotides (nt) (H1-I) and 26 nt (H1-II) downstream of typical TATA boxes. A putative 20-bp conserved enhancer element upstream of each TATA box closely resembles the consensus sequence associated with the nucleosomal histone-encoding genes in V. carteri [Müller et al., Gene 93 (1990) 167-175] and suggests stringent regulation. Accordingly, transcription of H1 was shown to be restricted to late embryogenesis, when new flagella are produced. We discuss the inferred accessory role of histone H1 proteins in stabilizing axonemal microtubules, as has been recently observed in sea urchin flagella [Multigner et al., Nature 360 (1992) 33-39].

  14. Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

    PubMed Central

    Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

    2012-01-01

    The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

  15. The twisted Gene Encodes Drosophila Protein O-Mannosyltransferase 2 and Genetically Interacts With the rotated abdomen Gene Encoding Drosophila Protein O-Mannosyltransferase 1

    PubMed Central

    Lyalin, Dmitry; Koles, Kate; Roosendaal, Sigrid D.; Repnikova, Elena; Van Wechel, Laura; Panin, Vladislav M.

    2006-01-01

    The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw1, corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8–17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using

  16. Nuclear gyrB encodes a functional subunit of the Plasmodium falciparum gyrase that is involved in apicoplast DNA replication.

    PubMed

    Raghu Ram, E V S; Kumar, Ambrish; Biswas, Subir; Kumar, Ashutosh; Chaubey, Sushma; Siddiqi, Mohammad Imran; Habib, Saman

    2007-07-01

    The DNA replication machinery of the Plasmodium falciparum apicoplast is a validated drug target. Nuclear-encoded gyrase subunits are predicted to play a critical role in maintaining DNA topology during the D-loop/bi-directional ori replication process of the parasite. We show the presence of P. falciparum gyrase subunits in parasite lysates by using antibodies generated against recombinant gyrase A and B. The ATPase activity of PfGyrB was inhibited by novobiocin that also caused parasite death in culture. Reduction of apicoplast/nuclear DNA ratio in the presence of novobiocin indicated that the drug targets apicoplast DNA replication. Molecular modeling of gyrase A and B subunits revealed extensive fold conservation with the Escherichia coli counterparts as well as the presence of a long disordered loop adjacent to the ATPase domain of PfGyrB. Our results have implications for development of PfGyrB as a drug target against malaria.

  17. A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

    PubMed

    Díaz-Magaña, Amada; Alva-Murillo, Nayeli; Chávez-Moctezuma, Martha P; López-Meza, Joel E; Ramírez-Díaz, Martha I; Cervantes, Carlos

    2015-07-01

    The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA-  mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA-  mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA-  mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

  18. Development and Application of Real-Time PCR Assays for Quantification of Genes Encoding Tetracycline Resistance

    PubMed Central

    Yu, Zhongtang; Michel, Frederick C.; Hansen, Glenn; Wittum, Thomas; Morrison, Mark

    2005-01-01

    We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). The assays were validated using known numbers of sample-derived tet gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log tet gene copies. New tet gene variants were also identified from cloned tet amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three tet gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of tet genes than the swine manures. The composts of swine manures had substantially reduced tet gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on tet gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of tet genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of tet genes, which should be useful for ecological studies of antibiotic resistance. PMID:16269727

  19. Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus.

    PubMed Central

    Bischoff, D S; Slavicek, J M

    1997-01-01

    A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) gene has been identified that encodes a homolog to the granulovirus (GV) enhancin proteins that are capable of enhancing the infection of other baculoviruses. Enhancin genes have been identified and sequenced for three species of GVs but have not been found in any other nuclear polyhedrosis virus to date. The LdMNPV enhancin gene is located between 67.6 and 70.1 kbp on the viral genome. Northern and primer extension analyses of viral RNAs indicate that the enhancin gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The LdMNPV enhancin exhibits 29% amino acid identity to the enhancin proteins of the Trichoplusia ni, Pseudaletia unipuncta, and Helicoverpa armigera GVs. All four proteins contain a conserved zinc-binding domain characteristic of metalloproteases. A recombinant virus (enhancin::cat) was constructed in which the LdMNPV enhancin gene was inactivated by insertion mutagenesis in order to ascertain the effect of the enhancin protein on viral potency. The bioassay results indicate that disruption of the enhancin gene in the LdMNPV results in a reduction in viral potency. PMID:9343163

  20. Distribution of Genes Encoding Resistance to Macrolides, Lincosamides, and Streptogramins among Staphylococci

    PubMed Central

    Lina, Gerard; Quaglia, Alain; Reverdy, Marie-Elisabeth; Leclercq, Roland; Vandenesch, François; Etienne, Jerome

    1999-01-01

    The relative frequency of 10 determinants of resistance to macrolides, lincosamides, and streptogramins was investigated by PCR in a series of 294 macrolide-, lincosamide-, and/or streptogramin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci isolated in 1995 from 32 French hospitals. Resistance was mainly due to the presence of ermA or ermC genes, which were detected in 259 strains (88%), in particular those resistant to methicillin (78% of the strains). Macrolide resistance due to msrA was more prevalent in coagulase-negative staphylococci (14.6%) than in S. aureus (2.1%). Genes related to linA/linA′ and conferring resistance to lincomycin were detected in one strain of S. aureus and seven strains of coagulase-negative staphylococci. Resistance to pristinamycin and quinupristin-dalfopristin was phenotypically detected in 10 strains of S. aureus and in three strains of coagulase-negative staphylococci; it was always associated with resistance to type A streptogramins encoded by vat or vatB genes and occurred in association with erm genes. The vga gene conferring decreased susceptibility to type A streptogramins was present alone in three strains of coagulase-negative staphylococci and in combination with erm genes in 10 strains of coagulase-negative staphylococci. A combination of vga-vgb-vat and ermA genes was found in a single strain of S. epidermidis. PMID:10223914

  1. Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins.

    PubMed Central

    Goldman, M H; Pezzotti, M; Seurinck, J; Mariani, C

    1992-01-01

    We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed. PMID:1392607

  2. Phylogenetic and evolutionary analysis of NBS-encoding genes in Rosaceae fruit crops.

    PubMed

    Xu, Qiang; Wen, Xiaopeng; Deng, Xiuxin

    2007-07-01

    Phylogenetic relationships of the nucleotide binding site (NBS)-encoding resistance gene homologues (RGHs) among 12 species in five genera of Rosaceae fruit crops were evaluated. A total of 228 Rosaceous RGHs were deeply separated into two distinct clades, designated as TIR (sequences within this clade containing a Toll Interleukin-1 Receptor domain) and NonTIR (sequences lacking a TIR domain). Most Rosaceous RGH genes were phylogenetically distinct from Arabidopsis, Rice or Pine genes, except for a few Rosaceous members which grouped closely with Arabidopsis genes. Within Rosaceae, sequences from multiple species were often phylogenetically clustered together, forming heterogenous groups, however, apple- and chestnut rose-specific groups really exist. Gene duplication followed by sequence divergence were proposed as the mode for the evolution of a large number of distantly or closely related RGH genes in Rosaceae, and this mode may play a role in the generation of new resistance specificity. Positively selected sites within NBS-coding region were detected and thus nucleotide variation within NBS domain may function in determining disease resistance specificity. This study also discusses the synteny of a genomic region that encompass powdery mildew resistance locus among Malus, Prunus and Rosa, which may have potential use for fruit tree disease breeding and important gene cloning.

  3. Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

    PubMed

    Merico, A; Rodrigues, F; Côrte-Real, M; Porro, D; Ranzi, B M; Compagno, C

    2001-06-30

    The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

  4. The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase.

    PubMed

    Li, Yanhong; Sun, Mingchi; Huang, Shengshu; Yu, Hai; Chokhawala, Harshal A; Thon, Vireak; Chen, Xi

    2007-09-21

    Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.

  5. Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers.

    PubMed

    Li, X A; Kokame, K; Okubo, K; Shimokado, K; Tsukamoto, Y; Miyata, T; Kato, H; Yutani, C

    1999-12-23

    This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.

  6. Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms.

    PubMed

    Emadali, Anouk; Hoghoughi, Neda; Duley, Samuel; Hajmirza, Azadeh; Verhoeyen, Els; Cosset, Francois-Loic; Bertrand, Philippe; Roumier, Christophe; Roggy, Anne; Suchaud-Martin, Céline; Chauvet, Martine; Bertrand, Sarah; Hamaidia, Sieme; Rousseaux, Sophie; Josserand, Véronique; Charles, Julie; Templier, Isabelle; Maeda, Takahiro; Bruder-Costa, Juliana; Chaperot, Laurence; Plumas, Joel; Jacob, Marie-Christine; Bonnefoix, Thierry; Park, Sophie; Gressin, Remy; Tensen, Cornelis P; Mecucci, Cristina; Macintyre, Elizabeth; Leroux, Dominique; Brambilla, Elisabeth; Nguyen-Khac, Florence; Luquet, Isabelle; Penther, Dominique; Bastard, Christian; Jardin, Fabrice; Lefebvre, Christine; Garnache, Francine; Callanan, Mary B

    2016-06-16

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN.

  7. Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms

    PubMed Central

    Emadali, Anouk; Hoghoughi, Neda; Duley, Samuel; Hajmirza, Azadeh; Verhoeyen, Els; Cosset, Francois-Loic; Bertrand, Philippe; Roumier, Christophe; Roggy, Anne; Suchaud-Martin, Céline; Chauvet, Martine; Bertrand, Sarah; Hamaidia, Sieme; Rousseaux, Sophie; Josserand, Véronique; Charles, Julie; Templier, Isabelle; Maeda, Takahiro; Bruder-Costa, Juliana; Chaperot, Laurence; Plumas, Joel; Jacob, Marie-Christine; Bonnefoix, Thierry; Park, Sophie; Gressin, Remy; Tensen, Cornelis P.; Mecucci, Cristina; Macintyre, Elizabeth; Leroux, Dominique; Brambilla, Elisabeth; Nguyen-Khac, Florence; Luquet, Isabelle; Penther, Dominique; Bastard, Christian; Jardin, Fabrice; Lefebvre, Christine; Garnache, Francine

    2016-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN. PMID:27060168

  8. Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

    PubMed

    Lahti, R; Pitkäranta, T; Valve, E; Ilta, I; Kukko-Kalske, E; Heinonen, J

    1988-12-01

    Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.

  9. Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease

    NASA Astrophysics Data System (ADS)

    Mo, Zhaolan; Guo, Dongsheng; Mao, Yunxiang; Ye, Xuhong; Zou, Yuxia; Xiao, Peng; Hao, Bin

    2010-01-01

    We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain. This prtV gene encodes a putative protein of 918 amino acids, and is highly homologous to the V. cholerae prtV gene. We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar, lower protease activity against azocasein, and lower activity for four glycosidases. This prtV mutant strain also had increased activity for two esterases in its extracellular products, as analyzed by the API ZYM system. In addition, the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes. Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish. We propose that prtV plays an important role in the pathogenesis of V. anguillarum.

  10. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    PubMed Central

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution. Images PMID:2895100

  11. Molecular characterization of the gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae.

    PubMed

    Balas, D; Fernández-Moreira, E; De La Campa, A G

    1998-06-01

    The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced. The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of gyrA. The bend center was mapped and located between the -35 and -10 regions of the promoter. Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended -10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed.

  12. Molecular Characterization of the Gene Encoding the DNA Gyrase A Subunit of Streptococcus pneumoniae

    PubMed Central

    Balas, Delia; Fernández-Moreira, Esteban; De La Campa, Adela G.

    1998-01-01

    The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced. The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of gyrA. The bend center was mapped and located between the −35 and −10 regions of the promoter. Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended −10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed. PMID:9603872

  13. The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

    PubMed Central

    Rexach, Jesus; Fernández, Emilio; Galván, Aurora

    2000-01-01

    A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO2 conditions. PMID:10948261

  14. Genetic variability in the sable (Martes zibellina L.) with respect to genes encoding blood proteins

    SciTech Connect

    Kashtanov, S.N.; Kazakova, T.I.

    1995-02-01

    Electrophoresis of blood proteins was used to determine, for the first time, the level of genetic variability of certain loci in the sable (Martes zibellina L., Mustelidae). Variation of 23 blood proteins encoded by 25 genes was analyzed. Polymorphism was revealed in six genes. The level of heterozygosity was estimated at 0.069; the proportion of polymorphic loci was 24%. Data on the history of the sable population maintained at the farm, on geographical distribution of natural sable populations, and on the number of animals selected for reproduction in captivity is presented. The great number of animals studies and the extensive range of natural sable populations, on the basis of which the population maintained in captivity was obtained, suggest that the results of this work can be used for estimating the variability of the gene pool of sable as a species. 9 refs., 2 figs., 1 tab.

  15. Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

    PubMed

    Villamizar, Genis A Castillo; Nacke, Heiko; Daniel, Rolf

    2017-01-01

    The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

  16. Identification and Characterization of Multi-gene Family Encoding Germin-like Proteins in Cultivated Peanut (Arachis hypogaea L.)

    USDA-ARS?s Scientific Manuscript database

    Germins and germin-like proteins (GLPs) play diversified roles in plant development and basic defense. In this study, 36 EST-clones encoding GLPs were identified. Sequence similarity analysis demonstrated that the peanut genome possessed multi-gene family encoding at least 8 GLPs, named AhGLP1 to Ah...

  17. Cloning of the genes encoding two murine and human cochlear unconventional type I myosins

    SciTech Connect

    Crozet, F.; El Amraoui, Z.; Blanchard, S.

    1997-03-01

    Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I{beta} and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) an SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that the genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p13 and 19p13.2- 19p1.3.3, respectively. 45 refs., 5 figs., 2 tabs.

  18. Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

    PubMed Central

    Shi, Kevin Y.; Mori, Eiichiro; Nizami, Zehra F.; Lin, Yi; Kato, Masato; Xiang, Siheng; Wu, Leeju C.; Ding, Ming; Yu, Yonghao; Gall, Joseph G.; McKnight, Steven L.

    2017-01-01

    The toxic proline:arginine (PRn) poly-dipeptide encoded by the (GGGGCC)n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PRn poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PRn poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PRn-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PRn poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PRn poly-dipeptide toxicity in the context of a prominent form of ALS. PMID:28069952

  19. Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

    PubMed Central

    Zuberi, A R; Bischoff, D S; Ordal, G W

    1991-01-01

    The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes. Images PMID:1898932

  20. PCR detection of enzyme-encoding genes in Leuconostoc mesenteroides strains of wine origin.

    PubMed

    Mtshali, Phillip Senzo; Divol, Benoit; du Toit, Maret

    2012-04-01

    Fifteen isolates of lactic acid bacteria originating from South African grape and wine samples were identified as Leuconostoc mesenteroides subsp. mesenteroides through the taxonomic analysis of their 16S rDNA gene sequences. These isolates were further tested for the presence of genes coding for enzymes of oenological relevance using PCR detection technique. A type strain of Leuc. mesenteroides (NCDO 529(T)) was also incorporated for comparative analysis. From the PCR detection results, the estA, prtP, alsD, alsS, metK, metC and metB genes were present in all the strains tested. The bgl and gshR genes encoding β-glucosidase and glutathione reductase, respectively, were not detected in some strains. On the other hand, none of the tested strains possessed the genes encoding phenolic acid decarboxylase (padA), citrate permease (citP), citrate lyase (citD, citE and citF) and arginine deiminase pathway enzymes (arcA, arcB and arcC). The verification of PCR-generated fragments was performed by sequencing. GenBank database was used to search for homologous DNA sequences. Neighbour-joining trees based on nucleotide sequences of alsS, estA, metK and mleA genes were also constructed in order to study the phylogenetic relationship between Leuc. mesenteroides strains and closely related species. The phylogenetic analyses revealed that there are genetic heterogeneities between strains of Leuc. mesenteroides species. In conclusion, this study has improved our knowledge on the genetics of oenological strains of Leuc. mesenteroides and their genetic potential to contribute to certain wine aroma compounds.

  1. Nuclear pore complexes and regulation of gene expression.

    PubMed

    Raices, Marcela; D'Angelo, Maximiliano A

    2017-01-11

    Nuclear pore complexes (NPCs), are large multiprotein channels that penetrate the nuclear envelope connecting the nucleus to the cytoplasm. Accumulating evidence shows that besides their main role in regulating the exchange of molecules between these two compartments, NPCs and their components also play important transport-independent roles, including gene expression regulation, chromatin organization, DNA repair, RNA processing and quality control, and cell cycle control. Here, we will describe the recent findings about the role of these structures in the regulation of gene expression.

  2. A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max

    USDA-ARS?s Scientific Manuscript database

    Background Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. CodA is a conditionally lethal dominant gene encoding cy...

  3. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    PubMed Central

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  4. The role of the nuclear receptor CAR as a coordinate regulator of hepatic gene expression in defense against chemical toxicity.

    PubMed

    Yamamoto, Yukio; Kawamoto, Takeshi; Negishi, Masahiko

    2003-01-01

    The nuclear receptor CAR (constitutive active receptor) mediates the induction of transcription of cytochrome P450 (CYP) genes by phenobarbital (PB) and PB-type inducers. A recent study using CAR-null mice has shown that CAR regulates not only the CYP genes but also other genes encoding various drug/steroid-metabolizing enzymes. In addition to coordinating these enzymes, CAR plays other roles in hepatic gene expression: CAR represses various genes including carnitine palmitoyltransferase 1a and phosphoenolpyruvate carboxykinase 1 in response to PB, and the receptor regulates the constitutive expression of genes such as squalene epoxidase. On the other hand, induction of certain genes such as amino levulinate synthase 1 by PB is not regulated by CAR. Here we describe diverse roles of CAR in hepatic gene expression with a particular focus on endogenous substances such as cholesterol, bilirubin, and steroid hormones.

  5. Characterization of a murine gene encoding a developmentally regulated cytoplasmic dual-specificity mitogen-activated protein kinase phosphatase.

    PubMed Central

    Dickinson, Robin J; Williams, David J; Slack, David N; Williamson, Jill; Seternes, Ole-Morten; Keyse, Stephen M

    2002-01-01

    Mitogen-activated protein kinases (MAPKs) play a vital role in cellular growth control, but far less is known about these signalling pathways in the context of embryonic development. Duration and magnitude of MAPK activation are crucial factors in cell fate decisions, and reflect a balance between the activities of upstream activators and specific MAPK phosphatases (MKPs). Here, we report the isolation and characterization of the murine Pyst3 gene, which encodes a cytosolic dual-specificity MKP. This enzyme selectively interacts with, and is catalytically activated by, the 'classical' extracellular signal-regulated kinases (ERKs) 1 and 2 and, to a lesser extent, the stress-activated MAPK p38alpha. These properties define the ability of this enzyme to dephosphorylate and inactivate ERK1/2 and p38alpha, but not JNK (c-Jun N-terminal kinase) in vivo. When expressed in mammalian cells, the Pyst3 protein is predominantly cytoplasmic. Furthermore, leptomycin B causes a complete redistribution of the protein to the nucleus, implicating a CRM (chromosomal region maintenance)1/exportin 1-dependent nuclear export signal in determining the subcellular localization of this enzyme. Finally, whole-mount in situ hybridization studies in mouse embryos reveal that the Pyst3 gene is expressed specifically in the placenta, developing liver and in migratory muscle cells. Our results suggest that this enzyme may have a critical role in regulating the activity of MAPK signalling during early development and organogenesis. PMID:11988087

  6. A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris.

    PubMed

    Park, Chang-Jin; Shin, Yun-Chul; Lee, Boo-Ja; Kim, Ki-Jeong; Kim, Jeong-Kook; Paek, Kyung-Hee

    2006-01-01

    Plant WRKY transcription factors were previously implicated in the alteration of gene expression in response to various pathogens. The WRKY proteins constitute a large family of plant transcription factors, whose precise functions have yet to be elucidated. Using a domain-specific differential display procedure, we isolated a WRKY gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) or Xanthomonas campestris pv . vesicatoria (Xcv). The full-length cDNA of CaWRKY-a (Capsicum annuum WRKY-a) encodes a putative polypeptide of 546 amino acids, containing two WRKY domains with a zinc finger motif. The expression of CaWRKY-a could be rapidly induced by not only chemical elicitor such as salicylic acid (SA) or ethephon but also wounding treatments. The nuclear localization of CaWRKY-a was determined in transient expression system using tobacco BY-2 cells by polyethylene glycol (PEG)-mediated transformation experiment. With oligonucleotide molecules containing the putative W-box sequences as a probe, we confirmed that CaWRKY-a protein had W-box-binding activity. These results suggest that CaWRKY-a might be involved as a transcription factor in plant defense-related signal transduction pathways.

  7. Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

    PubMed

    Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

    2015-12-04

    Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

  8. Cloning of the rice seed alpha-globulin-encoding gene: sequence similarity of the 5'-flanking region to those of the genes encoding wheat high-molecular-weight glutenin and barley D hordein.

    PubMed

    Nakase, M; Hotta, H; Adachi, T; Aoki, N; Nakamura, R; Masumura, T; Tanaka, K; Matsuda, T

    1996-05-08

    A genomic clone encoding the rice endosperm major globulin (alpha-globulin) with an apparent molecular mass of 26 kDa was isolated, and its nucleotide (nt) sequence and transcription start point (tsp) were determined. The tsp was identical to that of the gene encoding the wheat high-molecular-weight (HMW) glutenin subunit. The consensus '-300 element' and an A + T-rich sequence exist upstream from the TATA box in the 5'-flanking region. A nt sequence of about 130 bp in the 5'-flanking region was found to be markedly homologous to those of the genes encoding the wheat HMW glutenin subunit and barley D hordein.

  9. Full-genome identification and characterization of NBS-encoding disease resistance genes in wheat.

    PubMed

    Bouktila, Dhia; Khalfallah, Yosra; Habachi-Houimli, Yosra; Mezghani-Khemakhem, Maha; Makni, Mohamed; Makni, Hanem

    2015-02-01

    Host resistance is the most economical, effective and ecologically sustainable method of controlling diseases in crop plants. In bread wheat, despite the high number of resistance loci that have been cataloged to date, only few have been cloned, underlying the need for genomics-guided investigations capable of providing a prompt and acute knowledge on the identity of effective resistance genes that can be used in breeding programs. Proteins with a nucleotide-binding site (NBS) encoded by the major plant disease resistance (R) genes play an important role in the responses of plants to various pathogens. In this study, a comprehensive analysis of NBS-encoding genes within the whole wheat genome was performed, and the genome scale characterization of this gene family was established. From the recently published wheat genome sequence, we used a data mining and automatic prediction pipeline to identify 580 complete ORF candidate NBS-encoding genes and 1,099 partial-ORF ones. Among complete gene models, 464 were longer than 200 aa, among them 436 had less than 70 % of sequence identity to each other. This gene models set was deeply characterized. (1) First, we have analyzed domain architecture and identified, in addition to typical domain combinations, the presence of particular domains like signal peptides, zinc fingers, kinases, heavy-metal-associated and WRKY DNA-binding domains. (2) Functional and expression annotation via homology searches in protein and transcript databases, based on sufficient criteria, enabled identifying similar proteins for 60 % of the studied gene models and expression evidence for 13 % of them. (3) Shared orthologous groups were defined using NBS-domain proteins of rice and Brachypodium distachyon. (4) Finally, alignment of the 436 NBS-containing gene models to the full set of scaffolds from the IWGSC's wheat chromosome survey sequence enabled high-stringence anchoring to chromosome arms. The distribution of the R genes was found balanced

  10. The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene.

    PubMed

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S; King, Glenn F

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.

  11. Role of sequence encoded κB DNA geometry in gene regulation by Dorsal

    PubMed Central

    Mrinal, Nirotpal; Tomar, Archana; Nagaraju, Javaregowda

    2011-01-01

    Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network. PMID:21890896

  12. The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression

    SciTech Connect

    Lindner, I.; Ehlers, B. . E-mail: ehlersb@rki.de; Noack, S.; Dural, G.; Yasmum, N.; Bauer, C.; Goltz, M.

    2007-01-20

    The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1{sub h}, ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1{sub h} and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

  13. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1989-04-01

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has the same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.

  14. The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene

    PubMed Central

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S.; King, Glenn F.

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome. PMID:22928020

  15. Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

    PubMed Central

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

    2016-01-01

    Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

  16. Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae.

    PubMed

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko; Yaoi, Katsuro

    2016-03-04

    Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-D-xylopyranose-(1 → 6)-D-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase encoding gene in Gracilaria/Gracilariopsis lemaneiformis

    NASA Astrophysics Data System (ADS)

    Xueying, Ren; Zhenghong, Sui; Xuecheng, Zhang

    2006-04-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene ( gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  18. A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms1[OPEN

    PubMed Central

    Schipper, Bert; Beekwilder, Jules

    2016-01-01

    A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments. PMID:27707890

  19. Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase.

    PubMed

    Tan, Fui-Ching; Cheng, Qi; Saha, Kaushik; Heinemann, Ilka U; Jahn, Martina; Jahn, Dieter; Smith, Alison G

    2008-03-01

    UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

  20. Core-Gene-Encoded Peptide Regulating Virulence-Associated Traits in Streptococcus mutans

    PubMed Central

    Kim, Jeong Nam; Stanhope, Michael J.

    2013-01-01

    Recently, high-coverage genome sequence of 57 isolates of Streptococcus mutans, the primary etiological agent of human dental caries, was completed. The SMU.1147 gene, encoding a 61-amino-acid (61-aa) peptide, was present in all sequenced strains of S. mutans but absent in all bacteria in current databases. Reverse transcription-PCR revealed that SMU.1147 is cotranscribed with scnK and scnR, which encode the histidine kinase and response regulator, respectively, of a two-component system (TCS). The C terminus of the SMU.1147 gene product was tagged with a FLAG epitope and shown to be expressed in S. mutans by Western blotting with an anti-FLAG antibody. A nonpolar mutant of SMU.1147 formed less biofilm in glucose-containing medium and grew slower than did the wild-type strain under aerobic and anaerobic conditions, at low pH, or in the presence of H2O2. Mutation of SMU.1147 dramatically reduced genetic competence and expression of comX and comY, compared to S. mutans UA159. The competence defect of the SMU.1147 mutant could not be overcome by addition of sigX-inducing peptide (XIP) in defined medium or by competence-stimulating peptide (CSP) in complex medium. Complementation with SMU.1147 on a plasmid restored all phenotypes. Interestingly, mutants lacking either one of the TCS components and a mutant lacking all three genes behaved like the wild-type strain for all phenotypes mentioned above, but all mutant strains grew slower than UA159 in medium supplemented with 0.3 M NaCl. Thus, the SMU.1147-encoded peptide affects virulence-related traits and dominantly controls quorum-sensing pathways for development of genetic competence in S. mutans. PMID:23603743

  1. Diversity of Beetle Genes Encoding Novel Plant Cell Wall Degrading Enzymes

    PubMed Central

    Pauchet, Yannick; Wilkinson, Paul; Chauhan, Ritika; ffrench-Constant, Richard H.

    2010-01-01

    Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent “disappearance” of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology. PMID:21179425

  2. Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

    PubMed Central

    Wang, P; Denoya, C D; Morgenstern, M R; Skinner, D D; Wallace, K K; Digate, R; Patton, S; Banavali, N; Schuler, G; Speedie, M K; Reynolds, K A

    1996-01-01

    We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid. PMID:8955309

  3. The Caenorhabditis elegans unc-93 gene encodes a putative transmembrane protein that regulates muscle contraction

    PubMed Central

    1992-01-01

    unc-93 is one of a set of five interacting genes involved in the regulation or coordination of muscle contraction in Caenorhabditis elegans. Rare altered-function alleles of unc-93 result in sluggish movement and a characteristic "rubber band" uncoordinated phenotype. By contrast, null alleles cause no visibly abnormal phenotype, presumably as a consequence of the functional redundancy of unc-93. To understand better the role of unc-93 in regulating muscle contraction, we have cloned and molecularly characterized this gene. We isolated transposon- insertion alleles and used them to identify the region of DNA encoding the unc-93 protein. Two unc-93 proteins differing at their NH2 termini are potentially encoded by transcripts that differ at their 5' ends. The putative unc-93 proteins are 700 and 705 amino acids in length and have two distinct regions: the NH2 terminal portion of 240 or 245 amino acids is extremely hydrophilic, whereas the rest of the protein has multiple potential membrane-spanning domains. The unc-93 transcripts are low in abundance and the unc-93 gene displays weak codon usage bias, suggesting that the unc-93 protein is relatively rare. The unc-93 protein has no sequence similarity to proteins listed in current data- bases. Thus, unc-93 is likely to encode a novel membrane-associated muscle protein. We discuss possible roles for the unc-93 protein either as a component of an ion transport system involved in excitation- contraction coupling in muscle or in coordinating muscle contraction between muscle cells by affecting the functioning of gap junctions. PMID:1313436

  4. Divergence among genes encoding the elongation factor Tu of Yersinia Species.

    PubMed

    Isabel, Sandra; Leblanc, Eric; Boissinot, Maurice; Boudreau, Dominique K; Grondin, Myrian; Picard, François J; Martel, Eric A; Parham, Nicholas J; Chain, Patrick S G; Bader, Douglas E; Mulvey, Michael R; Bryden, Louis; Roy, Paul H; Ouellette, Marc; Bergeron, Michel G

    2008-11-01

    Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.

  5. A comparison of group II introns of plastid tRNALysUUU genes encoding maturase protein.

    PubMed

    Jankowiak, Kamila; Lesicka, Joanna; Pacak, Andrzej; Rybarczyk, Agnieszka; Szweykowska-Kulińska, Zofia

    2004-01-01

    All higher plant plastid genomes have six classes of tRNA genes containing introns. One of those is the tRNALysUUU gene, which encodes maturase protein. In the case of liverwort species from the genus Porella and mosses from the genus Plagiomnium, the maturase coding gene (matK) represents a truncated form of other plant matK genes: several subdomains of the reverse transcriptase-like domain and so-called domain X are not present in these ORFs. These ORFs probably represent pseudogenes of the matK gene. The analysis of codon usage within the matK gene revealed the presence of strong A/T pressure. The use of codons with the third letter being U or A varies from 71-93%. The comparison of maturase amino acid sequences at the family level shows a high identity between species. However, when liverwort and angiosperm maturase sequences are compared, the percentage of identity drops dramatically. The calculated values of the number of nucleotide substitutions vary considerably, even when liverwort species are compared pairwise. The phenetic tree of relationships between plant species on the basis of tRNALysUUU intron sequences concur with the generally accepted plant phylogeny.

  6. A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme

    SciTech Connect

    Yasunami, Michio; Chengsheng Chen; Yoshida, Akira )

    1991-09-01

    The human alcohol dehydrogenase gene family consists of five known loci (ADH1-ADH5), which have been mapped close together on chromosome 4 (4q21-25). ADH isozymes encoded by these genes are grouped in three distinct classes in terms of their enzymological properties. A moderate structural similarity is observed between the members of different classes. The authors isolated an additional member of the ADH gene family by means of cross-hybridization with the ADH2 (class I) cDNA probe. cDNA clones corresponding to this gene were derived from PCR-amplified libraries as well. The coding sequence of a 368-amino-acid-long open reading frame was interrupted by introns into eight exons and spanned approximately 17 kilobases on the genome. The gene contains a glucocorticoid response element at the 5{prime} region. The transcript was detected in the stomach and liver. The deduced amino acid sequence of the open reading frame showed about 60% positional identity with known human ADHs. This extent of homology is comparable to interclass similarity in the human ADH family. Thus, the newly identified gene, which is designated ADH6, governs the synthesis of an enzyme that belongs to another class of ADHs presumably with a distinct physiological role.

  7. Molecular characterization of the PEL1 gene encoding a putative phosphatidylserine synthase.

    PubMed

    Janitor, M; Jarosch, E; Schweyen, R J; Subík, J

    1995-10-01

    In the yeast Saccharomyces cerevisiae the PEL1 gene is essential for the viability of rho-/rhoo petite mutants, and its mutation in respiring cells results in a pleiotropic phenotype. Results of complementation analysis with different subclones of chromosomal DNA and re-sequencing of the YCL4w-YCL3w segment of chromsome III demonstrate that the coding region of the PEL1 gene corresponds to 1467 bp. The size of the PEL1 transcript in Northern blot analysis was estimated to be approximately 1.5 kb. Transcription initiation in wild-type cells was found to occur at the position -9 relative to the ATG. The PEL1 gene was moderately expressed irrespective of the state of the mitochondrial genome and the nature of the carbon sources. Disruption of the PEL1 gene was not lethal and resulted in the same phenotype as observed with the pel1 mutant, i.e. the cells were not able to survive ethidium bromide mutagenesis, were thermosensitive for growth on glucose at 37 degrees C and failed to grow on minimal glycerol medium. Although the Pel1 protein exhibits significant similarity to a family of phosphatidylserine synthases, the disrupted PEL1 gene was not complemented by the multicopy plasmid-borne CHO1 gene encoding an essential yeast phosphatidylserine synthase.

  8. A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene.

    PubMed

    Langfelder, Kim; Gattung, Stephanie; Brakhage, Axel A

    2002-07-01

    Aspergillus fumigatus is an important opportunistic human pathogenic fungus. In severely immunocompromised patients, the fungus causes life-threatening diseases, such as pneumonia and invasive aspergillosis. In order to obtain a better understanding of the key elements involved in A. fumigatus virulence and for identifying possible drug targets, it is essential to be able to generate gene-deletion strains. Until recently, the molecular techniques available did not provide a rapid method for gene deletion. A novel method described for A. nidulans was adapted for A. fumigatus. This method is quick and produces an increased homologous recombination efficiency. By using an Escherichia coli strain expressing the lambda red operon, it is possible to induce an in vivo recombination of a PCR fragment flanked by >50-bp regions with a cosmid containing the gene of interest. This produces cosmids in which the gene of interest has been replaced by a bi-functional marker. Such cosmids have large flanking regions surrounding the selectable marker pyrG of A. fumigatus used here, which result in high recombination efficiencies in A. fumigatus. Here, we identified a new ABC transporter-encoding gene in A. fumigatus, designated abcA. By using this method, an A. fumigatus knock-out mutant was generated, providing evidence that this method of generating gene deletions can also be used in A. fumigatus and significantly broadens our repertoire of molecular techniques to study A. fumigatus.

  9. Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression.

    PubMed Central

    Yang, C; Kaplan, H B

    1997-01-01

    Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene.