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Sample records for actively transcribed genes

  1. Replication-dependent histone genes are actively transcribed in differentiating and aging retinal neurons.

    PubMed

    Banday, Abdul Rouf; Baumgartner, Marybeth; Al Seesi, Sahar; Karunakaran, Devi Krishna Priya; Venkatesh, Aditya; Congdon, Sean; Lemoine, Christopher; Kilcollins, Ashley M; Mandoiu, Ion; Punzo, Claudio; Kanadia, Rahul N

    2014-01-01

    In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.

  2. The anti-tumor drug bleomycin preferentially cleaves at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-04-01

    The genome-wide pattern of DNA cleavage at transcription start sites (TSSs) for the anti-tumor drug bleomycin was examined in human HeLa cells using next-generation DNA sequencing. It was found that actively transcribed genes were preferentially cleaved compared with non-transcribed genes. The 143,600 identified human TSSs were split into non-transcribed genes (82,596) and transcribed genes (61,004) for HeLa cells. These transcribed genes were further split into quintiles of 12,201 genes comprising the top 20, 20-40, 40-60, 60-80, and 80-100 % of expressed genes. The bleomycin cleavage pattern at highly transcribed gene TSSs was greatly enhanced compared with purified DNA and non-transcribed gene TSSs. The top 20 and 20-40 % quintiles had a very similar enhanced cleavage pattern, the 40-60 % quintile was intermediate, while the 60-80 and 80-100 % quintiles were close to the non-transcribed and purified DNA profiles. The pattern of bleomycin enhanced cleavage had peaks that were approximately 200 bp apart, and this indicated that bleomycin was identifying the presence of phased nucleosomes at TSSs. Hence bleomycin can be utilized to detect chromatin structures that are present at actively transcribed genes. In this study, for the first time, the pattern of DNA damage by a clinically utilized cancer chemotherapeutic agent was performed on a human genome-wide scale at the nucleotide level.

  3. Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

    SciTech Connect

    Venema, J.; van Hoffen, A.; Karcagi, V.; Natarajan, A.T.; van Zeeland, A.A.; Mullenders, L.H. )

    1991-08-01

    The authors have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5{prime} part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3{prime} part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

  4. How eukaryotic genes are transcribed

    PubMed Central

    Venters, Bryan J.; Pugh, B. Franklin

    2009-01-01

    Summary Regulation of eukaryotic gene expression is far more complex than one might have imagined thirty years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: 1) a well-defined organization of nucleosomes and modification states at most genes, 2) regulatory networks of sequence-specific transcription factors, 3) chromatin remodeling coupled to promoter assembly of the general transcription factors and RNA polymerase II, and 4) phosphorylation states of RNA polymerase II coupled to chromatin modification states during transcription. The wealth of new insights arising from the tools of biochemistry, genomics, cell biology, and genetics is providing a remarkable view into the mechanics of gene regulation. PMID:19514890

  5. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome

    SciTech Connect

    Leadon, S.A. ); Copper, P.K. )

    1993-11-15

    Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribe genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, the authors examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. They found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. The results extend the generality of preferential repair in active genes to include damage other than bulky lesions.

  6. Enhanced DNA repair of bleomycin-induced 3'-phosphoglycolate termini at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-11-01

    The anti-tumour agent, bleomycin, cleaves DNA to give 3'-phosphoglycolate and 5'-phosphate termini. The removal of 3'-phosphoglycolate to give 3'-OH ends is a very important step in the DNA repair of these lesions. In this study, next-generation DNA sequencing was utilised to investigate the repair of these 3'-phosphoglycolate termini at the transcription start sites (TSSs) of genes in HeLa cells. The 143,600 identified human TSSs in HeLa cells comprised 82,596 non-transcribed genes and 61,004 transcribed genes; and the transcribed genes were divided into quintiles of 12,201 genes comprising the top 20%, 20-40%, 40-60%, 60-80%, 80-100% of expressed genes. Repair of bleomycin-induced 3'-phosphoglycolate termini was enhanced at actively transcribed genes. The top 20% and 20-40% quintiles had a very similar level of enhanced repair, the 40-60% quintile was intermediate, while the 60-80% and 80-100% quintiles were close to the low level of enhancement found in non-transcribed genes. There were also interesting differences regarding bleomycin repair on the sense and antisense strands of DNA at TSSs. The sense strand had highly enhanced repair between 0 and 250bp relative to the TSS, while for the antisense strand highly enhanced repair was between 150 and 450bp. Repair of DNA damage is a major mechanism of resistance to anti-tumour drugs and this study provides an insight into this process in human tumour cells.

  7. Electrophoretic separation of a class of nucleosomes enriched in HMG 14 and 17 and actively transcribed globin genes.

    PubMed Central

    Albanese, I; Weintraub, H

    1980-01-01

    Monomer nucleosomes from chick erythrocytes can be fractionated according to their electrophoretic mobility in (comparatively) high salt acrylamide gels. We show that the fractionation is based predominantly on differences in charge. The monomer heterogeneity persists even when the nucleosomes are trimmed down to 145 bp with Exo III or when H1 and H5 are removed. The slowest migrating monomers are associated with HMG 14 and 17; however, we do not believe that these proteins are entirely responsible for the altered mobility since the nucleosome heterogeneity persists even after removal of HMG 14 and 17. The DNA associated with the HMG 14 and 17 containing nucleosomes is shown to be enriched in actively transcribed globin sequences. Images PMID:6448987

  8. dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes.

    PubMed

    Lloret-Llinares, Marta; Pérez-Lluch, Sílvia; Rossell, David; Morán, Tomás; Ponsa-Cobas, Joan; Auer, Herbert; Corominas, Montserrat; Azorín, Fernando

    2012-10-01

    H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol llo(ser5) form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

  9. TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed

    PubMed Central

    Papantonis, Argyris; Kohro, Takahide; Baboo, Sabyasachi; Larkin, Joshua D; Deng, Binwei; Short, Patrick; Tsutsumi, Shuichi; Taylor, Stephen; Kanki, Yasuharu; Kobayashi, Mika; Li, Guoliang; Poh, Huay-Mei; Ruan, Xiaoan; Aburatani, Hiroyuki; Ruan, Yijun; Kodama, Tatsuhiko; Wada, Youichiro; Cook, Peter R

    2012-01-01

    Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories. PMID:23103767

  10. Assessment of preferential cleavage of an actively transcribed retroviral hybrid gene in murine cells by deoxyribonuclease I, bleomycin, neocarzinostatin, or ionizing radiation

    SciTech Connect

    Beckmann, R.P.; Agostino, M.J.; McHugh, M.M.; Sigmund, R.D.; Beerman, T.A.

    1987-08-25

    Preferential cleavage induced by bleomycin, neocarzinostatin, or ionizing radiation in a transcribed cellular gene was evaluated through comparisons with deoxyribonuclease I. The glucocorticoid-inducible LTL gene previously described served as the specific DNA target. A Southern blot analysis was used to specifically assess cleavage of the LTL gene in nuclei isolated from cells either treated or untreated with the synthetic glucocorticoid dexamethasone. Hypersensitivity of the gene to bleomycin or neocarzinostatin, which paralleled deoxyribonuclease I hypersensitivity, was evident only in nuclei isolated from dexamethasone-treated cells. Like deoxyribonuclease I, sites of dexamethasone-inducible drug hypersensitivity were coincident with the binding region for the glucocorticoid receptor found within the regulatory sequences of the LTL gene. In contrast, no hypersensitivity to ionizing radiation was evident. Although bleomycin and neocarzinostatin showed qualitatively similar preferences for the threshold LTL gene, quantitative evaluations of damage to total cellular DNA by filter elution showed that the relative specificity of bleomycin for the hypersensitive region was much less than that of either deoxyribonuclease I or neocarzinostatin.

  11. Conservation and Purifying Selection of Transcribed Genes Located in a Rice Centromere[W

    PubMed Central

    Fan, Chuanzhu; Walling, Jason G.; Zhang, Jianwei; Hirsch, Cory D.; Jiang, Jiming; Wing, Rod A.

    2011-01-01

    Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8 genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements. PMID:21856794

  12. Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

    PubMed Central

    Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

    2016-01-01

    The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. PMID:27559135

  13. Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

    PubMed Central

    Conconi, Antonio; Bespalov, Vyacheslav A.; Smerdon, Michael J.

    2002-01-01

    Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast. Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed. Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin. Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction. Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast. Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin. Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal. PMID:11782531

  14. Orientation-dependent influence of an intergenic enhancer on the promoter activity of the divergently transcribed mouse Shsp/alpha B-crystallin and Mkbp/HspB2 genes.

    PubMed

    Swamynathan, Shivalingappa K; Piatigorsky, Joram

    2002-12-20

    The mouse Shsp/alphaB-crystallin and Mkbp/HspB2 genes are closely linked and divergently transcribed. In this study, we have analyzed the contribution of the intergenic enhancer to Shsp/alphaB-crystallin and Mkbp/HspB2 promoter activity using dual-reporter vectors in transient transfection and transgenic mouse experiments. Deletion of the enhancer reduced Shsp/alphaB-crystallin promoter activity by 30- and 93-fold and Mkbp/HspB2 promoter activity by 6- and 10-fold in transiently transfected mouse lens alpha-TN4 and myoblast C2C12 cells, respectively. Surprisingly, inversion of the enhancer reduced Shsp/alphaB-crystallin promoter activity by 17-fold, but did not affect Mkbp/HspB2 promoter activity in the transfected cells. In contrast, enhancer activity was orientation-independent in combination with a heterologous promoter in transfected cells. Transgenic mouse experiments established the orientation dependence and Shsp/alphaB-crystallin promoter preference of the intergenic enhancer in its native context. The orientation dependence and preferential effect of the Shsp/alphaB-crystallin enhancer on the Shsp/alphaB-crystallin promoter provide an example of adaptive changes in gene regulation accompanying the functional diversification of duplicated genes during evolution.

  15. The structure of nucleosomal core particles within transcribed and repressed gene regions.

    PubMed Central

    Studitsky, V M; Belyavsky, A V; Melnikova, A F; Mirzabekov, A D

    1988-01-01

    The arrangement of histones along DNA in nucleosomal core particles within transcribed heat shock gene (hsp 70) region and repressed insertion within ribosomal genes of Drosophila was analysed by using protein-DNA crosslinking methods combined with hybridization tests. In addition, two-dimensional gel electrophoresis was employed to compare the overall nucleosomal shape and the nucleosomal DNA size. The arrangement of histones along DNA and general compactness of nucleosomes were shown to be rather similar in transcriptionally active and inactive genomic regions. On the other hand, nucleosomes within transcriptionally active chromatin are characterized by a larger size of nucleosomal DNA produced by micrococcal nuclease digestion and some peculiarity in electrophoretic mobility. Images PMID:3144704

  16. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  17. Infrequently transcribed long genes depend on the Set2/Rpd3S pathway for accurate transcription

    PubMed Central

    Li, Bing; Gogol, Madelaine; Carey, Mike; Pattenden, Samantha G.; Seidel, Chris; Workman, Jerry L.

    2007-01-01

    The presence of Set2-mediated methylation of H3K36 (K36me) correlates with transcription frequency throughout the yeast genome. K36me targets the Rpd3S complex to deacetylate transcribed regions and suppress cryptic transcription initiation at certain genes. Here, using a genome-wide approach, we report that the Set2–Rpd3S pathway is generally required for controlling acetylation at coding regions. When using acetylation as a functional readout for this pathway, we discovered that longer genes and, surprisingly, genes transcribed at lower frequency exhibit a stronger dependency. Moreover, a systematic screen using high-resolution tiling microarrays allowed us to identify a group of genes that rely on Set2–Rpd3S to suppress spurious transcripts. Interestingly, most of these genes are within the group that depend on the same pathway to maintain a hypoacetylated state at coding regions. These data highlight the importance of using the functional readout of histone codes to define the roles of specific pathways. PMID:17545470

  18. A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

    PubMed Central

    Diesch, Jeannine; Lesmana, Analia; Poortinga, Gretchen; Hein, Nadine; Lidgerwood, Grace; Cameron, Donald P.; Ellul, Jason; Goodall, Gregory J.; Wong, Lee H.; Dhillon, Amardeep S.; Hamdane, Nourdine; Rothblum, Lawrence I.; Pearson, Richard B.; Haviv, Izhak; Moss, Tom

    2015-01-01

    Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity). PMID:25452314

  19. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells.

    PubMed

    Minsky, Neri; Shema, Efrat; Field, Yair; Schuster, Meromit; Segal, Eran; Oren, Moshe

    2008-04-01

    Histone modifications have emerged as important regulators of transcription. Histone H2B monoubiquitination has also been implicated in transcription; however, better understanding of the biological significance of this modification in mammalian cells has been hindered by the lack of suitable reagents, particularly antibodies capable of specifically recognizing ubiquitinated H2B (ubH2B). Here, we report the generation of anti-ubH2B monoclonal antibodies using a branched peptide as immunogen. These antibodies provide a powerful tool for exploring the biochemical functions of H2B monoubiquitination at both a genome-wide and gene-specific level. Application of these antibodies in high resolution chromatin immunoprecipitation (ChIP)-chip experiments in human cells, using tiling arrays, revealed preferential association of ubiquitinated H2B with the transcribed regions of highly expressed genes. Unlike dimethylated H3K4, ubH2B was not associated with distal promoter regions. Furthermore, experimental modulation of the transcriptional activity of the tumour suppressor p53 was accompanied by rapid changes in the H2B ubiquitination status of its p21 target gene, attesting to the dynamic nature of this process. It has recently been demonstrated that the apparent extent of gene expression often reflects elongation rather than initiation rates; thus, our findings suggest that H2B ubiquitination is intimately linked with global transcriptional elongation in mammalian cells.

  20. Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

    PubMed Central

    Standring, D N; Venegas, A; Rutter, W J

    1981-01-01

    A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process. Images PMID:6796956

  1. Genetic Analysis of a Temporally Transcribed Chemotaxis Gene Cluster Caulobacter Crescentus

    PubMed Central

    Alley, MRK.; Gomes, S. L.; Alexander, W.; Shapiro, L.

    1991-01-01

    Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. Tn5 insertions causing a general chemotaxis phenotype, an inability to reverse swimming direction and to form large swarm colonies, have been mapped to an 8-kb region of the C. crescentus genome. These Tn5 mutations had different effects on the methyl-accepting chemotaxis proteins (MCP), and the activities of methyltransferase and methylesterase. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Using a cosmid library we isolated a clone that complemented SC1130. Complementation studies of the Tn5 mutants using derivatives of the cosmid clone showed that all the Tn5 insertions lie within a single operon that appears to encode many chemotaxis genes. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying β-galactosidase protein fusions to portions of the operon. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The transcription of the che operon occurred at a defined time in the cell cycle, prior to cell division. PMID:1660425

  2. Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of dsRNA fragments of rust pathogen genes in wheat seedlings through the barley stripe mosaic virus (BSMV) based host-induced gene silencing (HIGS) system can reduce the expression of the corresponding genes in the rust fungus. The highest levels of suppression have generally been observe...

  3. Bd oxidase homologue of photosynthetic purple sulfur bacterium Allochromatium vinosum is co-transcribed with a nitrogen fixation related gene.

    PubMed

    Dincturk, H Benan; Demir, Volkan; Aykanat, Tutku

    2011-02-01

    Purple sulfur bacteria, which are known to be the most ancient among anoxygenic phototrophs, play an important role in the global sulfur cycle. Allochromatium vinosum oxidizes reduced sulfur compounds such as hydrogen sulfide, elemental sulfur and thiosulfide. At low oxygen concentrations, A. vinosum can grow chemotrophically using oxygen as the terminal electron acceptor. Being also a nitrogen fixer, A. vinosum is faced with the paradox of co-existence of aerobic metabolism and nitrogen fixation. Due to growth difficulties, only a few studies have dealt with the aerobic metabolism of the organism and, until now, there has been no information about the genes involved in the respiratory metabolism of purple sulfur bacteria. In this article we show the first terminal oxidase gene for A. vinosum. The presence of a Bd type of quinol oxidase is necessary to protect nitrogenases against the inhibitory effects of oxygen. In this case, a nitrogen fixation related gene is part of the cyd operon and this gene is co-transcribed with cydAB genes. Bd oxidase of A. vinosum may be the earliest form of oxidase where the function of the enzyme is to scavenge the contaminant oxygen during nitrogen fixation. This may be an important clue about the early evolution of oxygenic photosynthesis, perhaps as a protective mechanism for nitrogen fixation.

  4. DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella conica (black morel).

    PubMed

    Wipf, D; Munch, J C; Botton, B; Buscot, F

    1996-09-01

    The internal transcribed spacer (ITS) of the gene coding for rRNA was sequenced in both directions with the gene walking technique in a black morel (Morchella conica) and a yellow morel (M. esculenta) to elucidate the ITS length discrepancy between the two species groups (750-bp ITS in black morels and 1,150-bp ITS in yellow morels.

  5. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    PubMed Central

    Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

    2016-01-01

    DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

  6. DNA Methylation within Transcribed Regions

    PubMed Central

    To, Taiko K.; Saze, Hidetoshi; Kakutani, Tetsuji

    2015-01-01

    DNA methylation within transcribed genes is commonly found in diverse animals and plants. Here, we provide an overview of recent advances and the remaining mystery regarding intragenic DNA methylation. PMID:26143255

  7. TSH stimulates 32P-labeling of thyroid nuclear HMG 14, a protein associated with actively transcribed chromatin

    SciTech Connect

    Cooper, E.; Palmer, R.J.; Spaulding, S.W.

    1982-04-01

    Thyroid slices were incubated with 32P with or without TSH. 32P-labeling of acid-soluble nuclear proteins was then examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. We found that TSH enhanced the labeling of the high mobility group protein HMG 14, a protein that is preferentially associated with actively transcribed chromatin. This observation suggests that changes in HMG 14 phosphorylation may be involved in mediating TSH-induced effects on the structure and function of active chromatin.

  8. Isolation and characterization of recombinant DNAs containing repeated elements of barley genome: identification of individual actively transcribed families of repeats

    SciTech Connect

    Prosnyak, M.I.; Kartel', N.A.; Ryskov, A.P.

    1986-05-01

    A bank of Escherichia coli clones containing fragments of barley nuclear DNA was obtained using plasmid pBR 322. Clones carrying repeated sequences of the plant genome were selected by means of colony and blot hybridization. Clones with actively transcribed sequences were selected by hybridization to complementary DNA synthesized by means of reverse transcription on a template of total barley poly(A)-containing RNA. Individual families of repeats, two of which contained transcriptionally active sequences of the barley genome, were identified by blot hybridization of recombinant plasmids containing labeled DNA fragments of the inserts of three different clones.

  9. Differential introduction of DNA damage and repair in mammalian genes transcribed by RNA polymerase I and II

    SciTech Connect

    Vos, J.H.; Wauthier, E.L. )

    1991-04-01

    The authors have developed a general quantitative method for comparing the levels of drug-induced DNA crosslinking in specific mammalian genes. They observed a dramatic difference between the efficiency of the removal of both psoralen monoadducts and interstrand crosslinks from the rRNA genes and the efficiency of their removal from the dihydrofolate reductase (DHFR) gene in cultured human and hamster cells. While 90% of the interstrand crosslinks were removed from the human DHFR gene in 48 h, less than 25% repair occurred in the rRNA genes. Similarly, in Chinese hamster ovary cells, 85% repair of interstrand crosslinks within 8 h in the DHFR gene versus only 20% repair in the rRNA genes. The preferential repair of the DHFR gene relative to that of the rRNA genes was also observed for psoralen monoadducts in cells from both mammalian species. In human-mouse hybrid cells, the active mouse rRNA genes were five times more susceptible to psoralen modification than are the silent rRNA human genes, but adduct removal was similarly inefficient for both classes. They conclude that the repair of chemical damage such as psoralen photadducts in an expressed mammalian gene may depend upon the class of transcription to which it belongs.

  10. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  11. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  12. A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways.

    PubMed Central

    van Tol, H; Stange, N; Gross, H J; Beier, H

    1987-01-01

    tRNA splicing enzymes had been identified in mammalian and plant cells long before homologous intron-containing tRNA genes were detected. The tRNATyr gene presented here is the first intron-containing, human tRNA gene for which transcription and pre-tRNA maturation has been studied in a homologous system. This gene is disrupted by a 20-bp long intron and encodes one of the two major human tRNAsTyr which have been purified and sequenced. A tRNATyr gene recently isolated from Nicotiana also contains an intron and codes for a functional, major cytoplasmic tRNATyr. Both tRNA genes are efficiently transcribed in a HeLa cell nuclear extract. Each of them produces two independent primary transcripts because of two initiation and termination sites, respectively. The maturation of the tRNATyr precursors proceeds along different pathways. The intervening sequence of the human pre-tRNATyr is excised first, followed by ligation of the tRNA halves and maturation of the flanks, as has been shown for all intron-containing tRNA genes transcribed in HeLa extract. The order of maturation steps is reversed for the plant pre-tRNATyr: processing of the flanking sequences precedes intron excision. This maturation pathway corresponds to that observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast. Images Fig. 1. Fig. 4. Fig. 5. Fig. 6. PMID:3502708

  13. Transcription promotes guanine to thymine mutations in the non-transcribed strand of an Escherichia coli gene.

    PubMed

    Klapacz, Joanna; Bhagwat, Ashok S

    2005-07-12

    Transcription of DNA opens the chromatin, causes topological changes in DNA and transiently exposes the two strands to different biochemical environments. Consequently, it has long been argued that transcription may promote damage to DNA and there are data in Escherichia coli and yeast supporting a correlation between high transcription and mutations. We examined the transcription-dependence of the reversion of a nonsense codon (TGA) in E. coli and found that there was a strong dependence of mutations on transcription in strains defective in the repair of 8-oxoguanine in DNA. Under conditions of high transcription there was a three to five-fold increase in mutations that changed TGA in the non-transcribed strand to a sense codon. Furthermore, in both mutY and mutM mutY backgrounds the mutations were overwhelmingly G:C to T:A. In contrast, when the TGA was in the transcribed strand in relation with the inducible promoter, high transcription decreased the rate of reversion. Similar results were obtained in a strain defective in the transcription-repair coupling factor, Mfd, suggesting that transcription dependent increase in base substitutions does not require transcription-dependent DNA repair. However, Mfd does modulate the magnitude of the mutagenic effect of transcription. These data are consistent with a model in which the non-transcribed strand is more susceptible to oxidative damage during transcription than the transcribed strand. These results suggest that the magnitudes of individual base substitutions and their relative numbers in other studies of mutational spectra may also be affected by transcription.

  14. 8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

    PubMed Central

    Sage, E; Drobetsky, E A; Moustacchi, E

    1993-01-01

    We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand. Images PMID:8440233

  15. The Nine Genes of the Nocardia lactamdurans Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region

    PubMed Central

    Enguita, Francisco J.; Coque, Juan Jose R.; Liras, Paloma; Martin, Juan F.

    1998-01-01

    The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp. PMID:9765587

  16. DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase.

    PubMed

    Sabouri, Nasim; McDonald, Karin R; Webb, Christopher J; Cristea, Ileana M; Zakian, Virginia A

    2012-03-15

    Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

  17. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys.

    PubMed

    Walters, William; Hyde, Embriette R; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A; Jansson, Janet K; Caporaso, J Gregory; Fuhrman, Jed A; Apprill, Amy; Knight, Rob

    2016-01-01

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for

  18. Positive and negative functional interactions between promoter elements from different classes of RNA polymerase III-transcribed genes.

    PubMed Central

    Parry, H D; Mattaj, I W

    1990-01-01

    Consensus tRNA gene promoter elements, A and B boxes, were introduced into the coding sequence of a Xenopus U6 gene. Combinations in which A and B boxes were coupled to wild-type or mutant U6 promoters were made. In this way information about both the functions of individual promoter elements and functional relationships between different classes of RNA polymerase III promoter element were obtained. Mutants in which the U6 PSE was non-functional were rescued by the presence of a B box, indicating a degree of functional relationship between these two elements. Moreover, the B box acted to increase the transcriptional activity and competitive strength of the wild-type U6 promoter. In contrast, no evidence was obtained to suggest that a tRNA A box can interact productively with U6 promoter elements in the absence of a B box. Data obtained suggest that the U6 PSE functions as an 'adaptor', being necessary to enable the basal U6 promoter to respond to upstream enhancement. Certain combinations of U6 and tRNA promoter elements are shown to be mutually antagonistic by a mechanism which is likely to involve blockage of transcription initiation. In summary, the U6 and tRNA promoters are shown to consist of functionally related, but distinct, promoter elements whose interactions shed new light on their normal roles in transcription. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:2323333

  19. cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

    PubMed Central

    Hofmann, A; Garfinkel, M D; Meyerowitz, E M

    1991-01-01

    The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region. PMID:1903838

  20. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob; Bik, Holly

    2015-12-22

    ABSTRACT

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection ofThaumarchaeotaand clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

    ImportanceWe continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to

  1. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    PubMed Central

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy

    2015-01-01

    ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers

  2. Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes

    PubMed Central

    Stoytcheva, Zoia R.; Vladimirov, Vladimir; Douet, Vanessa; Stoychev, Ilko; Berry, Marla J.

    2009-01-01

    Selenoprotein H is a redox-sensing DNA binding protein that upregulates genes involved in antioxidant responses. Given the known links between oxidative stress and heavy metals, we investigated the potential for regulation of selenoprotein H by metals. In silico analysis of the selenoprotein H genes from nine species reveals multiple predicted metal response elements (MREs). To validate MRE function, we investigated the effects of zinc or cadmium addition and metal-responsive transcription factor 1 (MTF-1) knockout on selenoprotein H mRNA levels. Chromatin immunoprecipitation was used to directly assess physical binding of the transcription factor to MREs in the human and mouse selenoprotein H genes. The results reported herein show that selenoprotein H is a newly identified target for MTF-1. Further, whereas nearly all prior studies of MREs focused on those located in promoters, we demonstrate binding of MTF-1 to MREs located downstream of the transcription start sites in the human and murine selenoprotein H genes. Finally, we identified MREs in downstream sequences in 15 additional MTF-1 regulated genes lacking promoter MREs, and demonstrated MTF-1 binding in three of these genes. This regulation via sequences downstream of promoters highlights a new direction for identifying previously unrecognized target genes for MTF-1. PMID:19913599

  3. Quantification of diatom gene expression in the sea by selecting uniformly transcribed mRNA as the basis for normalization.

    PubMed

    Kang, Lee-Kuo; Tsui, Feng-Hsiu; Chang, Jeng

    2012-09-01

    To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.

  4. Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene.

    PubMed Central

    Linton, J P; Yen, J Y; Selby, E; Chen, Z; Chinsky, J M; Liu, K; Kellems, R E; Crouse, G F

    1989-01-01

    The mouse dihydrofolate reductase gene (dhfr) is a housekeeping gene expressed under the control of a promoter region embedded in a CpG island--a region rich in unmethylated CpG dinucleotides. A divergent transcription unit exists immediately upstream of the dhfr gene which is coamplified with dhfr in some but not all methotrexate-resistant cell lines. We show that the promoter region for this gene pair consists of two bidirectional promoters, a major and minor promoter, which are situated within a 660-base-pair region upstream of the dhfr ATG translation initiation codon. The major promoter controls over 90% of dhfr transcription, while the minor promoter directs the transcription of the remaining dhfr mRNAs. The major promoter functions bidirectionally, transcribing a divergent 4.0-kilobase poly(A) mRNA (class A) in the direction opposite that of dhfr transcription. The predicted protein product of this mRNA is 105 kilodaltons. The minor promoter also functions bidirectionally, directing the transcription of at least two divergent RNAs (class B). These RNAs, present in quantities approximately 1/10 to 1/50 that of the class A mRNAs, are 4.4- and 1.6-kilobase poly(A) mRNAs. cDNAs representing both class A and class B mRNAs have been cloned from a mouse fibroblast cell line which has amplified the dhfr locus (3T3R500). DNA sequence analysis of these cDNAs reveals that the class A and class B mRNAs share, for the most part, the same exons. On the basis of S1 nuclease protection analysis of RNA preparations from several mouse tissues, both dhfr and divergent genes showed similar levels of expression but did show some specificity in start site utilization. Computer homology searches have revealed sequence similarity of the divergent transcripts with bacterial genes involved in DNA mismatch repair, and we therefore have named the divergently transcribed gene Rep-1. Images PMID:2674679

  5. Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene

    SciTech Connect

    Lommel, L.; Hanawalt, P.C. )

    1993-02-01

    People that suffer from xeroderma pigmentosum (XP) are sun sensitive and experience elevated incidences of cancer, particularly skin cancers on sun-light exposed parts of their bodies. Cultured cells from XP patients are found to be subtantially more sensitive to lethal and mutagenic effects of ultraviolet (UV) radiation than are cells from unaffected individuals. Using the cells from XP individuals, researchers study the roles that cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts play in UV resistance. The results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs form the transcribed strands of expressed genes is essential for UV resistance. 36 refs., 5 figs., 1 tab.

  6. Brief exposures of human body lice to sub-lethal amounts of ivermectin over transcribes detoxification genes involved in tolerance

    PubMed Central

    Yoon, K. S.; Strycharz, J. P.; Baek, J. H.; Sun, W.; Kim, J.H.; Kang, J.S.; Pittendrigh, B. R.; Lee, S. H.; Clark, J. M.

    2011-01-01

    Transcriptional profiling results, using our non-invasive induction assay [short exposure intervals (2–5 h) to sub-lethal amounts of insecticides (genes from body lice are identified by quantitative real-time PCR analyses. Of the cytochrome P450 monooxygenase and ATP binding cassette transporter genes induced by ivermectin, CYP6CJ1, CYP9AG1, CYP9AG2 and PhABCC4 were respectively most significantly over-expressed, had high basal expression levels and were most closely related to genes from other organisms that metabolized insecticides, including ivermectin. Injection of dsRNAs against either CYP9AG2 or PhABCC4 into non-induced female lice reduced their respective transcript level and resulted in increase sensitivity to ivermectin, indicating that these two genes are involved in the xenobiotic metabolism of ivermectin and in the production of tolerance. PMID:21895817

  7. A complete alpha1,3-galactosyltransferase gene is present in the human genome and partially transcribed.

    PubMed

    Lantéri, Marion; Giordanengo, Valérie; Vidal, Frédérique; Gaudray, Patrick; Lefebvre, Jean-Claude

    2002-12-01

    The synthesis of Galalpha1-3Gal-terminated oligosaccharides (alpha-Gal) epitopes has been interrupted during the course of evolution, starting with Old World primates. Partial sequences similar to the alpha1,3-galactosyltransferase (alpha1,3GalT) gene, which governs the synthesis of alpha-Gal epitopes, have been detected in the human genome and were found to correspond to pseudogenes. We completed the sequence of the human alpha1,3GalT pseudogene present on chromosome 9 and found it to be organized like the murine alpha1,3GalT gene. In human cell lines and several normal and tumor tissues we detected truncated transcripts corresponding to this pseudogene. Considering these mRNAs, translation of an open reading frame containing the first four translated exons but missing the two catalytic exons could predict a truncated alpha1,3GalT polypeptide that should be enzymatically inactive. We show that transcription of human alpha1,3GalT is prematurely terminated at the level of a strong transcriptional stop signal in the middle of intron VII. We were able to reproduce this effect in vitro by subcloning the implicated DNA region upstream from a reporter cDNA. The premature transcriptional arrest of human alpha1,3-GalT gene leads to an ectopic splicing event and to the connection of a short intronic sequence downstream from translated exons. Finally, we show that these truncated transcripts are overexpressed in cell lines with modifications of O-glycans.

  8. Identification of Giardia species and Giardia duodenalis assemblages by sequence analysis of the 5.8S rDNA gene and internal transcribed spacers.

    PubMed

    Cacciò, Simone M; Beck, Relja; Almeida, Andre; Bajer, Anna; Pozio, Edoardo

    2010-05-01

    PCR assays have been developed mainly to assist investigations into the epidemiology of Giardia duodenalis, the only species in the Giardia genus having zoonotic potential. However, a reliable identification of all species is of practical importance, particularly when water samples and samples from wild animals are investigated. The aim of the present work was to genotype Giardia species and G. duodenalis assemblages using as a target the region spanning the 5.8S gene and the 2 flanking internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene. Primers were designed to match strongly conserved regions in the 3' end of the small subunit and in the 5' end of the large subunit ribosomal genes. The corresponding region (about 310 bp) was amplified from 49 isolates of both human and animal origin, representing all G. duodenalis assemblages as well as G. muris and G. microti. Sequence comparison and phylogenetic analysis showed that G. ardeae, G. muris, G. microti as well as the 7 G. duodenalis assemblages can be easily distinguished. Since the major subgroups within the zoonotic assemblages A and B can be identified by sequence analysis, this assay is also informative for molecular epidemiological studies.

  9. MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

    PubMed

    Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A M

    2013-01-01

    Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

  10. PAX8 is transcribed aberrantly in cervical tumors and derived cell lines due to complex gene rearrangements.

    PubMed

    López-Urrutia, Eduardo; Pedroza-Torres, Abraham; Fernández-Retana, Jorge; De Leon, David Cantu; Morales-González, Fermín; Jacobo-Herrera, Nadia; Peralta-Zaragoza, Oscar; García-Mendez, Jorge; García-Castillo, Verónica; Bautista-Isidro, Osvaldo; Pérez-Plasencia, Carlos

    2016-07-01

    The transcription factor PAX8, a member of the paired box-containing gene family with an important role in embryogenesis of the kidney, thyroid gland and nervous system, has been described as a biomarker in tumors of the thyroid, parathyroid, kidney and thymus. The PAX8 gene gives rise to four isoforms, through alternative mRNA splicing, but the splicing pattern in tumors is not yet established. Cervical cancer has a positive expression of PAX8; however, there is no available data determining which PAX8 isoform or isoforms are present in cervical cancer tissues as well as in cervical carcinoma-derived cell lines. Instead of a differential pattern of splicing isoforms, we found numerous previously unreported PAX8 aberrant transcripts ranging from 378 to 542 bases and present in both cervical carcinoma-derived cell lines and tumor samples. This is the first report of PAX8 aberrant transcript production in cervical cancer. Reported PAX8 isoforms possess differential transactivation properties; therefore, besides being a helpful marker for detection of cancer, PAX8 isoforms can plausibly exert differential regulation properties during carcinogenesis.

  11. Topoisomerase 1-dependent deletions initiated by incision at ribonucleotides are biased to the non-transcribed strand of a highly activated reporter

    PubMed Central

    Cho, Jang-Eun; Kim, Nayun; Jinks-Robertson, Sue

    2015-01-01

    DNA polymerases incorporate ribonucleoside monophosphates (rNMPs) into genomic DNA at a low level and such rNMPs are efficiently removed in an error-free manner by ribonuclease (RNase) H2. In the absence of RNase H2 in budding yeast, persistent rNMPs give rise to short deletions via a mutagenic process initiated by Topoisomerase 1 (Top1). We examined the activity of a 2-bp, rNMP-dependent deletion hotspot [the (TG)2 hotspot] when on the transcribed or non-transcribed strand (TS or NTS, respectively) of a reporter placed in both orientations near a strong origin of replication. Under low-transcription conditions, hotspot activity depended on whether the (TG)2 sequence was part of the newly synthesized leading or lagging strand of replication. In agreement with an earlier study, deletions occurred at a much higher rate when (TG)2 was on the nascent leading strand. Under high-transcription conditions, however, hotspot activity was not dependent on replication direction, but rather on whether the (TG)2 sequence was on the TS or NTS of the reporter. Deletion rates were several orders of magnitude higher when (TG)2 was on the NTS. These results highlight the complex interplay between replication and transcription in regulating Top1-dependent genetic instability. PMID:26271994

  12. Characterization of internal transcribed spacer (ITS1)-ITS2 region of ribosomal RNA gene from 25 species of Culicoides biting midges (Diptera: Ceratopogonidae) in Japan.

    PubMed

    Matsumoto, Y; Yanase, T; Tsuda, T; Noda, H

    2009-09-01

    We determined nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS)1-5.8S-ITS2a-2S-ITS2 region in 103 individuals of 25 Culicoides species (Diptera: Ceratopogonidae) from 11 locations in Japan. Ribosomal RNA genes, 5.8S and 2S rDNA, were highly conserved among the species with few variations. The ITS2a region showed length variation among species. Both ITS1 and ITS2 showed highly varied sequences among species. The noticeable indel regions among ITS1 sequences are present in some Culicoides species, separating species into two types having long or short ITS1 region. However, Culicoides cylindratus Kitaoka possesses both types of ITS1 in each individual; these results seem to indicate that the ITS1-long type was the prototype and the short type was produced through deletion in many Culicoides species. One species, belonging to subgenus Avaritia, possessed an Avaritia-specific sequence in ITS1 and phylogenetically formed a monophyletic group. Geographical genotypes in a species were not clear. Species-specific sequence features were observed, enabling molecular identification of Culicoides species.

  13. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

  14. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  15. The gene coding for 3-deoxy-manno-octulosonic acid transferase and the rfaQ gene are transcribed from divergently arranged promoters in Escherichia coli.

    PubMed Central

    Clementz, T

    1992-01-01

    The gene kdtA in Escherichia coli codes for 3-deoxy-D-manno-octulosonic acid transferase, the enzyme responsible for attachment of the two 3-deoxy-D-manno-octulosonic acid residues that constitute the link between lipid A and the core oligosaccharide of the lipopolysaccharide. Cloning and subsequent sequencing of the region upstream of kdtA revealed an open reading frame identified as the first gene (rfaQ) in an rfa gene cluster. The kdtA and rfaQ transcripts were identified, and the 5' ends of the transcripts were mapped by primer extension. Two main, divergently arranged promoters were found. These promoters generated transcripts with 5' ends separated by 289 bases. That the two divergent transcripts from the identified promoters represent the kdtA and rfaQ transcripts was confirmed by fusing different parts of the intergenic region between the promoterless lacZ and phoA genes in promoter-screening plasmid pCB267. Images PMID:1447141

  16. Construction and expression in vivo of an internally deleted mouse alpha-fetoprotein gene: presence of a transcribed Alu-like repeat within the first intervening sequence.

    PubMed Central

    Young, P R; Scott, R W; Hamer, D H; Tilghman, S M

    1982-01-01

    An alpha-fetoprotein (AFP) 'minigene' was constructed by joining the first two exons along with 0.9 kilobases of 5' flanking sequence of the mouse AFP gene to its last exon and 0.4 kilobases of 3' flanking sequence. This 'minigene' was tested for activity by inserting it into an SV40 vector and infecting African green monkey kidney cells. Correct initiation, termination, polyadenylation and splicing were observed. Furthermore a 220 nucleotide transcript was detected and mapped to a mouse Alu-like or B1 repeat on the opposite strand to that encoding the AFP gene in the first intervening sequence. Images PMID:6179043

  17. Evolution of the mouse H-2K region: a hot spot of mutation associated with genes transcribed in embryos and/or germ cells.

    PubMed

    Yeom, Y I; Abe, K; Artzt, K

    1992-03-01

    Active gene transcription is known to promote genetic change in neighboring DNA. We reasoned that the change would be readily heritable if transcription was occurring in germ cells or early embryonic cells before the germ cells are set aside. The H-2K region of the major histocompatibility complex (MHC) provides a good vehicle for testing this hypothesis because it is replete with such genes. We have compared the amount of polymorphism in 240 kb of DNA contiguous with H-2K and 150 kb of DNA flanking a homologous duplicated region in t-haplotypes and inbred strains. Using 90 probes and three restriction enzymes, we find a staggering difference in the amount of polymorphism in the H-2K region vs. the duplicated region (26% vs. 0%) of t-haplotypes. The disparity in the rate of divergence between the two regions indicates that the spatial distribution of genes and their expression pattern might be important factors in sequence evolution. Since t-haplotypes normally show extremely limited variability among themselves due to their recent divergence from a single ancestor, these results imply that the mutation rate in the H-2K region is unusually high. This is in apparent contradiction to the current view that the MHC loci have evolved at the same rate as other loci. The implications for the evolution of the H-2K gene are discussed.

  18. Nucleosomes are depleted at the VSG expression site transcribed by RNA polymerase I in African trypanosomes.

    PubMed

    Figueiredo, Luisa M; Cross, George A M

    2010-01-01

    In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.

  19. Seeing What They Meant: Transcribing as a Route To Noticing.

    ERIC Educational Resources Information Center

    Lynch, Tony

    2001-01-01

    Describes a reflective noticing activity in which pairs of adult learners of English for academic purposes transcribe their own performances of a routine classroom speaking task. Analysis of the process and product of these cycles of work suggest that collaborative transcribing and editing can encourage learners to focus on their form in output in…

  20. Genotyping of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes.

    PubMed

    Li, Kai; He, Ai; Cai, Wei Ping; Tang, Xiao Ping; Zheng, Xiao Ying; Li, Zhuo Ya; Zhan, Xi Mei

    2013-01-01

    Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.

  1. Characterization of tissue expression and full-length coding sequence of a novel human gene mapping at 3q12.1 and transcribed in oligodendrocytes.

    PubMed

    Fayein, Nicole-Adeline; Stankoff, Bruno; Auffray, Charles; Devignes, Marie-Dominique

    2002-05-01

    Macro-array differential hybridization of a collection of 5058 human gene transcripts represented in an IMAGE infant brain cDNA library has led to the identification of transcripts displaying preferential or specific expression in brain (Genome Res. 9 (1999) 195; http://idefix.upr420.vjf.cnrs.fr/IMAGE). Most of these genes correspond to as yet undescribed functions. Detailed characterization of the expression, sequence, and genome assignment of one of these genes named C3orf4, is reported here. The full-length sequence of the transcript was obtained by 5' extension RT-PCR. The gene transcript (2.8 kb) encodes a 253 amino acid long protein, with four transmembrane domains. The position of the C3orf4 gene was determined at 3q12.1 thanks to the draft sequence of the human genome. It is composed of five exons spanning more than 7 kb. No TATAA box but a CpG island was found upstream of the beginning of the gene. Northern blot analysis and in situ hybridization revealed a predominant expression in myelinated structures such as corpus callosum and spinal cord. RT-PCR showed expression of the C3orf4 gene in rat optic nerve and cultured oligodendrocytes, the myelinating cells of the central nervous system, but not in astrocytes. This work supports further investigations aimed at determining the role of the C3orf4 gene in myelinating cells.

  2. Rhoptry-associated protein (rap-1) genes in the sheep pathogen Babesia sp. Xinjiang: Multiple transcribed copies differing by 3' end repeated sequences.

    PubMed

    Niu, Qingli; Marchand, Jordan; Yang, Congshan; Bonsergent, Claire; Guan, Guiquan; Yin, Hong; Malandrin, Laurence

    2015-07-30

    Sheep babesiosis occurs mainly in tropical and subtropical areas. The sheep parasite Babesia sp. Xinjiang is widespread in China, and our goal is to characterize rap-1 (rhoptry-associated protein 1) gene diversity and expression as a first step of a long term goal aiming at developing a recombinant subunit vaccine. Seven different rap-1a genes were amplified in Babesia sp. Xinjiang, using degenerate primers designed from conserved motifs. Rap-1b and rap-1c gene types could not be identified. In all seven rap-1a genes, the 5' regions exhibited identical sequences over 936 nt, and the 3' regions differed at 28 positions over 147 nt, defining two types of genes designated α and β. The remaining 3' part varied from 72 to 360 nt in length, depending on the gene. This region consists of a succession of two to ten 36 nt repeats, which explains the size differences. Even if the nucleotide sequences varied, 6 repeats encoded the same stretch of amino acids. Transcription of at least four α and two β genes was demonstrated by standard RT-PCR.

  3. Primers ITS1, ITS2 and ITS4 detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi.

    PubMed

    Korabecná, M; Liska, V; Fajfrlík, K

    2003-01-01

    Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.

  4. Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

    PubMed

    Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Ippolito, Domenica; Ventre, Giuseppe; Zhang, Ying; Saccone, Giuseppe

    2014-01-01

    The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

  5. Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes

    PubMed Central

    2014-01-01

    The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) and differential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function. PMID:25472628

  6. [Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus].

    PubMed

    Slugina, M A; Torres Minho, K; Filiushin, M A

    2014-01-01

    Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.

  7. The human Pim-1 gene is selectively transcribed in different hemato-lymphoid cell lines in spite of a G + C-rich housekeeping promoter.

    PubMed Central

    Meeker, T C; Loeb, J; Ayres, M; Sellers, W

    1990-01-01

    The expression of the Pim-1 proto-oncogene was studied by using the K562, Daudi, and Jurkat cell lines. In K562, Pim-1 mRNA levels were more than 20-fold higher than in Daudi and 50-fold higher than in Jurkat. Nuclear run-on assay data correlated directly with the steady-state mRNA levels, suggesting that the rate of transcription was responsible for the selective expression of this gene. Furthermore, the half-life of Pim-1 mRNA was shown to be 47 min in K562, 71 min in Daudi, and 35 min in Jurkat. This indicated that selective Pim-1 mRNA expression did not depend on posttranscriptional regulation. Therefore, 1.7 kilobases of the Pim-1 promoter was sequenced and studied in detail. The sequence showed that the region from nucleotide -1 to -873 was G + C rich (71%). Study of promoter deletions defined two major functional regions, a proximal element (nucleotide -104 to -1) and a distal element (nucleotide -427 to -336). DNase I protection assays identified binding sites for the Sp1 and AP2 proteins in these elements. A possible new transcription factor binds at position -348 in the distal element. In our study of the 1.7-kilobase Pim-1 promoter, we found no differences between K562 and Jurkat that could explain large differences in transcription. Therefore, the Pim-1 promoter appears to function constitutively, and we conclude that distant elements must regulate the tissue-selective expression of this gene. Although the Pim-1 gene has a G + C-rich housekeeping promoter, expression is carefully regulated at the level of transcription. Images PMID:2181282

  8. The Ancestral Gene for Transcribed, Low-Copy Repeats in the Prader-Willi/Angleman Region Encodes a Large Protein Implicated in Protein Trafficking that is Deficient in Mice with Neuromuscular and

    SciTech Connect

    Ji, Y.

    1999-01-01

    Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

  9. Two self-splicing group I introns interrupt two late transcribed genes of prolate-headed Lactobacillus delbrueckii phage JCL1032.

    PubMed

    Riipinen, K A; Alatossava, T

    2004-10-01

    Two group I introns were detected from the late gene region of the prolate-headed phage JCL1032 of Lactobacillus delbrueckii. Introns JCL-I1 and JCL-I2 interrupt orf602 and orf1868 encoding a phage terminase large subunit (TerL, 69.7 kDa) and a putative tape measure protein (TMP, 202 kDa), respectively. The introns JCL-I1 (226 bp) and JCL-I2 (322 bp) were efficiently self-spliced in vivo. Both introns were classified to the subgroup IA1 having all the conserved structures necessary for splicing, but lacking the ability to encode endonucleases or other gene products. The introns JCL-I1 and JCL-I2 shared restricted nucleotide sequence similarity with each other and with the group I terL intron of Lb. delbrueckii phage LL-H. No match was found for JCL-I1 in the homology searches. Instead, the primary sequence from the joining region of P8 and P7 to P9 of the intron JCL-I2 was homologous to the group I intron of Bacillus mojavensis; the orf142 introns I1, I2 and I3 of Staphylococcus aureus phage Twort; the group I intron of phage Bastille (Bacillus thuringiensis); and to the group IA3 intron of Monomastix species.

  10. A Mechanistic Model for Cooperative Behavior of Co-transcribing RNA Polymerases

    PubMed Central

    Heberling, Tamra; Davis, Lisa; Gedeon, Jakub; Morgan, Charles; Gedeon, Tomáš

    2016-01-01

    In fast-transcribing prokaryotic genes, such as an rrn gene in Escherichia coli, many RNA polymerases (RNAPs) transcribe the DNA simultaneously. Active elongation of RNAPs is often interrupted by pauses, which has been observed to cause RNAP traffic jams; yet some studies indicate that elongation seems to be faster in the presence of multiple RNAPs than elongation by a single RNAP. We propose that an interaction between RNAPs via the torque produced by RNAP motion on helically twisted DNA can explain this apparent paradox. We have incorporated the torque mechanism into a stochastic model and simulated transcription both with and without torque. Simulation results illustrate that the torque causes shorter pause durations and fewer collisions between polymerases. Our results suggest that the torsional interaction of RNAPs is an important mechanism in maintaining fast transcription times, and that transcription should be viewed as a cooperative group effort by multiple polymerases. PMID:27517607

  11. RNA-mediated gene activation

    PubMed Central

    Jiao, Alan L; Slack, Frank J

    2014-01-01

    The regulation of gene expression by non-coding RNAs (ncRNAs) has become a new paradigm in biology. RNA-mediated gene silencing pathways have been studied extensively, revealing diverse epigenetic and posttranscriptional mechanisms. In contrast, the roles of ncRNAs in activating gene expression remains poorly understood. In this review, we summarize the current knowledge of gene activation by small RNAs, long non-coding RNAs, and enhancer-derived RNAs, with an emphasis on epigenetic mechanisms. PMID:24185374

  12. Transcribed dark matter: meaning or myth?

    PubMed Central

    Ponting, Chris P.; Belgard, T. Grant

    2010-01-01

    Genomic tiling arrays, cDNA sequencing and, more recently, RNA-Seq have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. These methods have led to greatly improved annotations of protein-coding genes, but have also identified transcription outside of annotated exons. One resultant issue that has aroused dispute is the balance of transcription of known exons against transcription outside of known exons. While non-genic ‘dark matter’ transcription was found by tiling arrays to be pervasive, it was seen to contribute only a small percentage of the polyadenylated transcriptome in some RNA-Seq experiments. This apparent contradiction has been compounded by a lack of clarity about what exactly constitutes a protein-coding gene. It remains unclear, for example, whether or not all transcripts that overlap on either strand within a genomic locus should be assigned to a single gene locus, including those that fail to share promoters, exons and splice junctions. The inability of tiling arrays and RNA-Seq to count transcripts, rather than exons or exon pairs, adds to these difficulties. While there is agreement that thousands of apparently non-coding loci are present outside of protein-coding genes in the human genome, there is vigorous debate of what constitutes evidence for their functionality. These issues will only be resolved upon the demonstration, or otherwise, that organismal or cellular phenotypes frequently result when non-coding RNA loci are disrupted. PMID:20798109

  13. Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Tam, Emily W T; Chen, Jonathan H K; Lau, Eunice C L; Ngan, Antonio H Y; Fung, Kitty S C; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2014-04-01

    Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

  14. Molecular cloning and characterization of cDNAs coding for apo-polysialoglycoprotein of rainbow trout eggs. Multiple mRNA species transcribed from multiple genes contain diverged numbers of exact 39-base (13-amino acid) repeats.

    PubMed

    Sorimachi, H; Emori, Y; Kawasaki, H; Kitajima, K; Inoue, S; Suzuki, K; Inoue, Y

    1988-11-25

    Polysialoglycoprotein (PSGP) of unfertilized eggs of rainbow trout (Salmo gairdneri) consists of tandem repeats (about 25) of a glycotridecapeptide, Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly (* denotes the attachment site of a polysialoglycan chain) (Kitajima, K., Inoue, Y., and Inoue, S. (1986) J. Biol. Chem. 261, 5262-5269). By using oligodeoxynucleotide probes based on the above sequence, we isolated a genomic clone for apoPSGP which contains 39-base pair repeats (5'-GACGACGCCACCTCTGAAGCT-GCGACCGGCCCGTCTGGC-3') encoding the tridecapeptide. Using a fragment of this genomic DNA as a probe, we next screened a cDNA library constructed with mRNA from immature ovaries of rainbow trout. Nucleotide sequencing analyses of cDNA clones thus obtained revealed that apoPSGP is encoded by multiple mRNA species consisting of diverged numbers (6-32) of the 39-base repeat encoding the tridecapeptide unit and homologous 5'- and 3'-bordering regions. The encoded protein consists of three distinct regions: the N-region consisting of a putative signal peptide and a pro-peptide, the R-region containing diverged numbers of the tandem repeat of 13-amino acid residues, and the C-region with six amino acid residues. Southern blot analysis showed that multiple mRNAs are transcribed from multiple genes for apoPSGP containing diverged numbers of the 39-base pair repeat. Thus, the genes for apoPSGP constitute a multigene family. Expression of the mRNAs is stage and organ specific, i.e. they are expressed only in immature ovaries and not in mature ovaries or in any other organ.

  15. Distanced Data: Transcribing Other People's Research Tapes

    ERIC Educational Resources Information Center

    Tilley, Susan A.; Powick, Kelly D.

    2004-01-01

    In this article, we report on our qualitative study involving eight individuals hired to transcribe research tapes in university contexts. We consider issues of data analysis and data trustworthiness and the implications for both when transcription is assigned to someone other than the researcher. We explore the challenges transcribers faced…

  16. A quick transcribing technique for oral data

    USGS Publications Warehouse

    Schleicher, David

    1972-01-01

    Stenographic techniques offer a means for transcribing oral data accurately and efficiently. In one such application, during five Appolo lunar missions, a rough but helpful transcript was produced within minutes. Similarly, lectures, conferences, and audio tapes can be accurately transcribed as promptly as necessary. Computer programs for translating shorthand notes are being developed; they will increase both speed and accuracy of translation.

  17. Novel genetic diversity within Anopheles punctimacula s.l.: phylogenetic discrepancy between the Barcode cytochrome c oxidase I (COI) gene and the rDNA second internal transcribed spacer (ITS2).

    PubMed

    Loaiza, Jose R; Scott, Marilyn E; Bermingham, Eldredge; Sanjur, Oris I; Rovira, Jose R; Dutari, Larissa C; Linton, Yvonne-Marie; Bickersmith, Sara; Conn, Jan E

    2013-10-01

    Anopheles punctimacula s.l. is a regional malaria vector in parts of Central America, but its role in transmission is controversial due to its unresolved taxonomic status. Two cryptic species, An. malefactor and An. calderoni, have been previously confused with this taxon, and evidence for further genetic differentiation has been proposed. In the present study we collected and morphologically identified adult female mosquitoes of An. punctimacula s.l. from 10 localities across Panama and one in Costa Rica. DNA sequences from three molecular regions, the three prime end of the mitochondrial cytochrome c oxidase I gene (3' COI), the Barcode region in the five prime end of the COI (5' COI), and the rDNA second internal transcribed spacer (ITS2) were used to test the hypothesis of new molecular lineages within An. punctimacula s.l. Phylogenetic analyses using the 3' COI depicted six highly supported molecular lineages (A-F), none of which was An. malefactor. In contrast, phylogenetic inference with the 5' COI demonstrated paraphyly. Tree topologies based on the combined COI regions and ITS2 sequence data supported the same six lineages as the 3' COI alone. As a whole this evidence suggests that An. punctimacula s.l. comprises two geographically isolated lineages, but it is not clear whether these are true species. The phylogenetic structure of the An. punctimacula cluster as well as that of other unknown lineages (C type I vs C type II; D vs E) appears to be driven by geographic partition, because members of these assemblages did not overlap spatially. We report An. malefactor for the first time in Costa Rica, but our data do not support the presence of An. calderoni in Panama.

  18. 7th International Workshop on the Identification of Transcribed Sequences. Beyond the Identification of Transcribed Sequences

    SciTech Connect

    Gardner, Kathleen

    1997-11-19

    The Seventh Annual Human Genome Conference: Beyond the Identification of Transcribed Sequences (BITS) was held November 16-19, 1997 at the Asilomar Conference Center in Monterey, California. The format for the meeting was a combination of oral presentations, group discussions and poster sessions. The original workshop was held to discuss methodologies for the identification of transcribed sequences in mammalian genomes. Over the years, the focus of the workshops has gradually shifted towards functional analysis, with the most dramatic change in emphasis at this meeting, as reflected in the modest change in the workshop title. Topics presented and discussed included: (1) large scale expression and mutational analysis in yeast, C. elegans, Drosophila and zebrafish, (2) comparative mapping of zebrafish, chicken and Fugu; (3) functional analysis in mouse using promoter traps, mutational analysis of biochemical pathways, and Cre/lox constructs; (4) construction of 5 foot end and complete cDNA libraries; (5) expression analysis in mammalian organisms by array screening and differential display; (6) genome organization as determined by detailed transcriptional mapping and genomic sequence analysis; (7) analysis of genomic sequence, including gene and regulatory sequence predictions, annotation of genomic sequence, development of expression databases and verification of sequence analysis predictions; and (8) structural/functional relationships as determined by RNA secondary structure analysis and evolutionary conservation of non-coding sequences.

  19. Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

    PubMed Central

    Lucchini, R; Sogo, J M

    1992-01-01

    The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer. Images PMID:1406621

  20. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells

    PubMed Central

    Arner, Erik; Daub, Carsten O.; Vitting-Seerup, Kristoffer; Andersson, Robin; Lilje, Berit; Drabløs, Finn; Lennartsson, Andreas; Rönnerblad, Michelle; Hrydziuszko, Olga; Vitezic, Morana; Freeman, Tom C.; Alhendi, Ahmad M. N.; Arner, Peter; Axton, Richard; Baillie, J. Kenneth; Beckhouse, Anthony; Bodega, Beatrice; Briggs, James; Brombacher, Frank; Davis, Margaret; Detmar, Michael; Ehrlund, Anna; Endoh, Mitsuhiro; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley; Goldowitz, Daniel; Guler, Reto; Ha, Thomas; Hara, Mitsuko; Herlyn, Meenhard; Ikawa, Tomokatsu; Kai, Chieko; Kawamoto, Hiroshi; Khachigian, Levon M.; Klinken, S. Peter; Kojima, Soichi; Koseki, Haruhiko; Klein, Sarah; Mejhert, Niklas; Miyaguchi, Ken; Mizuno, Yosuke; Morimoto, Mitsuru; Morris, Kelly J.; Mummery, Christine; Nakachi, Yutaka; Ogishima, Soichi; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Qin, Xian-Yang; Roy, Sugata; Sato, Hiroki; Savvi, Suzana; Saxena, Alka; Schwegmann, Anita; Sugiyama, Daisuke; Swoboda, Rolf; Tanaka, Hiroshi; Tomoiu, Andru; Winteringham, Louise N.; Wolvetang, Ernst; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Zabierowski, Susan; Zhang, Peter; Abugessaisa, Imad; Bertin, Nicolas; Diehl, Alexander D.; Fukuda, Shiro; Furuno, Masaaki; Harshbarger, Jayson; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Ishizu, Yuri; Itoh, Masayoshi; Kawashima, Tsugumi; Kojima, Miki; Kondo, Naoto; Lizio, Marina; Meehan, Terrence F.; Mungall, Christopher J.; Murata, Mitsuyoshi; Nishiyori-Sueki, Hiromi; Sahin, Serkan; Nagao-Sato, Sayaka; Severin, Jessica; de Hoon, Michiel J. L.; Kawai, Jun; Kasukawa, Takeya; Lassmann, Timo; Suzuki, Harukazu; Kawaji, Hideya; Summers, Kim M.; Wells, Christine; Hume, David A.; Forrest, Alistair R. R.; Sandelin, Albin; Carninci, Piero; Hayashizaki, Yoshihide

    2015-01-01

    Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation. PMID:25678556

  1. The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription

    PubMed Central

    Bonnet, Jacques; Wang, Chen-Yi; Baptista, Tiago; Vincent, Stéphane D.; Hsiao, Wei-Chun; Stierle, Matthieu; Kao, Cheng-Fu; Tora, László

    2014-01-01

    The SAGA (Spt–Ada–Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription. PMID:25228644

  2. Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

    SciTech Connect

    Niedojadlo, Janusz; Perret-Vivancos, Cecile; Kalland, Karl-Henning; Cmarko, Dusan; Cremer, Thomas; Driel, Roel van; Fakan, Stanislav

    2011-02-15

    The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

  3. Unusual features of the sequences of copies of the 16S-23S rRNA internal transcribed spacer regions of Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi arise from horizontal gene transfer events.

    PubMed

    Maslunka, Christopher; Gürtler, Volker; Seviour, Robert

    2015-02-01

    The highly variable nature of the internal transcribed spacer region (ITS) has been claimed to represent an ideal target for designing species-specific probes/primers capable of differentiating between closely related Acinetobacter species. However, several Acinetobacter species contain multiple ITS copies of variable lengths, and these include Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi. This study shows these length variations result from inter-genomic insertion/deletion events (indels) involving horizontal transfer of ITS fragments of other Acinetobacter species and possibly unrelated bacteria, as shown previously by us. In some instances, indel incorporation results in the loss of probe target sites in the recipient cell ITS. In other cases, some indel sequences contain target sites for probes designed from a single ITS sequence to target other Acinetobacter species. Hence, these can generate false positives. The largest of the indels that remove probe sites is 683 bp (labelled bay/i1-0), and it derives from the horizontal transfer of a complete ITS between A. bereziniae BCRC15423(T) and A. baylyi strain ADP1. As a consequence, ITS sequencing or fingerprinting cannot be used to distinguish between the 683 bp ITS in these two strains.

  4. To Know How a Gene Works, We Need to Redefine It First but then, More Importantly, to Let the Cell Itself Decide How to Transcribe and Process Its RNAs

    PubMed Central

    Jia, Yuping; Chen, Lichan; Ma, Yukui; Zhang, Jian; Xu, Ningzhi; Liao, Dezhong Joshua

    2015-01-01

    Recent genomic and ribonomic research reveals that our genome produces a stupendous amount of non-coding RNAs (ncRNAs), including antisense RNAs, and that many genes contain other gene(s) in their introns. Since ncRNAs either regulate the transcription, translation or stability of mRNAs or directly exert cellular functions, they should be regarded as the fourth category of RNAs, after ribosomal, messenger and transfer RNAs. These and other research advances challenge the current concept of gene and raise a question as to how we should redefine gene. We can either consider each tiny part of the classically-defined gene, such as each mRNA variant, as a “gene”, or, alternatively and oppositely, regard a whole genomic locus as a “gene” that may contain intron-embedded genes and produce different types of RNAs and proteins. Each of the two ways to redefine gene not only has its strengths and weaknesses but also has its particular concern on the methodology for the determination of the gene's function: Ectopic expression of complementary DNA (cDNA) in cells has in the past decades provided us with great deal of detail about the functions of individual mRNA variants, and will make the data less conflicting with each other if just a small part of a classically-defined gene is considered as a “gene”. On the other hand, genomic DNA (gDNA) will better help us in understanding the collective function of a genomic locus. In our opinion, we need to be more cautious in the use of cDNA and in the explanation of data resulting from cDNA, and, instead, should make delivery of gDNA into cells routine in determination of genes' functions, although this demands some technology renovation. PMID:26681921

  5. Tissue-specific activity of the pro-opiomelanocortin gene promoter

    SciTech Connect

    Jeannotte, L.; Trifiro, M.A.; Plante, R.K.; Chamberland, M.; Drouin, J.

    1987-11-01

    The pro-opiomelanocortin (POMC) gene is specifically expressed in corticotroph cells of the anterior pituitary. To define the POMC promoter sequences responsible for tissue-specific expression, we assessed POMC promoter activity by gene transfer into POMC-expressing pituitary tumor cells (AtT-20) and fibroblast L cells. The rat POMC promoter was only efficiently utilized and correctly transcribed in AtT-20 cells. 5'-End deletion analysis revealed two promoter regions for activity in AtT-20 cells. When tested by fusion to a heterologuous promoter, DNA fragments corresponding to both regions exhibited tissue-specific activity, suggesting the presence of at least two tissue-specific DNA sequence elements within the promoter. In summary, POMC promoter sequences from -480 to -34 base pairs appear sufficient to mimic the specificity of anterior pituitary expression.

  6. SUMO functions in constitutive transcription and during activation of inducible genes in yeast.

    PubMed

    Rosonina, Emanuel; Duncan, Sarah M; Manley, James L

    2010-06-15

    Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cell's ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

  7. Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

    PubMed Central

    de Souza, Sandro J.; Camargo, Anamaria A.; Briones, Marcelo R. S.; Costa, Fernando F.; Nagai, Maria Aparecida; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; de Fátima Sonati, Maria; Tajara, Eloiza H.; Valentini, Sandro R.; Acencio, Marcio; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Bengtson, Mário Henrique; Carraro, Dirce M.; Carvalho, Alex F.; Carvalho, Lúcia Helena; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Costa, Maria Cristina R.; Curcio, Cyntia; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Leite, Luciana C. C.; Maia, Gustavo; Majumder, Paromita; Marins, Mozart; Matsukuma, Adriana; Melo, Analy S. A.; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana Gilbert; Rahal, Paula; Rainho, Claudia A.; da Ro's, Nancy; de Sá, Renata G.; Sales, Magaly M.; da Silva, Neusa P.; Silva, Tereza C.; da Silva, Wilson; Simão, Daniel F.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Zalcberg, Heloisa; Brentani, Ricardo R.; Reis, Luis F. L.; Dias-Neto, Emmanuel; Simpson, Andrew J. G.

    2000-01-01

    Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html). PMID:11070084

  8. Modeling the Activity of Single Genes

    NASA Technical Reports Server (NTRS)

    Mjolsness, Eric; Gibson, Michael

    1999-01-01

    The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large

  9. Patterns of Gene-Specific and Total Transcriptional Activity during the Plasmodium falciparum Intraerythrocytic Developmental Cycle ▿ †

    PubMed Central

    Sims, Jennifer S.; Militello, Kevin T.; Sims, Peter A.; Patel, Vishal P.; Kasper, Jacob M.; Wirth, Dyann F.

    2009-01-01

    The relationships among gene regulatory mechanisms in the malaria parasite Plasmodium falciparum throughout its asexual intraerythrocytic developmental cycle (IDC) remain poorly understood. To investigate the level and nature of transcriptional activity and its role in controlling gene expression during the IDC, we performed nuclear run-on on whole-transcriptome samples from time points throughout the IDC and found a peak in RNA polymerase II-dependent transcriptional activity related to both the number of nuclei per parasite and variable transcriptional activity per nucleus over time. These differential total transcriptional activity levels allowed the calculation of the absolute transcriptional activities of individual genes from gene-specific nuclear run-on hybridization data. For half of the genes analyzed, sense-strand transcriptional activity peaked at the same time point as total activity. The antisense strands of several genes were substantially transcribed. Comparison of the transcriptional activity of the sense strand of each gene to its steady-state RNA abundance across the time points assayed revealed both correlations and discrepancies, implying transcriptional and posttranscriptional regulation, respectively. Our results demonstrate that such comparisons can effectively indicate gene regulatory mechanisms in P. falciparum and suggest that genes with diverse transcriptional activity levels and patterns combine to produce total transcriptional activity levels tied to parasite development during the IDC. PMID:19151330

  10. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  11. The internal transcribed spacer region of belonolaimus (nemata: belonolaimidae).

    PubMed

    Cherry, T; Szalanski, A L; Todd, T C; Powers, T O

    1997-03-01

    Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates suggest that the California isolate is a relatively recent introduction into the state.

  12. Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

    PubMed Central

    Weake, Vikki M.; Dyer, Jamie O.; Seidel, Christopher; Box, Andrew; Swanson, Selene K.; Peak, Allison; Florens, Laurence; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2011-01-01

    The Spt–Ada–Gcn5–acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5′ end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression. PMID:21764853

  13. Active and inactive genes localize preferentially in the periphery of chromosome territories

    PubMed Central

    1996-01-01

    The intranuclear position of a set of genes was analyzed with respect to the territories occupied by the whole chromosomes in which these genes are localized. Genes and their respective chromosome territories were simultaneously visualized in three-dimensionally preserved nuclei applying dual color fluorescence in situ hybridization. Three coding (DMD, MYH7, and HBB) and two noncoding sequences (D1Z2 and an anonymous sequence) were analyzed in four different cell types, including cells where DMD and MYH7 are actively transcribed. Spatial analysis by confocal laser scanning microscopy revealed that the genes are preferentially located in the periphery of chromosome territories. This positioning was independent from the activity of the genes. In contrast, the non-expressed anonymous fragment was found randomly distributed or localized preferentially in the interior of the corresponding chromosome territory. Furthermore, the distribution of the analyzed genes within the territorial peripheries was found to be highly characteristic for each gene, and, again, independent from its expression. The impact of these findings with regard to the three- dimensional arrangement of the linear DNA string within chromosome territories, as well as with respect to a putative nuclear subcompartment confining gene expression, are discussed. PMID:8947544

  14. Molecular Systematics of Dictyostelids: 5.8S Ribosomal DNA and Internal Transcribed Spacer Region Analyses▿

    PubMed Central

    Romeralo, María; Escalante, Ricardo; Sastre, Leandro; Lado, Carlos

    2007-01-01

    The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown. PMID:17056743

  15. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote

    PubMed Central

    Schübeler, Dirk; MacAlpine, David M.; Scalzo, David; Wirbelauer, Christiane; Kooperberg, Charles; van Leeuwen, Fred; Gottschling, Daniel E.; O'Neill, Laura P.; Turner, Bryan M.; Delrow, Jeffrey; Bell, Stephen P.; Groudine, Mark

    2004-01-01

    The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity. PMID:15175259

  16. A trypanosome metacyclic VSG gene promoter with two functionally distinct, life cycle stage-specific activities.

    PubMed

    Graham, S V; Wymer, B; Barry, J D

    1998-04-15

    In the mammalian bloodstream, African trypanosomes express the variant surface glycoprotein (VSG), continual switching of which allows evasion of the host immune response. Bloodstream VSG genes are transcribed from polycistronic bloodstream expression sites with promoters which are located 45-60 kb upstream. These promoters are not exclusively stage-regulated, being active in the insect midgut stage where VSG is not expressed. However, the metacyclic VSG (M-VSG) genes, a small subset activated when VSG synthesis begins in the metacyclic stage in the tsetse fly salivary glands, are transcriptionally activated specifically in that stage from promoters <3 kb upstream. Using deletion mapping and transient transfection, we show that the entire 1.22 M-VSG gene promoter region (171 bp) is required for full activity in metacyclic-derived trypanosomes. However, a subsidiary, bloodstream stage-specific activity is present in its 5' half which directs transcription initiation very close to the initiation site used in metacyclic-derived trypanosomes. Our results imply that the M-VSG gene promoter is longer and more complex than other VSG gene promoters.

  17. Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

    PubMed

    Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

    2015-01-01

    Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

  18. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei.

    PubMed Central

    Wolffe, A P

    1989-01-01

    Xenopus sperm chromatin lacks class III transcription complexes and somatic histone H1. Inactive class III genes in sperm chromatin are easily programmed with transcription complexes de novo and transcribed in Xenopus oocyte nuclear extract. In contrast, repressed class III genes in somatic chromatin are not transcribed in the oocyte nuclear extract. Class III genes that are initially inactive or repressed in both types of chromatin can be efficiently transcribed in a cell free preparation of Xenopus eggs. Chromatin mediated repression of class III genes in somatic nuclei is reversible in Xenopus egg extract, but not in the oocyte nuclear extract. Any inhibition of transcription attributed to chromatin assembly onto a gene, will therefore depend on the extract in which transcription is assayed. Images PMID:2915929

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

    PubMed

    Trček, Janja; Barja, François

    2015-03-02

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

  20. Insertions or Deletions (Indels) in the rrn 16S-23S rRNA Gene Internal Transcribed Spacer Region (ITS) Compromise the Typing and Identification of Strains within the Acinetobacter calcoaceticus-baumannii (Acb) Complex and Closely Related Members

    PubMed Central

    Maslunka, Christopher; Gifford, Bianca; Tucci, Joseph; Gürtler, Volker; Seviour, Robert J.

    2014-01-01

    To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2–13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species. PMID:25141005

  1. Transcribed pseudogene ψPPM1K generates endogenous siRNA to suppress oncogenic cell growth in hepatocellular carcinoma

    PubMed Central

    Chan, Wen-Ling; Yuo, Chung-Yee; Yang, Wen-Kuang; Hung, Shih-Ya; Chang, Ya-Sian; Chiu, Chien-Chih; Yeh, Kun-Tu; Huang, Hsien-Da; Chang, Jan-Gowth

    2013-01-01

    Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene ψPPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted ψPPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected ψPPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of ψPPM1K. Furthermore, expression of NEK8 in ψPPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of ψPPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth. PMID:23376929

  2. A genome-wide function of THSC/TREX-2 at active genes prevents transcription–replication collisions

    PubMed Central

    Santos-Pereira, José M.; García-Rubio, María L.; González-Aguilera, Cristina; Luna, Rosa; Aguilera, Andrés

    2014-01-01

    The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3′ end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription–replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis. PMID:25294824

  3. Total DNA transcription in vitro: a procedure to detect highly repetitive and transcribable sequences with tRNA-like structures

    SciTech Connect

    Endoh, H.; Okada, N.

    1986-01-01

    Total DNAs from various animals were transcribed in vitro in a HeLa cell extract, and it was found that one to several discrete RNAs were transcribed by RNA polymerase III. With tortoise (Geoclemys reevessi) and newt (Cynops pyrrhogaster), distinct 6.5S and 8S RNAs were transcribed from these respective DNAs. Representative phage clones carrying the 6.5S and 8S RNA genes were isolated from genomic libraries of these animals, and the sequences of these genes were determined. The 5' parts of highly repetitive and transcribable sequences of tortoise and newt were found to have close resemblance to tRNA/sub 1//sup Lys/ (rabbit) gene (78% homology) and a tRNA/sup Glu/ (Drosophila) gene (74% homology, not counting the aminoacyl stem region), respectively. The homologies extended to secondary structures, homologous nucleotides being located on similar secondary structures. It is proposed that many, if not all, highly repetitive and transcribable sequences detected by total DNA transcription have specific tRNA genes as their progenitors.

  4. Bone Regeneration Using Gene-Activated Matrices.

    PubMed

    D'Mello, Sheetal; Atluri, Keerthi; Geary, Sean M; Hong, Liu; Elangovan, Satheesh; Salem, Aliasger K

    2017-01-01

    Gene delivery to bone is a potential therapeutic strategy for directed, sustained, and regulated protein expression. Tissue engineering strategies for bone regeneration include delivery of proteins, genes (viral and non-viral-mediated delivery), and/or cells to the bone defect site. In addition, biomimetic scaffolds and scaffolds incorporating bone anabolic agents greatly enhance the bone repair process. Regional gene therapy has the potential of enhancing bone defect healing and bone regeneration by delivering osteogenic genes locally to the osseous lesions, thereby reducing systemic toxicity and the need for using supraphysiological dosages of therapeutic proteins. By implanting gene-activated matrices (GAMs), sustained gene expression and continuous osteogenic protein production in situ can be achieved in a way that stimulates osteogenesis and bone repair within osseous defects. Critical parameters substantially affecting the therapeutic efficacy of gene therapy include the choice of osteogenic transgene(s), selection of non-viral or viral vectors, the wound environment, and the selection of ex vivo and in vivo gene delivery strategies, such as GAMs. It is critical for gene therapy applications that clinically beneficial amounts of proteins are synthesized endogenously within and around the lesion in a sustained manner. It is therefore necessary that reliable and reproducible methods of gene delivery be developed and tested for their efficacy and safety before translating into clinical practice. Practical considerations such as the age, gender, and systemic health of patients and the nature of the disease process also need to be taken into account in order to personalize the treatments and progress towards developing a clinically applicable gene therapy for healing bone defects. This review discusses tissue engineering strategies to regenerate bone with specific focus on non-viral gene delivery systems.

  5. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

    PubMed

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-06-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

  6. Gene activation-associated long noncoding RNAs function in mouse preimplantation development

    PubMed Central

    Hamazaki, Nobuhiko; Uesaka, Masahiro; Nakashima, Kinichi; Agata, Kiyokazu; Imamura, Takuya

    2015-01-01

    In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development. PMID:25633350

  7. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    PubMed Central

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  8. Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator.

    PubMed Central

    Sheehan, B; Klarsfeld, A; Msadek, T; Cossart, P

    1995-01-01

    PrfA is a pleiotropic activator of virulence gene expression in the pathogenic bacterium Listeria monocytogenes. Several lines of evidence have suggested that a hierarchy of virulence gene activation by PrfA exists. This hypothesis was investigated by assessing the ability of PrfA to activate the expression of virulence gene fusions to lacZ in Bacillus subtilis. Expression of PrfA in this heterologous host was sufficient for activation of transcription at the hly, plcA, mpl, and actA promoters. Activation was most efficient at the divergently transcribed hly and plcA promoters. The putative PrfA binding site shared by these promoters is perfectly symmetrical and appears to represent the optimum sequence for target gene activation by PrfA. The activation of actA and mpl expression was considerably weaker and occurred more slowly than that observed at the hly and plcA promoters, suggesting that greater quantities of PrfA are required for productive interaction at these promoters. Interestingly, expression of an inlA-lacZ transcriptional fusion was very poorly activated by PrfA in B. subtilis, suggesting that other Listeria factors, in addition to PrfA, are required for PrfA-mediated activation at this promoter. Further support for the involvement of such factors was obtained by constructing and analyzing a prfA deletion mutant of L. monocytogenes. We observed that, in contrast to that of the other genes of the PrfA regulon, expression of inlA is only partially dependent on PrfA. PMID:7592422

  9. Transcribed processed pseudogenes in the human genome: an intermediate form of expressed retrosequence lacking protein-coding ability.

    PubMed

    Harrison, Paul M; Zheng, Deyou; Zhang, Zhaolei; Carriero, Nicholas; Gerstein, Mark

    2005-01-01

    Pseudogenes, in the case of protein-coding genes, are gene copies that have lost the ability to code for a protein; they are typically identified through annotation of disabled, decayed or incomplete protein-coding sequences. Processed pseudogenes (PPsigs) are made through mRNA retrotransposition. There is overwhelming genomic evidence for thousands of human PPsigs and also dozens of human processed genes that comprise complete retrotransposed copies of other genes. Here, we survey for an intermediate entity, the transcribed processed pseudogene (TPPsig), which is disabled but nonetheless transcribed. TPPsigs may affect expression of paralogous genes, as observed in the case of the mouse makorin1-p1 TPPsig. To elucidate their role, we identified human TPPsigs by mapping expressed sequences onto PPsigs and, reciprocally, extracting TPPsigs from known mRNAs. We consider only those PPsigs that are homologous to either non-mammalian eukaryotic proteins or protein domains of known structure, and require detection of identical coding-sequence disablements in both the expressed and genomic sequences. Oligonucleotide microarray data provide further expression verification. Overall, we find 166-233 TPPsigs ( approximately 4-6% of PPsigs). Proteins/transcripts with the highest numbers of homologous TPPsigs generally have many homologous PPsigs and are abundantly expressed. TPPsigs are significantly over-represented near both the 5' and 3' ends of genes; this suggests that TPPsigs can be formed through gene-promoter co-option, or intrusion into untranslated regions. However, roughly half of the TPPsigs are located away from genes in the intergenic DNA and thus may be co-opting cryptic promoters of undesignated origin. Furthermore, TPPsigs are unlike other PPsigs and processed genes in the following ways: (i) they do not show a significant tendency to either deposit on or originate from the X chromosome; (ii) only 5% of human TPPsigs have potential orthologs in mouse. This

  10. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  11. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food

    PubMed Central

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2015-01-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411. PMID:26981389

  12. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food.

    PubMed

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2016-03-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411.

  13. The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes

    PubMed Central

    Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos

    2011-01-01

    Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process via the distribution of transcribed nucleosome positions. Significantly, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and that this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer, and histone-histone dissociation quantitatively rationalizes our observations and unifies results obtained with other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression. PMID:22081017

  14. Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain-filling-related genes.

    PubMed

    Fan, Jing; Guo, Xiao-Yi; Li, Liang; Huang, Fu; Sun, Wen-Xian; Li, Yan; Huang, Yan-Yan; Xu, Yong-Ju; Shi, Jun; Lei, Yang; Zheng, Ai-Ping; Wang, Wen-Ming

    2015-06-01

    Rice false smut has become an increasingly serious disease in rice (Oryza sativa L.) production worldwide. The typical feature of this disease is that the fungal pathogen Ustilaginoidea virens (Uv) specifically infects rice flower and forms false smut ball, the ustiloxin-containing ball-like fungal colony, of which the size is usually several times larger than that of a mature rice seed. However, the underlying mechanisms of Uv-rice interaction are poorly understood. Here, we applied time-course microscopic and transcriptional approaches to investigate rice responses to Uv infection. The results demonstrated that the flower-opening process and expression of associated transcription factors, including ARF6 and ARF8, were inhibited in Uv-infected spikelets. The ovaries in infected spikelets were interrupted in fertilization and thus were unable to set seeds. However, a number of grain-filling-related genes, including seed storage protein genes, starch anabolism genes and endosperm-specific transcription factors (RISBZ1 and RPBF), were highly transcribed as if the ovaries were fertilized. In addition, critical defense-related genes like NPR1 and PR1 were downregulated by Uv infection. Our data imply that Uv may hijack host nutrient reservoir by activation of the grain-filling network because of growth and formation of false smut balls.

  15. Aym1, a mouse meiotic gene identified by virtue of its ability to activate early meiotic genes in the yeast Saccharomyces cerevisiae.

    PubMed

    Malcov, Mira; Cesarkas, Karen; Stelzer, Gil; Shalom, Sarah; Dicken, Yosef; Naor, Yaniv; Goldstein, Ronald S; Sagee, Shira; Kassir, Yona; Don, Jeremy

    2004-12-01

    Our understanding of the molecular mechanisms that operate during differentiation of mitotically dividing spermatogonia cells into spermatocytes lags way behind what is known about other differentiating systems. Given the evolutionary conservation of the meiotic process, we screened for mouse proteins that could specifically activate early meiotic promoters in Saccharomyces cerevisiae yeast cells, when fused to the Gal4 activation domain (Gal4AD). Our screen yielded the Aym1 gene that encodes a short peptide of 45 amino acids. We show that a Gal4AD-AYM1 fusion protein activates expression of reporter genes through the promoters of the early meiosis-specific genes IME2 and HOP1, and that this activation is dependent on the DNA-binding protein Ume6. Aym1 is transcribed predominantly in mouse primary spermatocytes and in gonads of female embryos undergoing the corresponding meiotic divisions. Aym1 immunolocalized to nuclei of primary spermatocytes and oocytes and to specific type A spermatogonia cells, suggesting it might play a role in the processes leading to meiotic competence. The potential functional relationship between AYM1 and yeast proteins that regulate expression of early meiotic genes is discussed.

  16. Phylogenetic analysis of Antrodia species and Antrodia camphorata inferred from internal transcribed spacer region.

    PubMed

    Chiu, Hua-Hsien

    2007-04-01

    The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.

  17. Assessment of Transcriptional Activity of Borrelia burgdorferi and Host Cytokine Genes During Early and Late Infection in a Mouse Model

    PubMed Central

    Feng, Sunlian; Barthold, Stephen W.

    2013-01-01

    Abstract Differential gene expression by Borrelia burgdorferi spirochetes during mammalian infection facilitates their dissemination as well as immune evasion. Modulation of gene transcription in response to host immunity has been documented with the outer surface protein C, but the influence of transcription of other genes is largely unknown. A low-density array (LDA) was developed to study transcriptional activity of 43 B. burgdorferi genes and 19 host genes that may be involved in various host–agent interactions. Gene transcription in heart, joint, and muscle tissue was compared in immunocompetent C3H and immunodeficient C3H-scid mice during early (3 weeks) and late (2 months) B. burgdorferi infection. Among all tissue types, levels of relative transcription of over 80% of B. burgdorferi genes tested were one- to nine-fold less in C3H mice compared to C3H-scid mice. At the later time point, all genes were transcribed in C3H-scid mice, whereas transcription of 16 genes out of 43 tested was not detected in analyzed tissues of C3H mice. Our data suggest that during infection of immunocompetent mice, a majority of B. burgdorferi genes tested are downregulated in response to acquired host immunity. LDA revealed variable patterns of host gene expression in different tissues and at different intervals in infected mice. Higher levels of relative expression for IL-10 during both early and late infection were detected in heart base, and it was unchanged in the tibiotarsal joint. Comparative analysis of B. burgdorferi and host genes transcriptional activity revealed that increased flaB mRNA during early infection was followed by increases of CCL7, CCL8, interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in all assessed tissue types. LDA represents a valuable approach for sensitive and quantitative gene transcription profiling and for understanding Lyme borreliosis. PMID:23930938

  18. Training and Availability of Braille Transcribers in the United States.

    ERIC Educational Resources Information Center

    Corn, Anne L.; Wall, Robert S.

    2002-01-01

    A survey of Braille transcribers in 40 states found Braille production systems rely on volunteers and hence Braille transcription is not considered a bona fide career. The majority cited low funding and resources as obstacles to change. Issues of certification, training, availability, and definition are discussed, along with recommendations.…

  19. Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    The relationships among transcription, recombination, DNA damage, and repair in mammalian cells were investigated. We monitored the effects of transcription on UV-induced intrachromosomal recombination between neomycin repeats including a promoterless allele and an inducible heteroallele regulated by the mouse mammary tumor virus promoter. Although transcription and UV light separately stimulated recombination, increasing transcription levels reduced UV-induced recombination. Preferential repair of UV damage in transcribed strands was shown in highly transcribed DNA, suggesting that recombination is stimulated by unrepaired UV damage and that increased DNA repair in highly transcribed alleles removes recombinogenic lesions. This study indicates that the genetic consequences of DNA damage depend on transcriptional states and provides a basis for understanding tissue- and gene-specific responses to DNA-damaging agents. Images PMID:8264606

  20. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  1. 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.

  2. Mechanism of activation of the mouse c-mos oncogene by the LTR of an intracisternal A-particle gene.

    PubMed Central

    Horowitz, M; Luria, S; Rechavi, G; Givol, D

    1984-01-01

    In the mouse myeloma XRPC-24 the DNA of an intracisternal A-particle (IAP) is inserted within the coding region of c-mos. This insertion splits the c-mos into a 3' rc-mos and a 5' rc-mos separated by approximately 4.7 kb of IAP DNA. The insertion is in a head-to-head orientation and brings the 5' LTR of the IAP in juxtaposition to the 3' rc-mos such that the IAP and the 3' rc-mos are transcribed in opposite directions. The intact c-mos gene is usually dormant, whereas the 3' rc-mos is actively transcribed and is capable of transforming NIH3T3 cells. In an effort to understand the nature of this activation we mapped the 5' ends of the 3' rc-mos mRNA present in XPRC-24. We found two main mRNA start sites, one mapping to the junction of the 3' rc-mos and the 5' LTR, and the other located 10 nucleotides upstream to this junction, within the 5' LTR. This result indicates that the 3' rc-mos in XRPC-24 was activated by insertion of a promoter provided by the LTR of an IAP genome. Furthermore, the 5' LTR appears to possess promoter activities in two directions. This conclusion was confirmed by the fact that this 5' LTR, in both orientations, was able to activate the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in the modular vector pSVOCAT. Images Fig. 2. Fig. 5. PMID:6098457

  3. RNA polymerase I transcription factors in active yeast rRNA gene promoters enhance UV damage formation and inhibit repair.

    PubMed

    Meier, Andreas; Thoma, Fritz

    2005-03-01

    UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.

  4. Vibrio parahaemolyticus CalR down regulates the thermostable direct hemolysin (TDH) gene transcription and thereby inhibits hemolytic activity.

    PubMed

    Zhang, Yiquan; Zhang, Ying; Gao, He; Zhang, Lingyu; Yin, Zhe; Huang, Xinxiang; Zhou, Dongsheng; Yang, Huiying; Yang, Wenhui; Wang, Li

    2017-03-04

    TDH, encoded by tdh gene, is a major virulent determinant of V. parahaemolyticus that controls various biological activities, such as hemolytic activity, cytotoxicity, and enterotoxicity. The hemolytic activity on Wagatsuma agar ascribed to TDH is called Kanagawa phenomenon (KP). All KP positive strains contain tdh1 and tdh2 genes, but tdh2 is predominantly responsible for KP. CalR is a regulatory protein that was originally identified as a repressor of swarming motility and T3SS1 gene expression in V. parahaemolyticus. In the present study, the regulation of tdh2 by CalR was investigated using a set of experiments including qRT-PCR, primer extension, LacZ fusion, hemolytic phenotype, EMSA, and DNase I footprinting assays. The results showed that His-CalR protected a single region from 224bp to 318bp upstream of tdh2 against DNase I digestion, and a transcriptional start site located at 42bp upstream of tdh2 was detected and its transcribed activity was inhibited by CalR. Moreover, the KP test results showed that the hemolytic activity of V. parahaemolyticus is also under negative control of CalR. The data demonstrated that CalR is a repressor of the tdh2 transcription and thereby inhibits the hemolytic activity of V. parahaemolyticus.

  5. Cancer genes: rare recombinants instead of activated oncogenes (a review).

    PubMed Central

    Duesberg, P H

    1987-01-01

    The 20 known transforming (onc) genes of retroviruses are defined by sequences that are transduced from cellular genes termed protooncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox, since such genes are clearly undesirable. The hypothesis predicts that viral onc genes and proto-onc genes are isogenic; that expression of proto-onc genes induces tumors; that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes; and that diploid tumors exist. As yet, none of these predictions is confirmed. Instead: Structural comparisons between viral onc genes, essential retroviral genes, and proto-onc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are recombinants put together from truncated viral and truncated proto-onc genes. Proto-onc genes are frequently expressed in normal cells. To date, not one activated proto-onc gene has been isolated that transforms diploid cells. Above all, no diploid tumors with activated proto-onc genes have been found. Moreover, the probability of spontaneous transformation in vivo is at least 10(9) times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore, the hypothesis that proto-onc genes are latent cellular oncogenes appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germ-line configuration of cellular genes generate viral and possibly cellular cancer genes. The clonal chromosome

  6. Activities of Human Gene Nomenclature Committee

    SciTech Connect

    2002-07-16

    The objective of this project, shared between NIH and DOE, has been and remains to enable the medical genetics communities to use common names for genes that are discovered by different gene hunting groups, in different species. This effort provides consistent gene nomenclature and approved gene symbols to the community at large. This contributes to a uniform and consistent understanding of genomes, particularly the human as well as functional genomics based on comparisons between homologous genes in related species (human and mice).

  7. Activation of Cryptic hop Genes from Streptomyces peucetius ATCC 27952 Involved in Hopanoid Biosynthesis.

    PubMed

    Ghimire, Gopal Prasad; Koirala, Niranjan; Sohng, Jae Kyung

    2015-05-01

    Genes encoding enzymes with sequence similarity to hopanoids biosynthetic enzymes of other organisms were cloned from the hopanoid (hop) gene cluster of Streptomyces peucetius ATCC 27952 and transformed into Streptomyces venezuelae YJ028. The cloned fragments contained four genes, all transcribed in one direction. These genes encode polypeptides that resemble polyprenyl diphosphate synthase (hopD), squalene-phytoene synthases (hopAB), and squalenehopene cyclase (hopE). These enzymes are sufficient for the formation of the pentacyclic triterpenoid lipid, hopene. The formation of hopene was verified by gas chromatography/ mass spectrometry.

  8. PcaU, a Transcriptional Activator of Genes for Protocatechuate Utilization in Acinetobacter†

    PubMed Central

    Gerischer, Ulrike; Segura, Ana; Ornston, L. Nicholas

    1998-01-01

    The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are qui and pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the pca-qui-pob cluster was pobR, which encodes a transcriptional activator that responds to p-hydroxybenzoate and activates transcription of pobA. The pca and qui genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from pcaI and prevented expression of the pca genes. Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated pcaU. Downstream from pcaU are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaU intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the pob operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The pob and pca systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking pca

  9. Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data.

    PubMed

    Conti, Anastasia; Carnevali, Davide; Bollati, Valentina; Fustinoni, Silvia; Pellegrini, Matteo; Dieci, Giorgio

    2015-01-01

    Of the ∼ 1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq data sets and unique Alu DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual Alu elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ∼ 1300 Alu loci corresponding to detectable transcripts, with ∼ 120 of them expressed in at least three cell lines. In vitro transcription of selected Alus did not reflect their in vivo expression properties, and required the native 5'-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for Alus nested within Pol II-transcribed genes. The ability to investigate Alu transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant Alu RNAs and the assessment of Alu expression alteration under pathological conditions.

  10. Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data

    PubMed Central

    Conti, Anastasia; Carnevali, Davide; Bollati, Valentina; Fustinoni, Silvia; Pellegrini, Matteo; Dieci, Giorgio

    2015-01-01

    Of the ∼1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq data sets and unique Alu DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual Alu elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ∼1300 Alu loci corresponding to detectable transcripts, with ∼120 of them expressed in at least three cell lines. In vitro transcription of selected Alus did not reflect their in vivo expression properties, and required the native 5′-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for Alus nested within Pol II-transcribed genes. The ability to investigate Alu transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant Alu RNAs and the assessment of Alu expression alteration under pathological conditions. PMID:25550429

  11. Transcriptome map of plant mitochondria reveals islands of unexpected transcribed regions

    PubMed Central

    2011-01-01

    Background Plant mitochondria contain a relatively large amount of genetic information, suggesting that their functional regulation may not be as straightforward as that of metazoans. We used a genomic tiling array to draw a transcriptomic atlas of Oryza sativa japonica (rice) mitochondria, which was predicted to be approximately 490-kb long. Results Whereas statistical analysis verified the transcription of all previously known functional genes such as the ones related to oxidative phosphorylation, a similar extent of RNA expression was frequently observed in the inter-genic regions where none of the previously annotated genes are located. The newly identified open reading frames (ORFs) predicted in these transcribed inter-genic regions were generally not conserved among flowering plant species, suggesting that these ORFs did not play a role in mitochondrial principal functions. We also identified two partial fragments of retrotransposon sequences as being transcribed in rice mitochondria. Conclusion The present study indicated the previously unexpected complexity of plant mitochondrial RNA metabolism. Our transcriptomic data (Oryza sativa Mitochondrial rna Expression Server: OsMES) is publicly accessible at [http://bioinf.mind.meiji.ac.jp/cgi-bin/gbrowse/OsMes/#search]. PMID:21627843

  12. 11p15-subband specific search for transcribed sequences using exon trapping

    SciTech Connect

    Loebbert, R.; Prawitt, D.; Monroe, D.

    1994-09-01

    Evidence from cytogenetic and molecular data suggest that the region 11p15 contains genes involved in different disorders, like Beckwith-Wiedemann syndrome (BWS), long QT syndrome (LQT), Usher syndrome type I and tumor development. Focusing on the subregion 11p15.1, we are isolating and characterizing new transcribed sequences. The applied strategy includes exon amplification and subsequent PCR screening of cDNA libraries. So far 100 YACs and 38 cosmid clones from 11p15.1-15.3 have been collected and are currently arrayed. 16 cosmids have been analyzed for transcribed sequences using the exon amplification scheme developed by Buckler et al. (1991). We were able to identify 18 exons that contain correct open reading frames and map back to the cosmid clones. A data base search revealed that two exons represent parts of known genes from this region (ST5 and AMPD3). Moreover, we identified one exon that represents an EGF-like repeat with homologies to various proteins. Using PCR and primers from the exon sequences, a fetal brain library, which has been arranged in the form of hierarchic arrayed phage pools, was screened. Up to now, two cDNA clones corresponding to different exons were isolated and are currently sequenced.

  13. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  14. Project to transcribe old ship logs provides important weather data

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2012-11-01

    Kathy Wendolkowski is a citizen scientist. It's a term that Wendolkowski considers far too lofty for what she claims is simply a happy addiction that she and others have for transcribing old logs from naval ship and other vessels. They perform this task to glean the regularly recorded weather data from those logs for the benefit of science. For Wendolkowski, though, greater satisfaction comes from reading what the logs also reveal about the daily lives of the sailors as well as any accompanying historical drama.

  15. The human NTT gene: Identification of a novel 17-kb noncoding nuclear RNA expressed in activated CD4{sup +} T cells

    SciTech Connect

    Liu, A.Y.; Torchia, B.S.; Migeon, B.R.

    1997-01-15

    We describe the cloning and characterization of the NTT gene (noncoding transcript in T cells), identified by differential display RT-PCR based on the differential presence of its transcript in a subset of activated, human CD4{sup +} T-cell clones. The full-length cDNA and genomic sequences were cloned and found to produce a 17-kb transcript that is polyadenylated, but is not spliced. Consistent with the transcript`s nuclear predominance, NTT has no open reading frame larger than 270 bp. It is transcribed in a select subset of CD4{sup +} T-cell clones propagated in vitro. Its transcription can also be induced in freshly isolated T cells by in vitro activation with PHA or with PMA and ionomycin. In vivo, NTT transcripts are found only in activated, but not resting, T cells. Transcripts were absent in a variety of lymphoid cell lines and transformed lines from other tissues. NTT is a new member of the small group of genes including XIST (X{sub i}-specific transcript), H19, and IPW (imprinted gene in the Prader-Willi syndrome region), which are transcribed but not translated, and may have a role in the regulation of neighboring genes. XIST, H19, and IPW exhibit monoallelic expression, but both NTT alleles are expressed in CD4{sup +} T-cell clones. Southern blot and fluorescence in situ hybridization analyses show that NTT is a single-copy gene residing in chromosome 6q23-q24, near the interferon-{gamma} receptor gene (IFN-{gamma}R). Their proximity and shared expression pattern suggest a possible functional relationship. 57 refs., 10 figs., 1 tab.

  16. Correspondence between resting state activity and brain gene expression

    PubMed Central

    Wang, Guang-Zhong; Belgard, T. Grant; Mao, Deng; Chen, Leslie; Berto, Stefano; Preuss, Todd M.; Lu, Hanzhang; Geschwind, Daniel H.; Konopka, Genevieve

    2015-01-01

    SUMMARY The relationship between functional brain activity and gene expression has not been fully explored in the human brain. Here, we identify significant correlations between gene expression in the brain and functional activity by comparing fractional Amplitude of Low Frequency Fluctuations (fALFF) from two independent human fMRI resting state datasets to regional cortical gene expression from a newly generated RNA-seq dataset and two additional gene expression datasets to obtain robust and reproducible correlations. We find significantly more genes correlated with fALFF than expected by chance, and identify specific genes correlated with the imaging signals in multiple expression datasets in the default mode network. Together, these data support a population-level relationship between regional steady state brain gene expression and resting state brain activity. PMID:26590343

  17. Evolution of Brain Active Gene Promoters in Human Lineage Towards the Increased Plasticity of Gene Regulation.

    PubMed

    Gunbin, Konstantin V; Ponomarenko, Mikhail P; Suslov, Valentin V; Gusev, Fedor; Fedonin, Gennady G; Rogaev, Evgeny I

    2017-02-24

    Adaptability to a variety of environmental conditions is a prominent feature of Homo sapiens. We hypothesize that this feature can be explained by evolutionary changes in gene promoters active in the brain prefrontal cortex leading to a more flexible gene regulation network. The genotype-dependent range of gene expression can be broader in humans than in other higher primates. Thus, we searched for specific signatures of evolutionary changes in promoter architectures of multiple hominid genes, including the genes active in human cortical neurons that may indicate an increase of variability of gene expression rather than just changes in the level of expression, such as downregulation or upregulation of the genes. We performed a whole-genome search for genetic-based alterations that may impact gene regulation "flexibility" in a process of hominids evolution, such as (i) CpG dinucleotide content, (ii) predicted nucleosome-DNA dissociation constant, and (iii) predicted affinities for TATA-binding protein (TBP) in gene promoters. We tested all putative promoter regions across the human genome and especially gene promoters in active chromatin state in neurons of prefrontal cortex, the brain region critical for abstract thinking and social and behavioral adaptation. Our data imply that the origin of modern man has been associated with an increase of flexibility of promoter-driven gene regulation in brain. In contrast, after splitting from the ancestral lineages of H. sapiens, the evolution of ape species is characterized by reduced flexibility of gene promoter functioning, underlying reduced variability of the gene expression.

  18. The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.

    PubMed Central

    Verhage, R; Zeeman, A M; de Groot, N; Gleig, F; Bang, D D; van de Putte, P; Brouwer, J

    1994-01-01

    The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs. Images PMID:8065346

  19. Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease

    PubMed Central

    Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia; Gootenberg, Jonathan S.; Zhang, Feng; Konermann, Silvana

    2015-01-01

    We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct sgRNA constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14 15 bp target sequences and MS2 binding loops, can activate gene expression using an active Cas9 nuclease, without inducing DSBs. We use these ‘dead RNAs’ to perform orthogonal gene knockout and transcriptional activation in human cells. PMID:26436575

  20. 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.

  1. Constitutive androstane receptor activation evokes the expression of glycolytic genes.

    PubMed

    Yarushkin, Andrei A; Kazantseva, Yuliya A; Prokopyeva, Elena A; Markova, Diana N; Pustylnyak, Yuliya A; Pustylnyak, Vladimir O

    2016-09-23

    It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in a mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation.

  2. Photobleaching reveals complex effects of inhibitors on transcribing RNA polymerase II in living cells

    SciTech Connect

    Fromaget, Maud; Cook, Peter R. . E-mail: peter.cook@path.ox.ac.uk

    2007-08-15

    RNA polymerase II transcribes most eukaryotic genes. Photobleaching studies have revealed that living Chinese hamster ovary cells expressing the catalytic subunit of the polymerase tagged with the green fluorescent protein contain a large rapidly exchanging pool of enzyme, plus a smaller engaged fraction; genetic complementation shows this tagged polymerase to be fully functional. We investigated how transcriptional inhibitors - some of which are used therapeutically - affect the engaged fraction in living cells using fluorescence loss in photobleaching; all were used at concentrations that have reversible effects. Various kinase inhibitors (roscovitine, DRB, KM05283, alsterpaullone, isoquinolinesulfonamide derivatives H-7, H-8, H-89, H-9), proteasomal inhibitors (lactacystin, MG132), and an anti-tumour agent (cisplatin) all reduced the engaged fraction; an intercalator (actinomycin D), two histone deacetylase inhibitors (trichostatin A, sodium butyrate), and irradiation with ultra-violet light all increased it. The polymerase proves to be both a sensitive sensor and effector of the response to these inhibitors.

  3. Nucleotide sequence of the internal transcribed spacers and 5.8S region of ribosomal DNA in Pinus pinea L.

    PubMed

    Marrocco, R; Gelati, M T; Maggini, F

    1996-01-01

    The nucleotide sequence of the first internal transcribed spacer (ITS1) belonging to different ribosomal RNA genes from Pinus pinea are reported. The analyzed ITS1 can be distinguished on the basis of their length, being one 2631 bp and the other 271 bp long. Nucleotide comparison of these regions did not show appreciable sequence homology. The larger ITS1 contains five tandem arranged subrepeats with size ranging between 219 bp and 237 bp. The nucleotide sequence of the 5.8S and the ITS2 regions belonging to the larger ribosomal RNA gene are also reported.

  4. A yeast transcription system for the 5S rRNA gene.

    PubMed Central

    van Keulen, H; Thomas, D Y

    1982-01-01

    A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA genes packaged in chromatin. Images PMID:7145700

  5. Transcriptional activation of RNA polymerase III-dependent genes by the human T-cell leukemia virus type 1 tax protein.

    PubMed Central

    Gottesfeld, J M; Johnson, D L; Nyborg, J K

    1996-01-01

    The human T-cell leukemia virus-encoded tax protein is a potent activator of many viral and cellular genes transcribed by RNA polymerase II. We find that both chromatin and cell extracts derived from human T-cell leukemia virus type 1-infected human T lymphocytes support higher levels of 5S rRNA and tRNA gene transcription than chromatin or extracts from uninfected T lymphocytes. The viral protein Tax was likely responsible for this higher level of class II gene transcription, as purified Tax was found to stimulate both genes when added to the uninfected cell extract or in reconstituted systems. Both limiting-component transcription assays and DNA binding assays identified the class III gene transcription factor TFIIIB as the principle target of Tax activity. Surprisingly, we find that Tax increases the effective concentration of active TFIIIB molecules. These data suggest that Tax stimulates RNA polymerase III-dependent gene expression by accelerating the rate and/or extent of transcription initiation complex assembly. PMID:8657153

  6. Carcinogen-induced trans activation of gene expression.

    PubMed Central

    Kleinberger, T; Flint, Y B; Blank, M; Etkin, S; Lavi, S

    1988-01-01

    We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later. Images PMID:2835673

  7. Carcinogen-induced trans activation of gene expression

    SciTech Connect

    Kleinberger, T.; Flint, Y.B.; Blank, M.; Etkin, S.; Lavi, S.

    1988-03-01

    The authors report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later.

  8. The Escherichia coli orthologue of the Salmonella ushB gene (ushB(c)) produces neither UDP-sugar hydrolase activity nor detectable protein, but has an identical sequence to that of Escherichia coli cdh.

    PubMed

    Schroder, W; Burger, M; Edwards, C; Douglas, M; Innes, D; Beacham, I R; Burns, D M

    2001-09-11

    Salmonella ushB, which encodes a membrane-bound UDP-sugar hydrolase, has an Escherichia coli orthologue (ushB(c)) which does not detectably produce this activity. In this report, we show that ushB(c) does not produce any detectable protein either, despite being transcribed normally. Remarkably, ushB(c) is shown to have 100% sequence identity with E. coli cdh, previously characterised as encoding an active CDP-diglyceride hydrolase, an apparent contradiction with implications regarding enzyme evolution. We suggest that a useful gene designation is cdh (ushB(c)) rather than either ushB(c) or cdh, alone.

  9. Dietary Methanol Regulates Human Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Sheshukova, Ekaterina V.; Kosorukov, Vyacheslav S.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  10. Analysis of Mammalian rDNA Internal Transcribed Spacers

    PubMed Central

    Coleman, Annette W.

    2013-01-01

    Nuclear rDNA Internal Transcribed Spacers, ITS1 and ITS2, are widely used for eukaryote phylogenetic studies from the ordinal level to the species level, and there is even a database for ITS2 sequences. However, ITS regions have been ignored in mammalian phylogenetic studies, and only a few rodent and ape sequences are represented in GenBank. The reasons for this dearth, and the remedies, are described here. We have recovered these sequences, mostly >1 kb in length, for 36 mammalian species. Sequence alignment and transcript folding comparisons reveal the rRNA transcript secondary structure. Mammalian ITS regions, though quite long, still fold into the recognizable secondary structure of other eukaryotes. The ITS2 in particular bears the four standard helix loops, and loops II and III have the hallmark characters universal to eukaryotes. Both sequence and insertions/deletions of transcript secondary structure helices observed here support the four superorder taxonomy of Placentalia. On the family level, major unique indels, neatly excising entire helices, will be useful when additional species are represented, resulting in significant further understanding of the details of mammalian evolutionary history. Furthermore, the identification of a highly conserved element of ITS1 common to warm-blooded vertebrates may aid in deciphering the complex mechanism of RNA transcript processing. This is the last major group of terrestrial vertebrates for which rRNA ITS secondary structure has been resolved. PMID:24260162

  11. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene.

    PubMed Central

    Fuqua, C; Burbea, M; Winans, S C

    1995-01-01

    The Agrobacterium Ti plasmid tra regulon was previously found to be positively regulated by the TraR protein in the presence of a diffusible N-acyl homoserine lactone designated Agrobacterium autoinducer (AAI). TraR and AAI are similar to LuxR from Vibrio fischeri and the Vibrio autoinducer (VAI), which regulate target bioluminescence (lux) genes in a cell density-dependent manner. We now show that tra genes are also regulated by a second protein, designated TraM, which acts to antagonize TraR-dependent activation. The traM gene is closely linked to traR, and the two genes are transcribed convergently. The predicted TraM proteins of two different Ti plasmids are 77% identical but are not significantly similar to other protein sequences in the database, and thus TraM may represent a novel regulatory protein. Null mutations in traM cause strongly increased conjugation, tra gene transcription, and AAI production. A functional copy of traM introduced into traM mutants decreased conjugation, tra gene transcription, and AAI synthesis. TraM inhibits transcription of traA, traI, and traM. Although traM was first identified by its octopine-inducible promoter, we now show that induction by octopine requires traR, strongly suggesting that TraR is the direct traM activator. PMID:7868612

  12. Physical activity in adulthood: genes and mortality

    PubMed Central

    Karvinen, Sira; Waller, Katja; Silvennoinen, Mika; Koch, Lauren G.; Britton, Steven L.; Kaprio, Jaakko; Kainulainen, Heikki; Kujala, Urho M.

    2015-01-01

    Observational studies report a strong inverse relationship between leisure-time physical activity and all-cause mortality. Despite suggestive evidence from population-based associations, scientists have not been able to show a beneficial effect of physical activity on the risk of death in controlled intervention studies among individuals who have been healthy at baseline. On the other hand, high cardiorespiratory fitness is known to be a strong predictor of reduced mortality, even more robust than physical activity level itself. Here, in both animals and/or human twins, we show that the same genetic factors influence physical activity levels, cardiorespiratory fitness, and risk of death. Previous observational follow-up studies in humans suggest that increasing fitness through physical activity levels could prolong life; however, our controlled interventional study with laboratory rats bred for low and high intrinsic fitness contrast with these findings. Also, we find no evidence for the suggested association using pairwise analysis among monozygotic twin pairs who are discordant in their physical activity levels. Based on both our animal and human findings, we propose that genetic pleiotropy might partly explain the frequently observed associations between high baseline physical activity and later reduced mortality in humans. PMID:26666586

  13. The Hansenula polymorpha MOX gene presents two alternative transcription start points differentially utilized and sensitive to respiratory activity.

    PubMed

    Genu, Victor; Gödecke, Stefanie; Hollenberg, Cornelis P; Pereira, Gonçalo G

    2003-06-01

    The peroxisomal methanol metabolism of Hansenula polymorpha depends on a group of genes that are coordinately regulated. Methanol oxidase (Mox) plays a key role in this pathway and its synthesis has been shown to be regulated at the transcriptional level. MOX expression is strongly repressed on glucose and activated on glycerol or methanol. In this study we have identified two MOX transcripts that are differentially expressed along MOX derepression. The first one, named l-MOX (for longer MOX), starts at position -425, is only weakly and transiently transcribed and is not translated into the Mox protein. The other is the true MOX mRNA, which initiates around position -25. Using a strain bearing multiple copies of MOX(Q1N) and a reporter gene fused to the MOX promoter, regulation of the two transcripts was investigated. Initiation of the true MOX correlates with repression of l-MOX and conditions that are repressive for MOX transcription, such as the inhibition of mitochondrial activity, lead to higher levels of l-MOX expression. This effect was first observed in a mox mutant (Q1N-M8) unable to grow on nonfermentable carbon sources. No function was detected for l-MOX, but its regulation follows a pattern similar to that of catalase, which is essential for methanol metabolism. This suggests that, l-MOX, although precisely regulated, seems to be a remnant of the evolution of the methanol metabolism network.

  14. Genetic differences in internal transcribed spacer 1 between Dermanyssus gallinae from wild birds and domestic chickens.

    PubMed

    Brännström, S; Morrison, D A; Mattsson, J G; Chirico, J

    2008-06-01

    We investigated the presence of the poultry red mite or the chicken mite, Dermanyssus gallinae De Geer, Acari: Dermanyssidae, in wild bird populations in four different geographical regions of Sweden. The mites identified as D. gallinae were compared genetically with D. gallinae from egg-producing poultry farms in the same regions. The small subunit (SSU) gene, the 5.8S ribosomal RNA (rRNA) gene and the two internal transcribed spacers (ITS) of the rRNA genes were used in the genetic analysis. All D. gallinae mites had identical SSU rRNA, 5.8S rRNA and ITS2 sequences independent of their origin. By contrast, we identified significant differences in the ITS1 sequences. Based on the differences in the ITS1 sequences, the mites could be divided into two genotypes, of wild and domesticated origin, with no variation within the groups. These results imply that wild bird populations are of low importance, if any, as natural reservoirs of D. gallinae in these four geographical regions of Sweden.

  15. Peroxisome proliferator-activated receptor alpha target genes.

    PubMed

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  16. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    PubMed Central

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well. PMID:20936127

  17. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  18. Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional alpha-aminoadipate activating and reducing enzyme.

    PubMed

    Hijarrubia, M J; Aparicio, J F; Casqueiro, J; Martín, J F

    2001-02-01

    A 5.2-kb NotI DNA fragment isolated from a genomic library of Acremonium chrysogenum by hybridization with a probe internal to the Penicillium chrysogenum lys2 gene, was able to complement an alpha-aminoadipate reductase-deficient mutant of P. chrysogenum (lysine auxotroph L-G-). Enzyme assays showed that the alpha-aminoadipate reductase activity was restored in all the transformants tested. The lys2-encoded enzyme catalyzed both the activation and reduction of alpha-aminoadipic acid to its semialdehyde, as shown by reaction of the product with p-dimethylaminobenzaldehyde. The reaction required NADPH, and was not observed in the presence of NADH. Sequence analysis revealed that the gene encodes a protein with relatively high similarity to members of the superfamily of acyladenylate-forming enzymes. The Lys2 protein contained all nine motifs that are conserved in the adenylating domain of this enzyme family, a peptidyl carrier domain, and a reduction domain. In addition, a new NADP-binding motif located at the N-terminus of the reduction domain that may form a Rossmann-like betaalphabeta-fold has been identified and found to be shared by all known Lys2 proteins. The lys2 gene was mapped to chromosome I (2.2 Mb, the smallest chromosome) of A. chrysogenum C10 (the chromosome that contains the "late" cephalosporin cluster) and is transcribed as a monocistronic 4.5-kb mRNA although at relatively low levels compared with the beta-actin gene.

  19. Tbx16 regulates hox gene activation in mesodermal progenitor cells

    PubMed Central

    Payumo, Alexander Y.; McQuade, Lindsey E.; Walker, Whitney J.; Yamazoe, Sayumi; Chen, James K.

    2016-01-01

    The transcription factor T-box 16 (Tbx16/Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. The mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic, and we describe here the application of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identify 124 Tbx16-regulated genes that are expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis, and somitogenesis. Unexpectedly, we observe that loss of Tbx16 function precociously activates posterior hox genes in MPCs, and overexpression of a single posterior hox gene is sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs. PMID:27376691

  20. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    PubMed Central

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  1. Human DJ-1-specific transcriptional activation of tyrosine hydroxylase gene.

    PubMed

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

    2010-12-17

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-L-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice.

  2. Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species.

    PubMed

    Aktas, Münir; Bendele, Kylie G; Altay, Kürsat; Dumanli, Nazir; Tsuji, Masayoshi; Holman, Patricia J

    2007-07-20

    The genomic region spanning the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was cloned and sequenced from sixteen Theileria isolates. Each Theileria species possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among ruminant Theileria species. The spacers were most polymorphic in the agent of tropical theileriosis, Theileria annulata, and were more conserved in two benign species, Theileria buffeli and Theileria sergenti Chitose. Phylogenetic analysis of the rDNA ITS1-5.8S rRNA gene-ITS2 region clearly separated each taxon, placing them in three clusters. One held T. annulata, Theileria parva, and Theileria mutans, with the latter two most closely related. The second held T. sergenti Ikeda, T. sergenti Chitose, and T. buffeli, with the latter two most closely related. The third cluster held the Theileria ovis isolates.

  3. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    PubMed

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  4. Medical Terminology of the Musculoskeletal System. Medical Records. Instructional Unit for the Medical Transcriber.

    ERIC Educational Resources Information Center

    Gosman, Minna L.

    Following an analysis of the task of transcribing as practiced in a health facility, this study guide was developed to teach the knowledge and skills required of a medical transcriber. The medical record department was identified as a major occupational area, and a task inventory for medical records was developed and used as a basis for a…

  5. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

    PubMed Central

    Grandori, C; Mac, J; Siëbelt, F; Ayer, D E; Eisenman, R N

    1996-01-01

    The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism. Images PMID:8861962

  6. Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

    PubMed Central

    McDonald, James E.; Larsen, Niels; Pennington, Andrea; Connolly, John; Wallis, Corrin; Rooks, David J.; Hall, Neil; McCarthy, Alan J.; Allison, Heather E.

    2016-01-01

    PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, ‘universal’ SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by ‘universal’ primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology. PMID:27276347

  7. [Cascade of gene activation in Landouzy Dejerine muscular dystrophy].

    PubMed

    Belayew, A

    2010-01-01

    Our laboratory studies the Landouzy Dejerine muscular dystrophy or FSHD, a genetic disease which affects 7 in 100,000 individuals. The genetic defect is a deletion on chromosome 4 that decreases the copy number of a repeated DNA element, disturbs chromatin structure and activates the expression of neighbouring genes. The originality of our team has been to identify a gene within the repeated element itself and to show its activation in FSHD muscle cells. This gene expresses DUX4, a transcription factor that targets tens of genes, some of which express other transcription factors which target other genes, leading to a general deregulation. This DUX4-mediated cascade recapitulates by itself the major pathological features of FSHD: muscle atrophy, differentiation defect, oxidative stress... The homologous DUX4c gene located 42 kb from the repeat array expresses a protein that triggers myoblast proliferation. Its high expression level in severe cases of FSHD most probably contributes to the pathology by interfering with myoblast fusion with the muscle fibers at the last steps of muscle regeneration. We are performing global analyses of proteins and metabolites in healthy and FSHD myotubes (collaboration R Wattiez and JM Colet, UMONS) to identify abnormalities and their links with DUX4 or DUX4C.

  8. The neurotensin gene is a downstream target for Ras activation.

    PubMed Central

    Evers, B M; Zhou, Z; Celano, P; Li, J

    1995-01-01

    Ras regulates novel patterns of gene expression and the differentiation of various eukaryotic cell types. Stable transfection of Ha-ras into the human colon cancer line CaCo2 results in the morphologic differentiation to a small bowel phenotype. The purpose of our study was to determine whether the Ras regulatory pathway plays a role in the expression of the neurotensin gene (NT/N), a terminally differentiated endocrine product specifically localized in the gastrointestinal tract to the adult small bowel. We found that CaCo2-ras cells, but not parental CaCo2, express high levels of the human NT/N gene and, moreover, that this increase in gene expression is regulated at the level of transcription. Transfection experiments using NT/N-CAT mutation constructs identify the proximal 200 bp of NT/N flanking sequence as sufficient for maximal Ras-mediated NT/N reporter gene induction. Furthermore, a proximal AP-1/CRE motif is crucial for this Ras-mediated NT/N activation. Wild-type Ha-ras induces NT/N gene expression, albeit at lower levels than activated Ras; a dominant-negative Raf blocks this NT/N induction, suggesting that Raf lies down-stream of Ras in this pathway. In addition, postconfluent cultures of CaCo2 cells, which are differentiated to a small bowel phenotype, express the NT/N gene by 6 d after reaching confluency; this increase of NT/N expression is associated with concomitant increases of cellular p21ras protein. We conclude that Ras (both wild-type and activated) enhances expression of the NT/N gene in the gut-derived CaCo2 cell line, suggesting an important role for the Ras signaling pathway in NT/N gene transcription. Our results underscore the possibility that tissue-specific genes (such as NT/N) expressed in distinct subpopulations of the gut may be subject to Ras regulation. Finally, we speculate that the NT/N gene and the CaCo2 and CaCo2-ras cell systems will provide unique models to further define the cellular mechanisms leading to mammalian

  9. Transcriptional activation of virulence genes of Rhizobium etli.

    PubMed

    Wang, Luyao; Lacroix, Benoît; Guo, Jianhua; Citovsky, Vitaly

    2017-01-09

    Recently, Rhizobium etli has emerged, in addition to Agrobacterium spp., as a prokaryotic species that encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we showed that the signal phenolic molecule acetosyringone (AS) induced R. etli vir gene expression both in R. etli and in A. tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter T-DNA. Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium, but not from non-host plants. Early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside of the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a versus pTiC58 of A. tumefaciens IMPORTANCE: The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into plant cell genome. In this study, we explore the transcriptional regulation and induction of virulence genes in R. etli and show similarities and differences

  10. RNA activation of haploinsufficient Foxg1 gene in murine neocortex

    PubMed Central

    Fimiani, Cristina; Goina, Elisa; Su, Qin; Gao, Guangping; Mallamaci, Antonello

    2016-01-01

    More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo. PMID:27995975

  11. Linear topology confers in vivo gene transfer activity to polyethylenimines.

    PubMed

    Brissault, B; Leborgne, C; Guis, C; Danos, O; Cheradame, H; Kichler, A

    2006-01-01

    Although polyethylenimines (PEIs) are frequently used transfection agents, it is still unclear which of their properties are required for efficient gene delivery. This is even more striking when working in vivo since some PEIs are able to generate significant gene expression, whereas others are not. To facilitate a rational development of compounds with improved transfection activities, studies aimed at identifying the properties involved in the transfection process seem indispensable. In the present work, we investigated how transfection with linear PEI of 22 kDa allows for high reporter gene expression in lungs after intravenous injection, whereas the branched PEI of 25 kDa does not. To this end, we synthesized L-PEI derivatives that are intermediates between linear and branched PEIs. Our results show that the topology plays a crucial role in obtaining in vivo reporter gene expression, whereas the content of primary, secondary, and tertiary amines is only of minor importance.

  12. H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

    PubMed Central

    Guillemette, Benoit; Drogaris, Paul; Lin, Hsiu-Hsu Sophia; Armstrong, Harry; Hiragami-Hamada, Kyoko; Imhof, Axel; Bonneil, Éric; Thibault, Pierre; Verreault, Alain; Festenstein, Richard J.

    2011-01-01

    Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me). PMID:21483810

  13. Heat shock factor 1 binds to and transcribes satellite II and III sequences at several pericentromeric regions in heat-shocked cells

    SciTech Connect

    Eymery, Angeline; Souchier, Catherine; Vourc'h, Claire; Jolly, Caroline

    2010-07-01

    Cells respond to stress by activating the synthesis of heat shock proteins (HSPs) which protect the cells against the deleterious effects of stress. This mechanism is controlled by the heat shock factor 1 (HSF1). In parallel to HSP gene transcription, in human cells, HSF1 also binds to and transcribes satellite III repeated sequences present in numerous copies in the 9q12 pericentromeric region of chromosome 9. These HSF1 accumulation sites are termed nuclear stress bodies (nSBs). In tumor cells, however, the number of nSBs is higher than the number of 9q12 copies, suggesting the existence of other HSF1 targets. In this paper, we were interested in characterizing these other HSF1 binding sites. We show that HSF1 indeed binds to the pericentromeric region of 14 chromosomes, thereby directing the formation of 'secondary nSBs'. The appearance of secondary nSBs depends on the number of satellite sequences present in the target locus, and on the cellular amount of HSF1 protein. Moreover, secondary nSBs also correspond to transcription sites, thus demonstrating that heat shock induces a genome-wide transcription of satellite sequences. Finally, by analyzing published transcriptomic data, we show that the derepression of these large heterochromatic blocks does not significantly affect the transcription of neighboring genes.

  14. TAL2, a helix-loop-helix gene activated by the (7;9)(q34;q32) translocation in human T-cell leukemia.

    PubMed Central

    Xia, Y; Brown, L; Yang, C Y; Tsan, J T; Siciliano, M J; Espinosa, R; Le Beau, M M; Baer, R J

    1991-01-01

    Tumor-specific alteration of the TAL1 gene occurs in almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL). We now report the identification of TAL2, a distinct gene that was isolated on the basis of its sequence homology with TAL1. The TAL2 gene is located 33 kilobase pairs from the chromosome 9 breakpoint of t(7;9)(q34;q32), a recurring translocation specifically associated with T-ALL. As a consequence of t(7;9)(q34;q32), TAL2 is juxtaposed with sequences from the T-cell receptor beta-chain gene on chromosome 7. TAL2 sequences are actively transcribed in SUP-T3, a T-ALL cell line that harbors the t(7;9)(q34;q32). The TAL2 gene product includes a helix-loop-helix protein dimerization and DNA binding domain that is especially homologous to those encoded by the TAL1 and LYL1 protooncogenes. Hence, TAL2, TAL1, and LYL1 constitute a discrete subgroup of helix-loop-helix proteins, each of which can potentially contribute to the development of T-ALL. Images PMID:1763056

  15. TAL2, a helix-loop-helix gene activated by the (7; 9)(q34; q32) translocation in human T-cell leukemia

    SciTech Connect

    Ying Xia; Brown, L.; Yang, C.Y.; Tsan, J.T.; Baer, R.J. ); Siciliano, M.J. ); Espinosa, R. III; Le Beau, M.M. )

    1991-12-15

    Tumor-specific alteration of the TAL1 gene occurs in almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL). The authors now report the identification of TAL2, a distinct gene that was isolated on the basis of its sequence homology with TAL1. The TAL2 gene is located 33 kilobase pairs from the chromosome 9 breakpoint of t(7;9)(q34;q32), a recurring translocation specifically associated with T-ALL. As a consequence of t(7;9)(q34;q32), TAL2 is juxtaposed with sequences from the T-cell receptor {beta}-chain gene on chromosome 7. TAL2 sequences are actively transcribed in SUP-T3, a T-ALL cell line that harbors the t(7;9)(q34;q32). The TAL2 gene product includes a helix-loop-helix protein dimerization and DNA binding domain that is especially homologous to those encoded by the TAL1 and LYL1 protooncogenes. Hence, TAL2, TAL1, and LYL1 constitute a discrete subgroup of helix-loop-helix proteins, each of which can potentially contribute to the development of T-ALL.

  16. Activity-Regulated Genes as Mediators of Neural Circuit Plasticity

    PubMed Central

    Leslie, Jennifer H.; Nedivi, Elly

    2011-01-01

    Modifications of neuronal circuits allow the brain to adapt and change with experience. This plasticity manifests during development and throughout life, and can be remarkably long lasting. Many electrophysiological and molecular mechanisms are common to the seemingly diverse types of activity-dependent functional adaptation that take place during developmental critical periods, learning and memory, and alterations to sensory map representations in the adult. Experience-dependent plasticity is triggered when neuronal excitation activates cellular signaling pathways from the synapse to the nucleus that initiate new programs of gene expression. The protein products of activity-regulated genes then work via a diverse array of cellular mechanisms to modify neuronal functional properties. They fine-tune brain circuits by strengthening or weakening synaptic connections or by altering synapse numbers. Their effects are further modulated by posttranscriptional regulatory mechanisms, often also dependent on activity, that control activity-regulated gene transcript and protein function. Thus, the cellular response to neuronal activity integrates multiple tightly coordinated mechanisms to precisely orchestrate long-lasting, functional and structural changes in brain circuits. PMID:21601615

  17. FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

    PubMed Central

    Muñoz-Centeno, Mari Cruz; Singh, Rakesh Kumar; Oreal, Vincent; Reddy, Gajjalaiahvari Ugander; Liang, Dun; Géli, Vincent; Gunjan, Akash; Chávez, Sebastián

    2010-01-01

    The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication. PMID:20502685

  18. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

  19. Sequence analysis of the ribosomal internal transcribed spacer DNA of the crayfish parasite Psorospermium haeckeli.

    PubMed

    Bangyeekhun, E; Ryynänen, H J; Henttonen, P; Huner, J V; Cerenius, L; Söderhäll, K

    2001-10-08

    Two morphotypes of the crayfish parasite Psorospermium haeckeli were isolated from 2 crayfish species of different geographical origin. The oval-shaped sporocysts were obtained from the epidermal and connective tissue beneath the carapace of the noble crayfish Astacus astacus from Sweden and Finland. Elongated spores were isolated from the abdominal muscle tissue of the red swamp crayfish Procambarus clarkii from USA. To compare genetic divergence of 2 morphotypes of the parasite, the ribosomal internal transcribed spacer (ITS) DNA (ITS 1 and ITS 2) and the 5.8S rRNA gene were cloned and sequenced. The analysed region is variable in length, with the ribosomal ITS sequence of the European morphotype longer than the North American one. Sequence diversity is found mainly in ITS 1 and ITS 2 regions, and there is 66% and 58% similarity between the 2 morphotypes, respectively. Thus, analysis of the ribosomal ITS DNA suggests that P. haeckeli forms obtained from Europe and North America are genetically diverse, which supports the previously reported morphological characteristics.

  20. Characterization of Dermanyssus gallinae (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions.

    PubMed

    Potenza, L; Cafiero, M A; Camarda, A; La Salandra, G; Cucchiarini, L; Dachà, M

    2009-10-01

    In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3'end of 18S and at the 5'start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.

  1. Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species.

    PubMed

    Ibáñez-Escribano, Alexandra; Nogal-Ruiz, Juan José; Arán, Vicente J; Escario, José Antonio; Gómez-Barrio, Alicia; Alderete, J F

    2014-04-01

    The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~83%. There was >40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.

  2. Genetic structure of wild emmer wheat populations as reflected by transcribed versus anonymous SSR markers.

    PubMed

    Peleg, Zvi; Fahima, Tzion; Abbo, Shahal; Krugman, Tamar; Saranga, Yehoshua

    2008-03-01

    Simple sequence repeat (SSR) markers have become a major tool in population genetic analyses. The anonymous genomic SSRs (gSSRs) have been recently supplemented with expressed sequence tag (EST) derived SSRs (eSSRs), which represent the transcribed regions of the genome. In the present study, we used 8 populations of wild emmer wheat (Triticum turgidum subsp. dicoccoides) to compare the usefulness of the two types of SSR markers in assessing allelic diversity and population structure. gSSRs revealed significantly higher diversity than eSSRs in terms of average number of alleles (14.92 vs. 7.4, respectively), polymorphic information content (0.87 vs. 0.68, respectively), and gene diversity (He; 0.55 vs. 0.38, respectively). Despite the overall differences in the level of diversity, Mantel tests for correlations between eSSR and gSSR pairwise genetic distances were found to be significant for each population as well as for all accessions jointly (RM=0.54, p=0.01). Various genetic structure analyses (AMOVA, PCoA, STRUCTURE, unrooted UPGMA tree) revealed a better capacity of eSSRs to distinguish between populations, while gSSRs showed a higher proportion of intrapopulation (among accessions) diversity. We conclude that eSSR and gSSR markers should be employed in conjunction to obtain a high inter- and intra-specific (or inter- and intra-varietal) distinctness.

  3. 5. international workshop on the identification of transcribed sequences

    SciTech Connect

    1995-12-31

    This workshop was held November 5--8, 1995 in Les Embiez, France. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on mapping the human genome. Attention is focused on the following topics: transcriptional maps; functional analysis; techniques; model organisms; and tissue specific libraries and genes. Abstracts are included of the papers that were presented.

  4. Metallothionein gene activation in the earthworm (Lumbricus rubellus).

    PubMed

    Höckner, M; Dallinger, R; Stürzenbaum, S R

    2015-05-08

    In order to cope with changing environmental conditions, organisms require highly responsive stress mechanisms. Heavy metal stress is handled by metallothioneins (MTs), the regulation of which is evolutionary conserved in insects and vertebrates and involves the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) positioned in the promoter of MT genes. However, in most invertebrate phyla, the transcriptional activation of MTs is different and the exact mechanism is still unknown. Interestingly, although MREs are typically present also in invertebrate MT gene promoters, MTF-1 is notably absent. Here we use Lumbricus rubellus, the red earthworm, to study the elusive mechanism of wMT-2 activation in control and Cd-exposed conditions. EMSA and DNase I footprinting approaches were used to pinpoint functional binding sites within the wMT-2 promoter region, which revealed that the cAMP responsive element (CRE) is a promising candidate which may act as a transcriptional activator of invertebrate MTs.

  5. "Active" cancer immunotherapy by anti-Met antibody gene transfer.

    PubMed

    Vigna, Elisa; Pacchiana, Giovanni; Mazzone, Massimiliano; Chiriaco, Cristina; Fontani, Lara; Basilico, Cristina; Pennacchietti, Selma; Comoglio, Paolo M

    2008-11-15

    Gene therapy provides a still poorly explored opportunity to treat cancer by "active" immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing ("cell autonomous activity," in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface ("bystander effect," in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer-based immunotherapy strategy.

  6. CRISPR RNA-guided activation of endogenous human genes.

    PubMed

    Maeder, Morgan L; Linder, Samantha J; Cascio, Vincent M; Fu, Yanfang; Ho, Quan H; Joung, J Keith

    2013-10-01

    Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.

  7. Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

    PubMed

    Boulanger, Alice; Moon, Kyung; Decker, Kimberly B; Chen, Qing; Knipling, Leslie; Stibitz, Scott; Hinton, Deborah M

    2015-02-10

    Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

  8. Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

    PubMed

    Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

    2015-08-03

    Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

  9. Identification of Medically Important Yeast Species by Sequence Analysis of the Internal Transcribed Spacer Regions

    PubMed Central

    Leaw, Shiang Ning; Chang, Hsien Chang; Sun, Hsiao Fang; Barton, Richard; Bouchara, Jean-Philippe; Chang, Tsung Chain

    2006-01-01

    Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods. PMID:16517841

  10. Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome.

    PubMed

    She, Richard; Chakravarty, Anupam K; Layton, Curtis J; Chircus, Lauren M; Andreasson, Johan O L; Damaraju, Nandita; McMahon, Peter L; Buenrostro, Jason D; Jarosz, Daniel F; Greenleaf, William J

    2017-04-04

    RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

  11. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    PubMed Central

    Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

    2012-01-01

    Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

  12. Reverse transcription-polymerase chain reaction detection of transcribed sequences on human chromosome 21

    SciTech Connect

    Cheng, J.F.; Zhu, Y. )

    1994-03-15

    Seventy-four pairs of oligonucleotides derived from sequence-tagged sites (STSs) on the long arm of human chromosome 21, specifically from bands 21q22.1 to 21q22.3, were used in reverse transcription-polymerase chain reactions (RT-PCR) to detect the presence of expressed sequences in a fetal brain. These STSs included 69 that had not been related to transcribed sequences and 5 that had detected two known genes and three previously isolated cDNA clones. Of the 69 STSs analyzed in RT-PCR, 25 allowed amplification of specific cDNA fragments. The sizes of amplified cDNA fragments match those amplified from either human genomic DNA or somatic hybrid cells containing human chromosome 21. Of the 11 cDNA analyzed in Northern blot hybridizations, 6 hybridized to specific RNA species. The rapid screening for cDNA using previously mapped STSs has provided insight into the distribution of expressed sequences in this region of chromosome 21. Northern blot analysis of the amplified cDNA fragments has revealed interesting candidate genes in two disease loci. The marker D21S267 was previously mapped in the Down syndrome region of chromosome 21, and the marker D21S113 is closely linked to progressive myoclonus epilepsy. The cDNA fragments amplified using the primer sequences derived from D21S267 and D21S113 hybridized to 7- and 6.5-kb transcripts, respectively, which seems to express predominantly in brain. 37 refs., 3 figs., 1 tab.

  13. Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation

    PubMed Central

    Tummala, Padmaja; Mali, Raghuveer S.; Guzman, Eduardo; Zhang, Xiao

    2010-01-01

    signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region. Conclusions High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene’s promoter complex. PMID

  14. Visualization and analysis of unfolded nucleosomes associated with transcribing chromatin.

    PubMed Central

    Bazett-Jones, D P; Mendez, E; Czarnota, G J; Ottensmeyer, F P; Allfrey, V G

    1996-01-01

    We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U-shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three-dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer. PMID:8628657

  15. Transcribing and digitizing eighteenth- and nineteenth-century letters for a historical digital repository.

    PubMed

    Dunster, Emily S; Kipnis, Daniel G; Angelo, F Michael

    2014-01-01

    In fall 2011, the Scott Memorial Library purchased 53 letters belonging to an 1841 graduate of Jefferson Medical College, John Plimpton Green. The library staff transcribed and digitized the letters, creating an online collection in the university's institutional repository, Jefferson Digital Commons. This article will detail the process of transcribing and digitizing the collection along with sharing statistics and the benefits of this project to global researchers.

  16. Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene.

    PubMed

    Chen, Yuting; Lu, Wenqing; Gao, Na; Long, Yi; Shao, Yanjiao; Liu, Meizhen; Chen, Huaqing; Ye, Shixin; Ma, Xueyun; Liu, Mingyao; Li, Dali

    2017-02-01

    The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

  17. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery

    PubMed Central

    Chakravarthy, Harini; Ormsbee, Briana D.; Mallanna, Sunil K.; Rizzino, Angie

    2011-01-01

    The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.—Chakravarthy, H., Ormsbee, B. D., Mallanna, S. K., Rizzino, A. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery. PMID:20876214

  18. Novel Cationic Lipids with Enhanced Gene Delivery and Antimicrobial Activity

    PubMed Central

    Fein, David E.; Bucki, Robert; Byfield, Fitzroy; Leszczynska, Katarzyna; Janmey, Paul A.

    2010-01-01

    Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D2S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D2S. D2S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at ∼5 μM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D2S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D2S can improve transfection activity over common lipofection reagents, and D2S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation. PMID:20573781

  19. The Voice Transcription Technique: Use of Voice Recognition Software to Transcribe Digital Interview Data in Qualitative Research

    ERIC Educational Resources Information Center

    Matheson, Jennifer L.

    2007-01-01

    Transcribing interview data is a time-consuming task that most qualitative researchers dislike. Transcribing is even more difficult for people with physical limitations because traditional transcribing requires manual dexterity and the ability to sit at a computer for long stretches of time. Researchers have begun to explore using an automated…

  20. Adaptation of muscle gene expression to changes in contractile activity

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Babij, P.; Thomason, D. B.; Wong, T. S.; Morrison, P. R.

    1987-01-01

    A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA].

  1. Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

    PubMed Central

    Santangelo, J D; Jones, D T; Woods, D R

    1991-01-01

    An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

  2. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  3. Sensation-seeking genes and physical activity in youth.

    PubMed

    Wilkinson, A V; Gabriel, K P; Wang, J; Bondy, M L; Dong, Q; Wu, X; Shete, S; Spitz, M R

    2013-03-01

    Many studies examining genetic influences on physical activity (PA) have evaluated the impact of single nucleotide polymorphisms (SNPs) related to the development of lifestyle-related chronic diseases, under the hypothesis that they would be associated with PA. However, PA is a multidetermined behavior and associated with a multitude of health consequences. Thus, examining a broader range of candidate genes associated with a broader range of PA correlates may provide new insights into the genetic underpinnings of PA. In this study, we focus on one such correlate - sensation-seeking behavior. Participants (N = 1130 Mexican origin youth) provided a saliva sample and data on PA and sensation-seeking tendencies in 2008-2009. Participants were genotyped for 630 functional and tagging variants in the dopamine, serotonin and cannabinoid pathways. Overall 30% of participants (males - 37.6% and females - 22.0%) reported ≥60 min of PA on 5 of 7 days. After adjusting for gender, age and population stratification, and applying the Bayesian False Discovery Probability approach for assessing noteworthiness, four gene variants were significantly associated with PA. In a multivariable model, being male, having higher sensation-seeking tendencies and at least one copy of the minor allele for SNPs in angiotensin I-converting enzyme gene [ACE; rs8066276 odds ratio (OR) = 1.44; P = 0.012] and tryptophan hydroxylase 2 gene (TPH2; rs11615016 OR = 1.73; P = 0.021) were associated with increased likelihood of meeting PA recommendations. Participants with at least one copy of the minor allele for SNPs in synaptosomal-associated protein 25 gene (SNAP25; rs363035 OR = 0.53; P = 0.005) and cannabinoid receptor 1 gene (CNR1; rs6454672 OR = 0.62; P = 0.022) have decreased likelihood of meeting PA recommendations. Our findings extend current knowledge of the complex relationship between PA and possible genetic underpinnings.

  4. Structural Determination of a Transcribing RNA Polymerase II Complex

    DTIC Science & Technology

    2000-05-01

    eukaryote- TFIIH, whose helicase activities melt DNA transcription initiation factor TFIIE, as re- specific subunits Rpbl0 and Rpbl2. Contact of...from a DNA template. Despite challenges involved in this project, a major achievement is at hand. Firstly, a mainchain model of RNA Polymerase II with...conducting research utilizing recombinant DNA technology, the investigator(s) adhered to current guidelines promulgated by the National Institutes of

  5. Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human CellsD⃞

    PubMed Central

    Valgardsdottir, Rut; Chiodi, Ilaria; Giordano, Manuela; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2005-01-01

    Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs. PMID:15788562

  6. Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: spontaneous activation of NF-kappaB and CBF1.

    PubMed Central

    Miyazawa, K.; Mori, A.; Yamamoto, K.; Okudaira, H.

    1998-01-01

    The involvement of IL-6 in the pathogenesis of rheumatoid arthritis (RA) has been recently demonstrated. In the present study, we investigated the cellular and molecular mechanisms involved in the spontaneous IL-6 production by the fibroblast-like synoviocytes (FLSs) obtained from patients with RA. Cloned FLSs were established from the bulk cultures of FLSs by the limiting dilution method. Some FLS clones spontaneously produced large amounts of IL-6, whereas others produced low amounts of it. Neither anti-human TNF-alpha nor IL-1 antibody affected spontaneous IL-6 production of these FLS clones, suggesting that IL-6 production of the FLSs was endogenously up-regulated. A luciferase reporter plasmid containing the human IL-6 promoter region was significantly transcribed when transfected into the IL-6 high-producing clones, indicating that the rheumatoid FLSs retained constitutive transcriptional activity of the IL-6 gene. Electrophoretic mobility shift assays revealed that the binding activity of p50 and p65 NF-kappaB subunits and CBF1 was significantly enhanced in the IL-6 high-producing clones compared with that of IL-6 low-producing clones and cultured sarcoma cells, suggesting that spontaneous activation of NF-kappaB and CBF1 may lead to the constitutive transcription of the IL-6 gene by rheumatoid FLSs. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:9502421

  7. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin.

    PubMed

    Svensson, J Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C; Allshire, Robin C; Ekwall, Karl

    2015-06-01

    Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

  8. Identification of Aspergillus Species Using Internal Transcribed Spacer Regions 1 and 2

    PubMed Central

    Henry, Travis; Iwen, Peter C.; Hinrichs, Steven H.

    2000-01-01

    Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1–5.8S–ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA. PMID:10747135

  9. Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA.

    PubMed

    Goodwin, S B; Dunkle, L D; Zismann, V L

    2001-07-01

    ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.

  10. Three faces of recombination activating gene 1 (RAG1) mutations.

    PubMed

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  11. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    SciTech Connect

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-05-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. /sup 32/P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA.

  12. Identifying Novel Transcriptional and Epigenetic Features of Nuclear Lamina-associated Genes.

    PubMed

    Wu, Feinan; Yao, Jie

    2017-12-01

    Because a large portion of the mammalian genome is associated with the nuclear lamina (NL), it is interesting to study how native genes resided there are transcribed and regulated. In this study, we report unique transcriptional and epigenetic features of nearly 3,500 NL-associated genes (NL genes). Promoter regions of active NL genes are often excluded from NL-association, suggesting that NL-promoter interactions may repress transcription. Active NL genes with higher RNA polymerase II (Pol II) recruitment levels tend to display Pol II promoter-proximal pausing, while Pol II recruitment and Pol II pausing are not correlated among non-NL genes. At the genome-wide scale, NL-association and H3K27me3 distinguishes two large gene classes with low transcriptional activities. Notably, NL-association is anti-correlated with both transcription and active histone mark levels among genes not significantly enriched with H3K9me3 or H3K27me3, suggesting that NL-association may represent a novel gene repression pathway. Interestingly, an NL gene subgroup is not significantly enriched with H3K9me3 or H3K27me3 and is transcribed at higher levels than the rest of NL genes. Furthermore, we identified distal enhancers associated with active NL genes and reported their epigenetic features.

  13. Trans-activation function of a 3 prime truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

    SciTech Connect

    Takada, Shinako; Koike, Katsuro )

    1990-08-01

    To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3{prime} end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.

  14. G-quadruplex structures formed by expanded hexanucleotide repeat RNA and DNA from the neurodegenerative disease-linked C9orf72 gene efficiently sequester and activate heme.

    PubMed

    Grigg, Jason C; Shumayrikh, Nisreen; Sen, Dipankar

    2014-01-01

    The expansion of a (G(4)C(2))n repeat within the human C9orf72 gene has been causally linked to a number of neurodegenerative diseases, most notably familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies have shown that the repeat expansion alters gene function in four ways, disrupting the gene's normal cellular roles and introducing toxic gain of function at the level of both DNA and RNA. (G(4)C(2))n DNA, as well as the RNA transcribed from it, are found to fold into four-stranded G-quadruplex structures. It has been shown that the toxicity of the RNA G-quadruplexes, often localized in intracellular RNA foci, lies in their ability to sequester many important RNA binding proteins. Herein we propose that a distinct toxic property of such RNA and DNA G-quadruplexes from the C9orf72 gene may arise from their ability to bind and oxidatively activate cellular heme. We show that G-quadruplexes formed by both (G(4)C(2))(4) RNA and DNA not only complex tightly with heme but also enhance its intrinsic peroxidase and oxidase propensities. By contrast, the antisense (C(4)G(2))(4) RNA and DNA neither bind heme nor influence its oxidative activity. Curiously, the ability of C9orf72 DNA and transcripts to bind and activate heme mirror similar properties that have been reported for the Aβ peptide and its oligomers in Alzheimer's disease neurons. It is therefore conceivable that C9orf72 RNA G-quadruplex tangles play roles in sequestering intracellular heme and promoting oxidative damage in ALS and FTD analogous to those proposed for Aβ peptide and its tangles in Alzheimer's Disease. Given that neurodegenerative diseases in general are characterized by mitochondrial and respiratory malfunctions, the role of C9orf72 DNA and RNA in heme sequestration as well as its inappropriate activation in ALS and FTD neurons may warrant examination.

  15. Coexpression of two closely linked avian genes for purine nucleotide synthesis from a bidirectional promoter.

    PubMed Central

    Gavalas, A; Dixon, J E; Brayton, K A; Zalkin, H

    1993-01-01

    Two avian genes encoding essential steps in the purine nucleotide biosynthetic pathway are transcribed divergently from a bidirectional promoter element. The bidirectional promoter, embedded in a CpG island, directs coexpression of GPAT and AIRC genes from distinct transcriptional start sites 229 bp apart. The bidirectional promoter can be divided in half, with each half retaining partial activity towards the cognate gene. GPAT and AIRC genes encode the enzymes that catalyze step 1 and steps 6 plus 7, respectively, in the de novo purine biosynthetic pathway. This is the first report of genes coding for structurally unrelated enzymes of the same pathway that are tightly linked and transcribed divergently from a bidirectional promoter. This arrangement has the potential to provide for regulated coexpression comparable to that in a prokaryotic operon. Images PMID:8336716

  16. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

  17. Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana

    PubMed Central

    He, Chongsheng; Huang, Hai; Xu, Lin

    2013-01-01

    Polycomb group (PcG) proteins act in an evolutionarily conserved epigenetic pathway that regulates chromatin structures in plants and animals, repressing many developmentally important genes by modifying histones. PcG proteins can form at least two multiprotein complexes: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2, respectively). The functions of Arabidopsis thaliana PRCs have been characterized in multiple stages of development and have diverse roles in response to environmental stimuli. Recently, the mechanism that precisely regulates Arabidopsis PcG activity was extensively studied. In this review, we summarize recent discoveries in the regulations of PcG at the three different layers: the recruitment of PRCs to specific target loci, the polyubiquitination and degradation of PRC2, and the antagonism of PRC2 activity by the Trithorax group proteins. Current knowledge indicates that the powerful activity of the PcG pathway is strictly controlled for specific silencing of target genes during plant development and in response to environmental stimuli. PMID:24312106

  18. Transcribed sequences in the human genome to be held in San Francisco, November 7 and 8, 1992. Final report, September 1, 1992--August 31, 1993

    SciTech Connect

    Gardiner, K.

    1993-11-01

    The Second International Workshop on the Identification of Transcribed Sequences was held in San Francisco on November 7--8, 1992. The purpose of the workshop was to discuss and evaluate techniques for developing a complete transcriptional map of the human genome. Such a map requires the positions, sequences, and expression patterns of all genes. This goal is being approached from two different directions, each with strengths and weaknesses. One method is to identify the transcribed sequences from genomic DNA of a given region; the other is to systematically sequence and map cDNAs. The cDNA approach yields sequence information rapidly, but mapping each cDNA is a technical challenge. In the first approach, the map locations of genomic sequences are known at the outset, and the challenge is to identify exons. The efficient construction of a transcriptional map will require a diverse array of techniques.

  19. Detection of multiple infections by Monocystis strains in a single earthworm host using ribosomal internal transcribed spacer sequence variation.

    PubMed

    Velavan, T P; Schulenburg, H; Michiels, N K

    2010-01-01

    Monocystis sp. are sporocyst-forming apicomplexan parasites common in seminal vesicles of the earthworm Lumbricus terrestris where they may account for temporary castration. This study describes the internal transcribed spacer (ITS) region of the ribosomal cistron of Monocystis sp. This region, including ITS-1, the 5.8S ribosomal RNA gene, and ITS-2, was PCR amplified, cloned, and sequenced for Monocystis sp. isolated from the seminal vesicles of several wild-caught L. terrestris. Our analysis revealed substantial polymorphisms, also within single host organisms, indicating intra-host diversity of parasites. These genetic markers are the first that allow distinction of Monocystis sp. genotypes, opening new avenues for the study of parasite diversity within and between hosts.

  20. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery.

    PubMed

    Chakravarthy, Harini; Ormsbee, Briana D; Mallanna, Sunil K; Rizzino, Angie

    2011-01-01

    The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.

  1. Inhibition of retinoic acid-induced activation of 3' human HOXB genes by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters.

    PubMed Central

    Faiella, A; Zappavigna, V; Mavilio, F; Boncinelli, E

    1994-01-01

    Most homeobox genes belonging to the Hox family are sequentially activated in embryonal carcinoma cells upon treatment with retinoic acid. Genes located at the 3' end of each one of the four Hox clusters are activated first, whereas upstream Hox genes are activated progressively later. This activation has been extensively studied for human HOX genes in the NT2/D1 cell line and shown to take place at the transcriptional level. To understand the molecular mechanisms of sequential HOX gene activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a significant inhibition of the activation of the upstream HOXB genes, as well as of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this inhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional level. These data suggest a cascade model of progressive activation of Hox genes, with a 3'-to-5' polarity. Images PMID:7911240

  2. Hormonal activity of polycyclic musks evaluated by reporter gene assay.

    PubMed

    Mori, Taiki; Iida, Mitsuru; Ishibashi, Hiroshi; Kohra, Shinya; Takao, Yuji; Takemasa, Takehiro; Arizono, Koji

    2007-01-01

    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

  3. Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription.

    PubMed

    Angermayr, Michaela; Oechsner, Ulrich; Gregor, Kerstin; Schroth, Gary P; Bandlow, Wolfhard

    2002-10-01

    The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to define elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6). Nucleotide sequences 5' of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription. We show that the core promoter of AKY2 is constitutively kept free of nucleosomes. Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA. DNA structure analysis by computer and by mutation identified two kinks flanking an interstitial stretch of 65 bp of moderately bent core promoter DNA. Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the flanks allowing unimpeded access of the basal transcription machinery to the core promoter. The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation.

  4. Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting

    PubMed Central

    2014-01-01

    Background Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes. Results We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping. Conclusions We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference

  5. A Bayesian Framework for the Classification of Microbial Gene Activity States

    PubMed Central

    Disselkoen, Craig; Greco, Brian; Cook, Kaitlyn; Koch, Kristin; Lerebours, Reginald; Viss, Chase; Cape, Joshua; Held, Elizabeth; Ashenafi, Yonatan; Fischer, Karen; Acosta, Allyson; Cunningham, Mark; Best, Aaron A.; DeJongh, Matthew; Tintle, Nathan

    2016-01-01

    Numerous methods for classifying gene activity states based on gene expression data have been proposed for use in downstream applications, such as incorporating transcriptomics data into metabolic models in order to improve resulting flux predictions. These methods often attempt to classify gene activity for each gene in each experimental condition as belonging to one of two states: active (the gene product is part of an active cellular mechanism) or inactive (the cellular mechanism is not active). These existing methods of classifying gene activity states suffer from multiple limitations, including enforcing unrealistic constraints on the overall proportions of active and inactive genes, failing to leverage a priori knowledge of gene co-regulation, failing to account for differences between genes, and failing to provide statistically meaningful confidence estimates. We propose a flexible Bayesian approach to classifying gene activity states based on a Gaussian mixture model. The model integrates genome-wide transcriptomics data from multiple conditions and information about gene co-regulation to provide activity state confidence estimates for each gene in each condition. We compare the performance of our novel method to existing methods on both simulated data and real data from 907 E. coli gene expression arrays, as well as a comparison with experimentally measured flux values in 29 conditions, demonstrating that our method provides more consistent and accurate results than existing methods across a variety of metrics. PMID:27555837

  6. Structure-activity relationship in cationic lipid mediated gene transfection.

    PubMed

    Niculescu-Duvaz, Dan; Heyes, James; Springer, Caroline J

    2003-07-01

    Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.

  7. GeneSet2miRNA: finding the signature of cooperative miRNA activities in the gene lists

    PubMed Central

    Antonov, Alexey V.; Dietmann, Sabine; Wong, Philip; Lutter, Dominik; Mewes, Hans W.

    2009-01-01

    GeneSet2miRNA is the first web-based tool which is able to identify whether or not a gene list has a signature of miRNA-regulatory activity. As input, GeneSet2miRNA accepts a list of genes. As output, a list of miRNA-regulatory models is provided. A miRNA-regulatory model is a group of miRNAs (single, pair, triplet or quadruplet) that is predicted to regulate a significant subset of genes from the submitted list. GeneSet2miRNA provides a user friendly dialog-driven web page submission available for several model organisms. GeneSet2miRNA is freely available at http://mips.helmholtz-muenchen.de/proj/gene2mir/. PMID:19420064

  8. Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

    PubMed

    Hesberg, Christine; Hänsch, Robert; Mendel, Ralf R; Bittner, Florian

    2004-04-02

    Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

  9. Activation of tissue plasminogen activator gene transcription by Neovastat, a multifunctional antiangiogenic agent.

    PubMed

    Gingras, Denis; Nyalendo, Carine; Di Tomasso, Geneviève; Annabi, Borhane; Béliveau, Richard

    2004-07-16

    We recently reported that Neovastat, an antiangiogenic drug that is currently undergoing Phase III clinical trials for the treatment of non-small cell lung cancer, may inhibit angiogenesis through an increase in tPA activity. Here, we show that Neovastat also stimulates tPA gene transcription in endothelial cells, in a TNFalpha-like manner. RT-PCR analysis and gene reporter assays using the human tPA promoter indicated that upregulation of the tPA gene transcription by both Neovastat and TNFalpha was correlated with the phosphorylation of JNK1/2 and of IkappaB and that SP600125 and BAY11-7082, inhibitors of JNK and IkappaK, respectively, inhibit the increase of tPA gene transcription induced by Neovastat and TNFalpha. These results suggest that Neovastat induces tPA gene transcription through activation of the JNK and NFkappaB signaling pathways, leading to an increase of tPA secretion by endothelial cells. This may lead to the localized destruction of the fibrin provisional matrix that is necessary for neovessel formation and thus contribute to the reported antiangiogenic properties of this compound.

  10. Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages

    PubMed Central

    Saavedra, Nicolás; Cuevas, Alejandro; Cavalcante, Marcela F.; Dörr, Felipe A.; Saavedra, Kathleen; Zambrano, Tomás; Abdalla, Dulcineia S. P.; Salazar, Luis A.

    2016-01-01

    Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques. PMID:27119082

  11. MBD3 Localizes at Promoters, Gene Bodies and Enhancers of Active Genes

    PubMed Central

    Shimbo, Takashi; Du, Ying; Grimm, Sara A.; Dhasarathy, Archana; Mav, Deepak; Shah, Ruchir R.; Shi, Huidong; Wade, Paul A.

    2013-01-01

    The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status. PMID:24385926

  12. Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

    PubMed Central

    De Vos, P; Lefebvre, A M; Miller, S G; Guerre-Millo, M; Wong, K; Saladin, R; Hamann, L G; Staels, B; Briggs, M R; Auwerx, J

    1996-01-01

    The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter. PMID:8770873

  13. Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers

    PubMed Central

    2012-01-01

    Background Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. Results We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. Conclusions This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research. PMID:22929540

  14. Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA

    PubMed Central

    Li, Dezhu; Sun, Yongzhen; Niu, Yunyun; Chen, Zhiduan; Luo, Hongmei; Pang, Xiaohui; Sun, Zhiying; Liu, Chang; Lv, Aiping; Deng, Youping; Larson-Rabin, Zachary; Wilkinson, Mike; Chen, Shilin

    2012-01-01

    Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification. PMID:22952830

  15. High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.

    PubMed

    Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

    2015-10-01

    Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin.

  16. T cell activation by concanavalin A in the presence of cyclosporin A: immunosuppressor withdrawal induces NFATp translocation and interleukin-2 gene transcription.

    PubMed

    Bemer, V; Truffa-Bachi, P

    1996-07-01

    Cyclosporin A (CSA), an immunosuppressive agent used in organ transplantation and to treat some autoimmune diseases, blocks the Ca2+-dependent steps involved in T cell receptor triggering leading to interleukin (IL)-2 production. Considering that the early steps of T cell activation are insensitive to CSA, we asked whether the initial activation achieved in presence of this immunosuppressor could affect the capacity of the T cell to respond to a mitogenic restimulation. We found that T cells activated by concanavalin A (ConA) for 48 h in the presence of CSA retain the capacity to proliferate in response to ConA once the immunosuppressor is removed. These cells are able to transcribe anew the IL-2 gene, without the requirement of new protein synthesis, and to up-regulate the alpha chain of the IL-2 receptor. Furthermore, we present the first direct evidence that the nuclear factor AP-1 is present in the nucleus of the T cells primed for 48 h in presence of CSA and that withdrawal of the immunosuppressor leads to the translocation of NFATp from the cytoplasm to the nucleus.

  17. Rcs signalling-activated transcription of rcsA induces strong anti-sense transcription of upstream fliPQR flagellar genes from a weak intergenic promoter: regulatory roles for the anti-sense transcript in virulence and motility.

    PubMed

    Wang, Qingfeng; Harshey, Rasika M

    2009-10-01

    In Salmonella enterica, an activated Rcs signalling system inhibits initiation of transcription of the flhD master operon. Under these conditions, where motility is shut down, microarray experiments showed an increased RNA signal for three flagellar genes -fliPQR- located upstream of rcsA. We show here that it is the anti-sense (AS) strand of these genes that is transcribed, originating at a weak promoter in the intergenic region between fliR and rcsA. RcsA is an auxiliary regulator for the Rcs system, whose transcription is dependent on the response regulator RcsB. Rcs-activated rightward transcription, but not translation, of rcsA is required for stimulation of leftward AS transcription. Our results implicate a combined action of RcsB and rcsA transcription in activating the AS promoter, likely by modulating DNA superhelicity in the intergenic region. We show that the AS transcript regulates many genes in the Rcs regulon, including SPI-1 and SPI-2 virulence and stress-response genes. In the wild-type strain the AS transcript is present in low amounts, independent of Rcs signalling. Here, AS transcription modulates complementary sense RNA levels and impacts swarming motility. It appears that the flagellar AS transcript has been co-opted by the Rcs system to regulate virulence.

  18. Behavioral science and the study of gene-nutrition and gene-physical activity interactions in obesity research.

    PubMed

    Faith, Myles S

    2008-12-01

    This report summarizes emerging opportunities for behavioral science to help advance the field of gene-environment and gene-behavior interactions, based on presentations at The National Cancer Institute (NCI) Workshop, "Gene-Nutrition and Gene-Physical Activity Interactions in the Etiology of Obesity." Three opportunities are highlighted: (i) designing potent behavioral "challenges" in experiments, (ii) determining viable behavioral phenotypes for genetics studies, and (iii) identifying specific measures of the environment or environmental exposures. Additional points are underscored, including the need to incorporate novel findings from neuroimaging studies regarding motivation and drive for eating and physical activity. Advances in behavioral science theory and methods can play an important role in advancing understanding of gene-brain-behavior relationships in obesity onset.

  19. A strategy to establish a gene-activated matrix on titanium using gene vectors protected in a polylactide coating.

    PubMed

    Kolk, Andreas; Haczek, Cornelia; Koch, Christian; Vogt, Stephan; Kullmer, Martin; Pautke, Christoph; Deppe, Herbert; Plank, Christian

    2011-10-01

    Bioactive implants are promising tools in regenerative medicine. Here we describe a versatile procedure for preparing a gene-activated matrix on titanium. Lyophilized copolymer-protected gene vectors (COPROGs) suspended in poly(d,l-lactide) (PDLLA) solutions in ethyl acetate were used to varnish solid surfaces. The gene-activated PDLLA surfaces were first established on polypropylene 96-well plates. Vector release from these surfaces in aqueous buffer, cell viability and gene transfer efficiency to NIH 3T3 fibroblasts was strongly dependent on the vector dose and its ratio to PDLLA film thickness. A detailed analysis of these relationships allowed establishing correlations which can be used to calculate suitable combinations of COPROGs and PDLLA yielding optimal gene transfer efficiency. This was verified with COPROG-activated PDLLA coatings on titanium foils. HEK 293 and mesenchymal stem cells expressed the BMP-2 gene comprised in the gene-activated surface in a manner that was consistent with the predicted dose-response and toxicity profiles found in NIH 3T3 cells. The systematic procedure presented here for identifying optimal coating compositions can be applied to any combination of vector type and coating material.

  20. Genetic diversity and molecular evolution of Naga King Chili inferred from internal transcribed spacer sequence of nuclear ribosomal DNA.

    PubMed

    Kehie, Mechuselie; Kumaria, Suman; Devi, Khumuckcham Sangeeta; Tandon, Pramod

    2016-02-01

    Sequences of the Internal Transcribed Spacer (ITS1-5.8S-ITS2) of nuclear ribosomal DNAs were explored to study the genetic diversity and molecular evolution of Naga King Chili. Our study indicated the occurrence of nucleotide polymorphism and haplotypic diversity in the ITS regions. The present study demonstrated that the variability of ITS1 with respect to nucleotide diversity and sequence polymorphism exceeded that of ITS2. Sequence analysis of 5.8S gene revealed a much conserved region in all the accessions of Naga King Chili. However, strong phylogenetic information of this species is the distinct 13 bp deletion in the 5.8S gene which discriminated Naga King Chili from the rest of the Capsicum sp. Neutrality test results implied a neutral variation, and population seems to be evolving at drift-mutation equilibrium and free from directed selection pressure. Furthermore, mismatch analysis showed multimodal curve indicating a demographic equilibrium. Phylogenetic relationships revealed by Median Joining Network (MJN) analysis denoted a clear discrimination of Naga King Chili from its closest sister species (Capsicum chinense and Capsicum frutescens). The absence of star-like network of haplotypes suggested an ancient population expansion of this chili.

  1. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer

    PubMed Central

    Ruegger, Paul M.; Clark, Robin T.; Weger, John R.; Braun, Jonathan; Borneman, James

    2014-01-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses. PMID:25034229

  2. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer.

    PubMed

    Ruegger, Paul M; Clark, Robin T; Weger, John R; Braun, Jonathan; Borneman, James

    2014-10-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses.

  3. Setting limits on homeotic gene function: restraint of Sex combs reduced activity by teashirt and other homeotic genes.

    PubMed Central

    Andrew, D J; Horner, M A; Petitt, M G; Smolik, S M; Scott, M P

    1994-01-01

    Each of the homeotic genes of the HOM or HOX complexes is expressed in a limited domain along the anterior-posterior axis. Each homeotic protein directs the formation of characteristic structures, such as wings or ribs. In flies, when a heat shock-inducible homeotic gene is used to produce a homeotic protein in all cells of the embryo, only some cells respond by altering their fates. We have identified genes that limit where the homeotic gene Sex combs reduced (Scr) can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is prevented from inducing prothoracic structures by the three bithorax complex (BX-C) homeotic genes. However, two of the BX-C homeotic genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), have no effect on the ability of Scr to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt (tsh) and in the last abdominal segment by the third BX-C gene, Abdominal-B (AbdB). Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function. Images PMID:7907545

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2008-09-01

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

  6. A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus

    PubMed Central

    Sun, Ren; Lin, Su-Fang; Gradoville, Lyndle; Yuan, Yan; Zhu, Fanxiu; Miller, George

    1998-01-01

    Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases. PMID:9724796

  7. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    PubMed Central

    Horlbeck, Max A; Gilbert, Luke A; Villalta, Jacqueline E; Adamson, Britt; Pak, Ryan A; Chen, Yuwen; Fields, Alexander P; Park, Chong Yon; Corn, Jacob E; Kampmann, Martin; Weissman, Jonathan S

    2016-01-01

    We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 PMID:27661255

  8. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    EPA Science Inventory

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  9. Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes.

    PubMed

    Tardáguila, Manuel; González-Gugel, Elena; Sánchez-Pacheco, Aurora

    2011-03-01

    Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displacement of the heterochromatin protein 1β (HP1β) to the nuclear periphery. Moreover, we found that T3-mediated pituitary gene transcription is associated with an increase in H3Ser10ph. Interestingly, the Aurora kinase B inhibitor ZM443979 abolishes the effect of T3 on H3Ser10ph, blocks HP1β delocalization, and significantly reduces ligand-dependent transactivation. Similar effects were shown when Aurora kinase B expression was abrogated in small interfering RNA assays. In an effort to understand the underlying mechanism by which T3 increases H3Ser10ph, we demonstrate that liganded thyroid hormone receptor directly interacts with Aurora kinase B, increasing its kinase activity. Moreover, using chromatin immunoprecipitation assays, we have shown that Aurora kinase B participates of a mechanism that displaces HP1β from promoter region, thus preparing the chromatin for the transcriptional activation of T3 regulated genes. Our findings reveal a novel role for Aurora kinase B during transcriptional initiation in GO/G1, apart from its well-known mitotic activity.

  10. Structure and transcription of the Drosophila mulleri alcohol dehydrogenase genes.

    PubMed

    Fischer, J A; Maniatis, T

    1985-10-11

    The D. melanogaster Adh gene is transcribed from two different promoters; a proximal (larval) promoter is active during late embryonic and larval stages, and a distal (adult) promoter is active primarily in third instar larvae and in adult flies (1). Genetic analyses suggest that several species of the mulleri subgroup (distant relatives of D. melanogaster) have two closely-linked Adh genes, Adh-1 and Adh-2, each of which expresses a different ADH protein (2). The temporal pattern of expression of Adh-1 and Adh-2 is similar to the expression of D. melanogaster Adh from the proximal and distal promoters (2,3,4). We are interested in the molecular basis for the pattern of Adh expression in the mulleri subgroup species and in the mechanism of the switch in Adh promoter utilization. For these reasons, we have studied the structure and transcription of the Adh locus of D. mulleri, a species of the mulleri subgroup. We show that the ADH-1 and ADH-2 proteins are expressed from two distinct genes separated by 2 kilobase pairs, and that Adh-1 and Adh-2 are transcribed in the expected temporal pattern. In addition, we find a pseudogene 1.2 kb upstream from Adh-2, which is transcribed in a temporal pattern similar to Adh-2.

  11. Molecular Identification of Two Strains of Phellinus sp. by Internal Transcribed Spacer Sequence Analysis

    PubMed Central

    2011-01-01

    Two species of cultivated Phellinus sp. were identified as P. baumii by internal transcribed spacer (ITS) sequence analysis. The fruit bodies of the examined strains were similar to those of naturally occurring strains, having a bracket-like form, yellow-to-orange color, and poroid hymenial surfaces. The DNA sequences of ITS region of both strains showed a homology of 99% with ITS1 to ITS2 sequences of P. (Inonotus) baumii strain PB0806. PMID:22783119

  12. Network activity-independent coordinated gene expression program for synapse assembly.

    PubMed

    Valor, Luis M; Charlesworth, Paul; Humphreys, Lawrence; Anderson, Chris N G; Grant, Seth G N

    2007-03-13

    Global biological datasets generated by genomics, transcriptomics, and proteomics provide new approaches to understanding the relationship between the genome and the synapse. Combined transcriptome analysis and multielectrode recordings of neuronal network activity were used in mouse embryonic primary neuronal cultures to examine synapse formation and activity-dependent gene regulation. Evidence for a coordinated gene expression program for assembly of synapses was observed in the expression of 642 genes encoding postsynaptic and plasticity proteins. This synaptogenesis gene expression program preceded protein expression of synapse markers and onset of spiking activity. Continued expression was followed by maturation of morphology and electrical neuronal networks, which was then followed by the expression of activity-dependent genes. Thus, two distinct sequentially active gene expression programs underlie the genomic programs of synapse function.

  13. Xenoestrogenic gene expression: structural features of active polycyclic aromatic hydrocarbons.

    PubMed

    Schultz, T Wayne; Sinks, Glendon D

    2002-04-01

    Estrogenicity was assessed using the Saccharomyces cerevisiae-based Lac-Z reporter assay and was reported as the logarithm of the inverse of the 50% molar beta-galactosidase activity (log[EC50(-1)]). In an effort to quantify the relationship between molecular structure of polycyclic aromatic hydrocarbons (PAHs) and estrogenic gene expression, a series of PAHs were evaluated. With noted exceptions, the results of these studies indicate that the initial two-dimensional structural warning for estrogenicity, the superpositioning of a hydroxylated aromatic system on the phenolic A-ring of 17-beta-estradiol, can be extended to the PAHs. This two-dimensional-alignment criterion correctly identified estrogenicity of 22 of the 29 PAHs evaluated. Moreover, the estrogenic potency of these compounds was directly related to the size of the hydrophobic backbone. The seven compounds classified incorrectly by this structural feature were either dihydroxylated naphthalenes or aromatic nitrogen-heterocyclic compounds; all such compounds were false positives. Results with dihydroxylated naphthalenes reveal derivatives that were nonestrogenic when superimposed on the phenolic A-ring of 17-beta-estradiol had the second hydroxyl group in the position of the C-ring or were catechol-like in structure. Structural alerts for nitrogen-heterocyclic compounds must take into account the position of the hydroxyl group and the in-ring nitrogen atom; compounds with the hydroxyl group and nitrogen atom involved with the same ring were observed to be nonactive.

  14. Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae

    SciTech Connect

    Takuma, Shinya; Nakashima, Noriyuki; Tantirungkij, Manee

    1991-12-31

    A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P steps were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

  15. 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.

  16. 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.

  17. Providencia stuartii genes activated by cell-to-cell signaling and identification of a gene required for production or activity of an extracellular factor.

    PubMed

    Rather, P N; Ding, X; Baca-DeLancey, R R; Siddiqui, S

    1999-12-01

    By utilizing reporter transposons, five Providencia stuartii genes that are activated by the accumulation of self-produced extracellular signals have been identified. These genes have been designated cma for conditioned medium activated. The presence of conditioned medium from stationary-phase cultures grown in rich media resulted in the premature activation of each gene in cells at early log phase, with activation values ranging from 6- to 26-fold. Preparation of conditioned medium from an M9 salts medium and fractionation by gel filtration chromatography resulted in fractions within the included volume which activated three of the cma fusions. In addition, depending on the reporter fusion, peak activity was found in different fractions. The partially purified factors activated in a dose-dependent manner. Characterization of the factors activating the cma fusions indicated that they were stable to heat, alkali, and acid. Furthermore, for each cma fusion, factor activity was not reproduced by the addition of homoserine lactone, homocysteine thiolactone, pyruvate, Casamino Acids, or alpha-ketoglutarate. The identities of three cma genes have been determined and revealed physiological roles in amino acid biosynthesis and nutrient import. To begin to address the pathways for production of or response to the extracellular factors, we have identified a locus, aarA, that is required for the activation of four cma fusions. The AarA product was required for factor activity in extracellular supernatants, indicating a possible role in biosynthesis or export.

  18. The great escape: Active genes on inactive sex chromosomes and their evolutionary implications.

    PubMed

    Sin, Ho-Su; Namekawa, Satoshi H

    2013-09-01

    Epigenetic mechanisms precisely regulate sex chromosome inactivation as well as genes that escape the silencing process. In male germ cells, DNA damage response factor RNF8 establishes active epigenetic modifications on the silent sex chromosomes during meiosis, and activates escape genes during a state of sex chromosome-wide silencing in postmeiotic spermatids. During the course of evolution, the gene content of escape genes in postmeiotic spermatids recently diverged on the sex chromosomes. This evolutionary feature mirrors the epigenetic processes of sex chromosomes in germ cells. In this article, we describe how epigenetic processes have helped to shape the evolution of sex chromosome-linked genes. Furthermore, we compare features of escape genes on sex chromosomes in male germ cells to escape genes located on the single X chromosome silenced during X-inactivation in females, clarifying the distinct evolutionary implications between male and female escape genes.

  19. Detecting microRNA activity from gene expression data

    PubMed Central

    2010-01-01

    Background MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. Results Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. Conclusions We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources. PMID:20482775

  20. Therapeutic antibody gene transfer: an active approach to passive immunity.

    PubMed

    Bakker, Joost M; Bleeker, Wim K; Parren, Paul W H I

    2004-09-01

    Advances in gene transfer approaches are enabling the possibility of applying therapeutic antibodies using DNA. In particular gene transfer in combination with electroporation is promising and can result in generating in vivo antibody concentrations in the low therapeutic range. However, several important problems need to be dealt with before antibody gene transfer can become a valuable supplement to the current therapies. As antibody production following gene transfer is difficult to control, the danger of inducing autoimmune conditions or uncontrollable side effects occurs in cases in which autologous antigens are targeted. It is suggested that the most promising area of application therefore appears to be infectious disease in which heterologous antigens are targeted and concerns for long-term antibody exposure are minimal. Finally, genes encoding fully human antibodies will enhance long-term expression and decrease problems linked to immunogenicity.

  1. High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Janse, Ingmar; Meima, Marion; Kardinaal, W. Edwin A.; Zwart, Gabriel

    2003-01-01

    For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed. PMID:14602623

  2. Gene editing activity on extrachromosomal arrays in C. elegans transgenics.

    PubMed

    Falgowski, Kerry A; Kmiec, Eric B

    2011-04-15

    Gene editing by modified single-stranded oligonucleotides is a strategy aimed at inducing single base changes into the genome, generating a permanent genetic change. The work presented here explores gene editing capabilities in the model organism Caenorhabditis elegans. Current approaches to gene mutagenesis in C. elegans have been plagued by non-specificity and thus the ability to induce precise, directed alterations within the genome of C. elegans would offer a platform upon which structure/function analyses can be carried out. As such, several in vivo assay systems were developed to evaluate gene editing capabilities in C. elegans. Fluorescence was chosen as the selectable endpoint as fluorescence can be easily detected through the transparent worm body even from minimal expression. Two tissue specific fluorescent expression vectors containing either a GFP or mCherry transgene were mutagenized to create a single nonsense mutation within the open reading frame of each respective fluorescent gene. These served as the target site to evaluate the frequency of gene editing on extrachromosomal array transgenic lines. Extrachromosomal arrays can carry hundreds of copies of the transgene, therefore low frequency events (like those in the gene editing reaction) may be detected. Delivery of the oligonucleotide was accomplished by microinjection into the gonads of young adult worms in an effort to induce repair of the mutated fluorescent gene in the F1 progeny. Despite many microinjections on the transgenic strains with varying concentrations of ODNs, no gene editing events were detected. This result is consistent with the previous research, demonstrating the difficulties encountered in targeting embryonic stem cells and the pronuclei of single-celled embryos.

  3. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts.

    PubMed

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-09-29

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil.

  4. H2-Producing Bacterial Community during Rice Straw Decomposition in Paddy Field Soil: Estimation by an Analysis of [FeFe]-Hydrogenase Gene Transcripts

    PubMed Central

    Baba, Ryuko; Asakawa, Susumu; Watanabe, Takeshi

    2016-01-01

    The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H2 production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H2 producers and/or H2 metabolic patterns dynamically change during rice straw decomposition in paddy soil. PMID:27319579

  5. Satellite-tagged transcribing sequences in Bubalus bubalis genome undergo programmed modulation in meiocytes: possible implications for transcriptional inactivation.

    PubMed

    Chattopadhyay, M; Gangadharan, S; Kapur, V; Azfer, M A; Prakash, B; Ali, S

    2001-09-01

    We cloned and sequenced a 1378 bp BamHI satellite DNA fraction from the water buffalo Bubalus bubalis and have studied its expression in different tissues. The GC-rich sequences of the resultant contig pDS5 crosshybridize only with bovid DNA and are not conserved evolutionarily. Typing of buffalo genomic DNA using pDS5 with several restriction enzymes revealed multilocus monomorphic bands. Similar typing of cattle, buffalo, goat, sheep, and gaur genomic DNA revealed variations in copy number and allele length giving rise to species-specific band patterns. Expression study of pDS5 in bubaline samples by RNA slot-blot, Northern blot, and RT-PCR showed various levels of signal in all the somatic tissues and germline cells except heart. A GenBank database search revealed homology of pDS5 sequences in the 5' region from nt 1-1261 with collagen gene. An AluI typing analysis of DNA from bubaline semen samples showed consistent loss of two bands. The presence of corresponding bands in somatic tissues suggests a sequence modulation within the pDS5 array in meiocytes during spermatogenesis, which is restored in the somatic cells after fertilization. Modulation of the satellite-tagged transcribing sequence in the meiocytes may be a mechanism of its inactivation.

  6. Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

    PubMed

    Booton, G C; Kaufman, L; Chandler, M; Oguto-Ohwayo, R; Duan, W; Fuerst, P A

    1999-03-01

    The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

  7. Conservation in the first internal transcribed spacer (ITS1) region of Hematodinium perezi (genotype III) from Callinectes sapidus .

    PubMed

    Pagenkopp Lohan, Katrina M; Small, Hamish J; Shields, Jeffrey D; Place, Allen R; Reece, Kimberly S

    2013-03-13

    Hematodinium spp. infections have been reported from blue crabs Callinectes sapidus in high-salinity waters of the USA from New Jersey to Texas. Recently, H. perezi (genotype III) has been proposed as the parasite species and genotype infecting blue crabs from Virginia; however, it is unknown whether this same genotype is present in blue crabs from other locations. To address this question, we collected 317 blue crabs from Massachusetts, Virginia, Georgia, Florida, Louisiana, and Texas to test for the presence of H. perezi (III) using a specific PCR assay targeting the first internal transcribed spacer (ITS1) region of the ribosomal RNA gene complex. To examine the genetic variation within H. perezi (III), ITS1 region sequences from the parasite in blue crabs from multiple locations were compared to each other and to those of H. perezi (III) found in alternate hosts from Virginia. In total, 34 distinct ITS1 sequence variants of the parasite were identified from blue crabs alone, and 38 distinct variants were identified when alternate hosts were included. However, a single ITS1 sequence variant appeared in all geographic regions and hosts, and also in blue crabs sampled from a previous study. The high similarity among all the ITS1 region sequences examined (>98%) and the observation of a single variant found throughout a large geographic range, strongly suggests that a single species and genotype of Hematodinium, specifically H. perezi (III), infects blue crabs from Virginia to Texas and multiple alternate host species in Virginia.

  8. Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Coutinho, João Paulo; Carvalho, Ana; Lima-Brito, José

    2014-11-26

    The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. In this study, co-dominant ITS PCR-RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ∼700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (HE) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR-RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR-RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area.

  9. Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction–restriction fragment length polymorphism

    PubMed Central

    Coutinho, João Paulo; Carvalho, Ana; Lima-Brito, José

    2015-01-01

    The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) techniques. In this study, co-dominant ITS PCR–RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ∼700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (HE) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR–RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR–RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area. PMID:25429047

  10. Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout

    USGS Publications Warehouse

    Rasmussen, C.; Purcell, M.K.; Gregg, J.L.; LaPatra, S.E.; Winton, J.R.; Hershberger, P.K.

    2010-01-01

    The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of ~740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

  11. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases.

    PubMed

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-03-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  12. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    PubMed Central

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-01-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals. PMID:26950874

  13. Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster.

    PubMed Central

    Chary, V K; de la Fuente, J L; Liras, P; Martin, J F

    1997-01-01

    Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase. PMID:9251185

  14. RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids

    PubMed Central

    Sin, Ho-Su; Barski, Artem; Zhang, Fan; Kartashov, Andrey V.; Nussenzweig, Andre; Chen, Junjie; Andreassen, Paul R.; Namekawa, Satoshi H.

    2012-01-01

    Sex chromosomes are uniquely subject to chromosome-wide silencing during male meiosis, and silencing persists into post-meiotic spermatids. Against this background, a select set of sex chromosome-linked genes escapes silencing and is activated in post-meiotic spermatids. Here, we identify a novel mechanism that regulates escape gene activation in an environment of chromosome-wide silencing in murine germ cells. We show that RNF8-dependent ubiquitination of histone H2A during meiosis establishes active epigenetic modifications, including dimethylation of H3K4 on the sex chromosomes. RNF8-dependent active epigenetic memory, defined by dimethylation of H3K4, persists throughout meiotic division. Various active epigenetic modifications are subsequently established on the sex chromosomes in post-meiotic spermatids. These RNF8-dependent modifications include trimethylation of H3K4, histone lysine crotonylation (Kcr), and incorporation of the histone variant H2AFZ. RNF8-dependent epigenetic programming regulates escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Kcr accumulates at transcriptional start sites of sex-linked genes activated in an RNF8-dependent manner, and a chromatin conformational change is associated with RNF8-dependent epigenetic programming. Furthermore, we demonstrate that this RNF8-dependent pathway is distinct from that which recognizes DNA double-strand breaks. Our results establish a novel connection between a DNA damage response factor (RNF8) and epigenetic programming, specifically in establishing active epigenetic modifications and gene activation. PMID:23249736

  15. Somatic Activation of rasK Gene in a Human Ovarian Carcinoma

    NASA Astrophysics Data System (ADS)

    Feig, L. A.; Bast, R. C.; Knapp, R. C.; Cooper, G. M.

    1984-02-01

    A tumor isolate from a patient with serous cystadenocarcinoma of the ovary contained an activated rasK gene detected by transfection of NIH/3T3 cells. In contrast, DNA from normal cells of the same patient lacked transforming activity, indicating that activation of this transforming gene was the consequence of somatic mutation in the neoplastic cells. The transforming gene product displayed an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that differed from the mobilities of rasK transforming proteins in other tumors, indicating that a previously undescribed mutation was responsible for activation of rasK in this ovarian carcinoma.

  16. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  17. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    SciTech Connect

    Xiao, Xiao; Gang, Yi; Wang, Honghong; Wang, Jiayin; Zhao, Lina; Xu, Li; Liu, Zhiguo

    2015-02-06

    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  18. Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian; Ulrich, Eldon L.; Zhao, Qin; Wrobel, Russell L.; Newman, Craig S.; Fox, Brian G.; Phillips, George N Jr; Markley, John L.; Sussman, Michael R.

    2005-01-01

    Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

  19. Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

    PubMed

    Osipovich, Anna B; White-Grindley, Erica K; Hicks, Geoffrey G; Roshon, Michael J; Shaffer, Christian; Moore, Jason H; Ruley, H Earl

    2004-01-01

    Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

  20. Activation of cryptic 3′ splice sites within introns of cellular genes following gene entrapment

    PubMed Central

    Osipovich, Anna B.; White-Grindley, Erica K.; Hicks, Geoffrey G.; Roshon, Michael J.; Shaffer, Christian; Moore, Jason H.; Ruley, H. Earl

    2004-01-01

    Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3′-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3′ splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3′ splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3′ splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3′ splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3′ splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3′ splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3′ processing and polyadenylation of cellular transcripts. PMID:15155860

  1. Transposable Elements Contribute to Activation of Maize Genes in Response to Abiotic Stress

    PubMed Central

    Makarevitch, Irina; Waters, Amanda J.; West, Patrick T.; Stitzer, Michelle; Hirsch, Candice N.; Ross-Ibarra, Jeffrey; Springer, Nathan M.

    2015-01-01

    Transposable elements (TEs) account for a large portion of the genome in many eukaryotic species. Despite their reputation as “junk” DNA or genomic parasites deleterious for the host, TEs have complex interactions with host genes and the potential to contribute to regulatory variation in gene expression. It has been hypothesized that TEs and genes they insert near may be transcriptionally activated in response to stress conditions. The maize genome, with many different types of TEs interspersed with genes, provides an ideal system to study the genome-wide influence of TEs on gene regulation. To analyze the magnitude of the TE effect on gene expression response to environmental changes, we profiled gene and TE transcript levels in maize seedlings exposed to a number of abiotic stresses. Many genes exhibit up- or down-regulation in response to these stress conditions. The analysis of TE families inserted within upstream regions of up-regulated genes revealed that between four and nine different TE families are associated with up-regulated gene expression in each of these stress conditions, affecting up to 20% of the genes up-regulated in response to abiotic stress, and as many as 33% of genes that are only expressed in response to stress. Expression of many of these same TE families also responds to the same stress conditions. The analysis of the stress-induced transcripts and proximity of the transposon to the gene suggests that these TEs may provide local enhancer activities that stimulate stress-responsive gene expression. Our data on allelic variation for insertions of several of these TEs show strong correlation between the presence of TE insertions and stress-responsive up-regulation of gene expression. Our findings suggest that TEs provide an important source of allelic regulatory variation in gene response to abiotic stress in maize. PMID:25569788

  2. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

    PubMed

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-03-03

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

  3. Differential regulation of Bvg-activated virulence factors plays a role in Bordetella pertussis pathogenicity.

    PubMed

    Kinnear, S M; Marques, R R; Carbonetti, N H

    2001-04-01

    Bordetella pertussis, the causative agent of whooping cough, regulates expression of many virulence factors via a two-component signal transduction system encoded by the bvgAS regulatory locus. It has been shown by transcription activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed within 10 min following a bvgAS-inducing signal, while prn is transcribed after 1 h and ptx is not transcribed until 2 to 4 h after induction. These genes therefore represent early, intermediate, and late classes of bvg-activated promoters, respectively. Although there have been many insightful studies into the mechanisms of BvgAS-mediated regulation, the role that differential regulation of virulence genes plays in B. pertussis pathogenicity has not been characterized. We provide evidence that alterations to the promoter regions of bvg-activated genes can alter the kinetic pattern of expression of these genes without changing steady-state transcription levels. In addition, B. pertussis strains containing these promoter alterations that express either ptx at an early time or fha at a late time demonstrate a significant reduction in their ability to colonize respiratory tracts in an intranasal mouse model of infection. These data suggest a role for differential regulation of bvg-activated genes, and therefore for the BvgAS regulatory system, in the pathogenicity of B. pertussis.

  4. Differential Regulation of Bvg-Activated Virulence Factors Plays a Role in Bordetella pertussis Pathogenicity

    PubMed Central

    Kinnear, Susan M.; Marques, Ryan R.; Carbonetti, Nicholas H.

    2001-01-01

    Bordetella pertussis, the causative agent of whooping cough, regulates expression of many virulence factors via a two-component signal transduction system encoded by the bvgAS regulatory locus. It has been shown by transcription activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed within 10 min following a bvgAS-inducing signal, while prn is transcribed after 1 h and ptx is not transcribed until 2 to 4 h after induction. These genes therefore represent early, intermediate, and late classes of bvg-activated promoters, respectively. Although there have been many insightful studies into the mechanisms of BvgAS-mediated regulation, the role that differential regulation of virulence genes plays in B. pertussis pathogenicity has not been characterized. We provide evidence that alterations to the promoter regions of bvg-activated genes can alter the kinetic pattern of expression of these genes without changing steady-state transcription levels. In addition, B. pertussis strains containing these promoter alterations that express either ptx at an early time or fha at a late time demonstrate a significant reduction in their ability to colonize respiratory tracts in an intranasal mouse model of infection. These data suggest a role for differential regulation of bvg-activated genes, and therefore for the BvgAS regulatory system, in the pathogenicity of B. pertussis. PMID:11254549

  5. Comparative evolution of S7 Intron 1 and ribosomal internal transcribed spacer in Coilia nasus (Clupeiformes: Engraulidae).

    PubMed

    Liu, Dong; Guo, Hong-Yi; Tang, Wen-Qiao; Yang, Jin-Quan

    2012-01-01

    Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.

  6. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions.

  7. ihfA Gene of the Bacterium Myxococcus xanthus and Its Role in Activation of Carotenoid Genes by Blue Light

    PubMed Central

    Moreno, Alberto J.; Fontes, Marta; Murillo, Francisco J.

    2001-01-01

    Myxococcus xanthus responds to blue light by producing carotenoids. Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. We had already reported the isolation of a carotenoid-less, Tn5-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes. Here, we show that ΩMR508::Tn5 affects all known light-inducible promoters in different ways. It blocks the activation of two of them by light but makes the activity of a third one light independent. The ΩMR508 locus has been cloned and sequenced. The mutation had occurred at the promoter of a gene we propose is the M. xanthus ortholog of ihfA. This encodes the α subunit of the histone-like integration host factor protein. An in-frame deletion within ihfA causes the same effects as the ΩMR508::Tn5 insertion. Like other IhfA proteins, the deduced amino acid sequence of M. xanthus IhfA shows much similarity to HU, another histone-like protein. Sequence comparison data, however, and the finding that the M. xanthus gene is preceded by gene pheT, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship. The M. xanthus ihfA gene shows some unusual features, both from structural and physiological points of view. In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of M. xanthus. PMID:11133949

  8. Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens.

    PubMed

    Chen, Jie; Moinard, Magalie; Xu, Jianping; Wang, Shouxian; Foulongne-Oriol, Marie; Zhao, Ruilin; Hyde, Kevin D; Callac, Philippe

    2016-01-01

    The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.

  9. Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens

    PubMed Central

    Chen, Jie; Moinard, Magalie; Xu, Jianping; Wang, Shouxian; Foulongne-Oriol, Marie; Zhao, Ruilin; Hyde, Kevin D.; Callac, Philippe

    2016-01-01

    The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated. PMID:27228131

  10. Regulation of Gene33 expression by insulin requires MEK-ERK activation.

    PubMed

    Keeton, Adam B; Xu, Jie; Franklin, J Lee; Messina, Joseph L

    2004-09-17

    Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK-ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.

  11. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

  12. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity

    PubMed Central

    Ahmed, Afsar U.; Williams, Bryan R. G.; Hannigan, Gregory E.

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  13. Laughter up-regulates the genes related to NK cell activity in diabetes.

    PubMed

    Hayashi, Takashi; Tsujii, Satoru; Iburi, Tadao; Tamanaha, Tamiko; Yamagami, Keiko; Ishibashi, Rieko; Hori, Miyo; Sakamoto, Shigeko; Ishii, Hitoshi; Murakami, Kazuo

    2007-12-01

    To elucidate the sustainable effects of laughter on gene expression, we recruited type 2 diabetic patients who were in-patient for receiving self-management education and examined time-dependent regulation for gene expression by laughter. Two-day experiment was performed. On one day, the patients watched comic video and laughed together with hospital staffs. On the other day, they participated in an inpatient diabetes educational program. Blood samples were collected before and 1.5, 4 h after watching comic video or spending lecture time, and changes in gene expression were comprehensively analyzed by microarray technique. Of the 41,000 genes analyzed, the laughter relatively up-regulated 39 genes, among which, 27 genes were relatively increased in the expression for all the observation period after watching comic video. By functional classification of these genes, 14 genes were found to be related to natural killer cell activity. No genes were included that are directly involved in blood glucose regulation, though successive suppression of postprandial blood glucose levels was observed. These results suggest that the laughter influences the expression of many genes classified into immune responses, and may contribute to amelioration of postprandial blood glucose elevation through a modulation of NK cell activity caused by up-regulation of relating genes.

  14. Upregulating endogenous genes by an RNA-programmable artificial transactivator

    PubMed Central

    Fimiani, Cristina; Goina, Elisa; Mallamaci, Antonello

    2015-01-01

    To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. PMID:26152305

  15. Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues

    PubMed Central

    2012-01-01

    Background Transposable elements (TEs) make up a large part of eukaryotic genomes. Due to their repetitive nature and to the fact that they harbour regulatory signals, TEs can be responsible for chromosomal rearrangements, movement of gene sequences and evolution of gene regulation and function. Retrotransposon ubiquity raises the question about their function in genomes and most are transcriptionally inactive due to rearrangements that compromise their activity. However, the activity of TEs is currently considered to have been one of the major processes in genome evolution. Findings We report on the characterization of a transcriptionally active gypsy-like retrotransposon (named Corky) from Quercus suber, in a comparative and quantitative study of expression levels in different tissues and distinct developmental stages through RT-qPCR. We observed Corky’s differential transcription levels in all the tissues analysed. Conclusions These results document that Corky’s transcription levels are not constant. Nevertheless, they depend upon the developmental stage, the tissue analysed and the potential occurring events during an individuals’ life span. This modulation brought upon by different developmental and environmental influences suggests an involvement of Corky in stress response and during development. PMID:22888907

  16. Impact of physical activity and doping on epigenetic gene regulation.

    PubMed

    Schwarzenbach, Heidi

    2011-10-01

    To achieve success in sports, many athletes consume doping substances, such as anabolic androgenic steroids and growth hormones, and ignore the negative influence of these drugs on their health. Apart from the unethical aspect of doping in sports, it is essential to consider the tremendous risk it represents to their physical condition. The abuse of pharmaceuticals which improve athletic performance may alter the expression of specific genes involved in muscle and bone metabolism by epigenetic mechanisms, such as DNA methylation and histone modifications. Moreover, excessive and relentless training to increase the muscle mass, may also have an influence on the health of the athletes. This stress releases neurotransmitters and growth factors, and may affect the expression of endogenous genes by DNA methylation, too. This paper focuses on the relationship between epigenetic mechanisms and sports, highlights the potential consequences of abuse of doping drugs on gene expression, and describes methods to molecularly detect epigenetic changes of gene markers reflecting the physiological or metabolic effects of doping agents.

  17. Activation and Characterization of a Cryptic Polycyclic Tetramate Macrolactam Biosynthetic Gene Cluster

    PubMed Central

    Luo, Yunzi; Huang, Hua; Liang, Jing; Wang, Meng; Lu, Lu; Shao, Zengyi; Cobb, Ryan E.; Zhao, Huimin

    2014-01-01

    Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of them have not been characterized. Here we apply a plug and play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three potential PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) which resembles iterative PKSs known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products. PMID:24305602

  18. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    NASA Astrophysics Data System (ADS)

    Golubnitchaya-Labudová, O.; Portele, A.; Vaçata, V.; Lubec, G.; Rink, H.; Höfer, M.

    1997-10-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the insert of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9.

  19. Lymphocyte activation gene 3 and coronary artery disease

    PubMed Central

    Golden, Diana; Kolmakova, Antonina; Sura, Sunitha; Vella, Anthony T.; Manichaikul, Ani; Wang, Xin-Qun; Bielinski, Suzette J.; Taylor, Kent D.; Chen, Yii-Der Ida; Rich, Stephen S.

    2016-01-01

    BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants. RESULTS: In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004). CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk. FUNDING: This work was

  20. Lymphocyte activation gene 3 and coronary artery disease.

    PubMed

    Golden, Diana; Kolmakova, Antonina; Sura, Sunitha; Vella, Anthony T; Manichaikul, Ani; Wang, Xin-Qun; Bielinski, Suzette J; Taylor, Kent D; Chen, Yii-Der Ida; Rich, Stephen S; Rodriguez, Annabelle

    2016-10-20

    BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants. RESULTS: In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004). CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk. FUNDING: This work was

  1. Correcting Transcription Factor Gene Sets for Copy Number and Promoter Methylation Variations

    PubMed Central

    Rathi, Komal S.; Gaykalova, Daria A.; Hennesey, Patrick; Califano, Joseph A.; Ochs, Michael F.

    2014-01-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, since some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation and demonstrate the improvement in inference of transcription factor activity in a simulated data set based on the background expression observed in normal head and neck tissue. PMID:25195578

  2. Correcting transcription factor gene sets for copy number and promoter methylation variations.

    PubMed

    Rathi, Komal S; Gaykalova, Daria A; Hennessey, Patrick; Califano, Joseph A; Ochs, Michael F

    2014-09-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, as some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation, and demonstrate the improvement in inference of transcription factor activity in a simulated dataset based on the background expression observed in normal head and neck tissue.

  3. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    DOEpatents

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  4. Aeromonas hydrophila Lateral Flagellar Gene Transcriptional Hierarchy

    PubMed Central

    Wilhelms, Markus; Gonzalez, Victor; Merino, Susana

    2013-01-01

    Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ54/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. PMID:23335410

  5. BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks.

    PubMed

    Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M Mallar; Banaschewski, Tobias; Barker, Gareth J; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W; Smolka, Michael N; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D

    2015-06-12

    During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function.

  6. Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

    PubMed

    Griesenbeck, Joachim; Wittner, Manuel; Charton, Romain; Conconi, Antonio

    2012-01-01

    In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

  7. Genomic signatures of germline gene expression.

    PubMed

    McVicker, Graham; Green, Phil

    2010-11-01

    Transcribed regions in the human genome differ from adjacent intergenic regions in transposable element density, crossover rates, and asymmetric substitution and sequence composition patterns. We tested whether these differences reflect selection or are instead a byproduct of germline transcription, using publicly available gene expression data from a variety of germline and somatic tissues. Crossover rate shows a strong negative correlation with gene expression in meiotic tissues, suggesting that crossover is inhibited by transcription. Strand-biased composition (G+T content) and A → G versus T → C substitution asymmetry are both positively correlated with germline gene expression. We find no evidence for a strand bias in allele frequency data, implying that the substitution asymmetry reflects a mutation rather than a fixation bias. The density of transposable elements is positively correlated with germline expression, suggesting that such elements preferentially insert into regions that are actively transcribed. For each of the features examined, our analyses favor a nonselective explanation for the observed trends and point to the role of germline gene expression in shaping the mammalian genome.

  8. Cluster Analysis of Tumor Suppressor Genes in Canine Leukocytes Identifies Activation State

    PubMed Central

    Daly, Julie-Anne; Mortlock, Sally-Anne; Taylor, Rosanne M.; Williamson, Peter

    2015-01-01

    Cells of the immune system undergo activation and subsequent proliferation in the normal course of an immune response. Infrequently, the molecular and cellular events that underlie the mechanisms of proliferation are dysregulated and may lead to oncogenesis, leading to tumor formation. The most common forms of immunological cancers are lymphomas, which in dogs account for 8%–20% of all cancers, affecting up to 1.2% of the dog population. Key genes involved in negatively regulating proliferation of lymphocytes include a group classified as tumor suppressor genes (TSGs). These genes are also known to be associated with progression of lymphoma in humans, mice, and dogs and are potential candidates for pathological grading and diagnosis. The aim of the present study was to analyze TSG profiles in stimulated leukocytes from dogs to identify genes that discriminate an activated phenotype. A total of 554 TSGs and three gene set collections were analyzed from microarray data. Cluster analysis of three subsets of genes discriminated between stimulated and unstimulated cells. These included 20 most upregulated and downregulated TSGs, TSG in hallmark gene sets significantly enriched in active cells, and a selection of candidate TSGs, p15 (CDKN2B), p18 (CDKN2C), p19 (CDKN1A), p21 (CDKN2A), p27 (CDKN1B), and p53 (TP53) in the third set. Analysis of two subsets suggested that these genes or a subset of these genes may be used as a specialized PCR set for additional analysis. PMID:27478369

  9. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  10. Identification of two peanut germin-like genes and the potential superoxide dismutase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Germin and germin-like protein (GLP) genes are members of large multigene families. These genes have been reported to play a role directly or indirectly in plant defense response. A number of GLPs have been demonstrated to have superoxidase dismutase (SOD) or oxalate oxidase (OxO) activity, leading ...

  11. Quantitative structure-activity relationships and docking studies of calcitonin gene-related peptide antagonists.

    PubMed

    Kyani, Anahita; Mehrabian, Mohadeseh; Jenssen, Håvard

    2012-02-01

    Defining the role of calcitonin gene-related peptide in migraine pathogenesis could lead to the application of calcitonin gene-related peptide antagonists as novel migraine therapeutics. In this work, quantitative structure-activity relationship modeling of biological activities of a large range of calcitonin gene-related peptide antagonists was performed using a panel of physicochemical descriptors. The computational studies evaluated different variable selection techniques and demonstrated shuffling stepwise multiple linear regression to be superior over genetic algorithm-multiple linear regression. The linear quantitative structure-activity relationship model revealed better statistical parameters of cross-validation in comparison with the non-linear support vector regression technique. Implementing only five peptide descriptors into this linear quantitative structure-activity relationship model resulted in an extremely robust and highly predictive model with calibration, leave-one-out and leave-20-out validation R(2) of 0.9194, 0.9103, and 0.9214, respectively. We performed docking of the most potent calcitonin gene-related peptide antagonists with the calcitonin gene-related peptide receptor and demonstrated that peptide antagonists act by blocking access to the peptide-binding cleft. We also demonstrated the direct contact of residues 28-37 of the calcitonin gene-related peptide antagonists with the receptor. These results are in agreement with the conclusions drawn from the quantitative structure-activity relationship model, indicating that both electrostatic and steric factors should be taken into account when designing novel calcitonin gene-related peptide antagonists.

  12. Influence of tetracycline on tetracycline-resistant heterotrophs and tet genes in activated sludge process.

    PubMed

    Yu, Jie; Liu, Dongfang; Li, Kexun

    2015-03-01

    The concentrations of tetracycline-intermediate resistant, tetracycline-resistant heterotrophic bacteria, and total heterotrophic bacteria were examined to assess the influence of tetracycline on tetracycline-resistant heterotrophs by the R2A agar cultivation method in the tetracycline fortified activated sludge process and in the natural background. Results showed that the percentages of both tetracycline-intermediate resistant and tetracycline-resistant heterotrophic bacteria in total heterotrophic bacteria were significantly increased, after tetracycline was fed to activated sludge for a 3 months period under four different operating conditions, as compared with the background. In order to investigate the mechanism of activated sludge resistance to tetracycline, polymerase chain reaction experiments were carried out to analyze the existence and evolution of tet genes in the presence of tetracycline. Results revealed that only tet A and tet B genes out of the 11 target tet genes were observed in tetracycline treated activated sludge while no tet gene was detected in background. This indicated that tet A gene could accumulate in activated sludge with slower and continuous influent, while the accumulation of tet B gene could be attributed to shorter hydraulic retention time. Therefore, it was proposed in this study that tetracycline-resistant genes created by efflux pumps spread earlier and quicker to encode resistance to tetracycline, which facilitated the increase in tetracycline-resistance.

  13. Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage.

    PubMed

    Thomas, David M; Francescutti-Verbeem, Dina M; Kuhn, Donald M

    2006-03-01

    Microglia are the resident antigen-presenting cells within the central nervous system (CNS), and they serve immune-like functions in protecting the brain against injury and invading pathogens. By contrast, activated microglia can secrete numerous reactants that damage neurons. The pathogenesis of various neurodegenerative diseases has been associated with microglial activation, but the signaling pathways that program a neuronally protective or destructive phenotype in microglia are not known. To increase the understanding of microglial activation, microarray analysis was used to profile the transcriptome of BV-2 microglial cells after activation. Microglia were activated by lipopolysaccharide, the HIV neurotoxic protein TAT, and dopamine quinone, each of which has been linked to dopamine neuronal damage. We identified 210 of 9882 genes whose expression was differentially regulated by all activators (116 increased and 94 decreased in expression). Gene ontology analysis assigned up-regulated genes to a number of specific biological processes and molecular functions, including immune response, inflammation, and cytokine/chemokine activity. Genes down-regulated in expression contribute to conditions that are permissive of microglial migration, lowered adhesion to matrix, lessened phagocytosis, and reduction in receptors that oppose chemotaxis and inflammation. These results elaborate a broad profile of microglial genes whose expression is altered by conditions associated with both neurodegenerative diseases and microglial activation.

  14. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  15. FTH1P3, a Novel H-Ferritin Pseudogene Transcriptionally Active, Is Ubiquitously Expressed and Regulated during Cell Differentiation

    PubMed Central

    Di Sanzo, Maddalena; Aversa, Ilenia; Santamaria, Gianluca; Gagliardi, Monica; Panebianco, Mariafranca; Biamonte, Flavia; Zolea, Fabiana; Faniello, Maria Concetta

    2016-01-01

    Ferritin, the major iron storage protein, performs its essential functions in the cytoplasm, nucleus and mitochondria. The variable assembly of 24 subunits of the Heavy (H) and Light (L) type composes the cytoplasmic molecule. In humans, two distinct genes code these subunits, both belonging to complex multigene families. Until now, one H gene has been identified with the coding sequence interrupted by three introns and more than 20 intronless copies widely dispersed on different chromosomes. Two of the intronless genes are actively transcribed in a tissue-specific manner. Herein, we report that FTH1P3, another intronless pseudogene, is transcribed. FTH1P3 transcript was detected in several cell lines and tissues, suggesting that its transcription is ubiquitary, as it happens for the parental ferritin H gene. Moreover, FTH1P3 expression is positively regulated during the cell differentiation process. PMID:26982978

  16. FTH1P3, a Novel H-Ferritin Pseudogene Transcriptionally Active, Is Ubiquitously Expressed and Regulated during Cell Differentiation.

    PubMed

    Di Sanzo, Maddalena; Aversa, Ilenia; Santamaria, Gianluca; Gagliardi, Monica; Panebianco, Mariafranca; Biamonte, Flavia; Zolea, Fabiana; Faniello, Maria Concetta; Cuda, Giovanni; Costanzo, Francesco

    2016-01-01

    Ferritin, the major iron storage protein, performs its essential functions in the cytoplasm, nucleus and mitochondria. The variable assembly of 24 subunits of the Heavy (H) and Light (L) type composes the cytoplasmic molecule. In humans, two distinct genes code these subunits, both belonging to complex multigene families. Until now, one H gene has been identified with the coding sequence interrupted by three introns and more than 20 intronless copies widely dispersed on different chromosomes. Two of the intronless genes are actively transcribed in a tissue-specific manner. Herein, we report that FTH1P3, another intronless pseudogene, is transcribed. FTH1P3 transcript was detected in several cell lines and tissues, suggesting that its transcription is ubiquitary, as it happens for the parental ferritin H gene. Moreover, FTH1P3 expression is positively regulated during the cell differentiation process.

  17. T7 RNA polymerase cannot transcribe through a highly knotted DNA template.

    PubMed Central

    Portugal, J; Rodríguez-Campos, A

    1996-01-01

    The ability of T7 RNA polymerase to transcribe a plasmid DNA in vitro in its linear, supercoiled, relaxed and knotted forms was analysed. Similar levels of transcription were found on each template with the exception of plasmids showing varying degrees of knotting (obtained using stoichiometric amounts of yeast topoisomerase II). A purified fraction of knotted DNA with a high number of nodes (crosses) was found to be refractory to transcription. The unknotting of the knotted plasmids, using catalytic amounts of topoisomerase II, restored their capacity as templates for transcription to levels similar to those obtained for the other topological forms. These results demonstrate that highly knotted DNA is the only topological form of DNA that is not a template for transcription. We suggest that the regulation of transcription, which depends on the topological state of the template, might be related to the presence of knotted DNA with different number of nodes. PMID:9016657

  18. Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA.

    PubMed

    Varela, Eduardo S; Lima, João P M S; Galdino, Alexsandro S; Pinto, Luciano da S; Bezerra, Walderly M; Nunes, Edson P; Alves, Maria A O; Grangeiro, Thalles B

    2004-01-01

    The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.

  19. Imidacloprid and Thiamethoxam Induced Mutations in Internal Transcribed Spacer 2 (ITS2) of Anopheles stephensi.

    PubMed

    Bhinder, Preety; Chaudhry, Asha; Barna, Bhupinder; Kaur, Satvinderjeet

    2012-05-01

    The present article deals with the polymerase chain reaction (PCR)-based genotoxicity evaluation of neonicotinoid pesticides, imidacloprid and thiamethoxam, by using the genome of a mosquito Anopheles stephensi taken as an experimental model. After treatment of the second instar larvae with LC(20) of the pesticides for 24 h, the induced nucleotide sequence variations in the internal transcribed spacer 2 (ITS2) of freshly hatched unfed control and treated individuals was studied from the sequence alignment data and the mutations in the form of insertion, deletion and substitution of bases were recorded. Measurable differences, indicative of the genetic damage due to imidacloprid and thiamethoxam were observed when ITS2 sequences of control and treated individuals were compared. It was found that imidacloprid-treated individual had 8 deletions, 29 insertions, 18 transitions and 33 transversions, whereas thiamethoxam-treated individual had 10 deletions, 8 insertions, 47 transitions and 68 transversions.

  20. Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows.

    PubMed

    Vargas-Bello-Pérez, E; Cancino-Padilla, N; Romero, J

    2016-12-01

    Molecular techniques are important tools for microbiological studies in different habitats, and the internal transcribed spacer (ITS) has been proved to be useful for analyzing fungal diversity. The aim of this study was to use the ITS region to generate ruminal yeast profile and to identify ruminal yeast. DNA from ruminal digesta was extracted to amplify the ribosomal ITS region. The profile from the PCR products was visualized and the excised bands from the profile were identified as the genera Millerozyma, Pichia, Rhizomucor and Hyphopichia. Overall, the ITS resulted to be a simple, fast and sensitive approach that allowed profiling and identification of ruminal yeast that have not been previously described (Millerozyma and Hyphopichia) in the rumen microbial community.

  1. Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure.

    PubMed

    Coleman, Annette W

    2015-03-01

    rRNA is one of the few universal features of life, making it uniquely suited to assess phylogenetic relationships. The processing of the initial polycistronic rRNA transcript is also a conserved process, involving numerous cleavage events and the generation of secondary structures. The secondary structure of the internal transcribed spacer (ITS) regions of nuclear rRNA transcripts are well known for a wide variety of eukaryotes and have been used to aid in the alignment of these sequences for phylogenetic comparisons. By contrast, study of the processing of the initial rRNA transcripts has been largely limited to yeast, mice, rats, and humans. Here I examine the known cleavage sites in the two ITS regions and their positions relative to the secondary structure. A better understanding of the conservation of secondary structures and cleavage sites within the ITS regions will improve evolutionary inferences based on these sequences.

  2. Ectopic Activation of Germline and Placental Genes Identifies Aggressive Metastasis-Prone Lung Cancers

    PubMed Central

    Rousseaux, Sophie; Debernardi, Alexandra; Jacquiau, Baptiste; Vitte, Anne-Laure; Vesin, Aurélien; Nagy-Mignotte, Hélène; Moro-Sibilot, Denis; Brichon, Pierre-Yves; Lantuejoul, Sylvie; Hainaut, Pierre; Laffaire, Julien; de Reyniès, Aurélien; Beer, David G.; Timsit, Jean-François; Brambilla, Christian; Brambilla, Elisabeth; Khochbin, Saadi

    2016-01-01

    Activation of normally silent tissue-specific genes and the resulting cell “identity crisis” are the unexplored consequences of malignant epigenetic reprogramming. We designed a strategy for investigating this reprogramming, which consisted of identifying a large number of tissue-restricted genes that are epigenetically silenced in normal somatic cells and then detecting their expression in cancer. This approach led to the demonstration that large-scale “off-context” gene activations systematically occur in a variety of cancer types. In our series of 293 lung tumors, we identified an ectopic gene expression signature associated with a subset of highly aggressive tumors, which predicted poor prognosis independently of the TNM (tumor size, node positivity, and metastasis) stage or histological subtype. The ability to isolate these tumors allowed us to reveal their common molecular features characterized by the acquisition of embryonic stem cell/germ cell gene expression profiles and the down-regulation of immune response genes. The methodical recognition of ectopic gene activations in cancer cells could serve as a basis for gene signature–guided tumor stratification, as well as for the discovery of oncogenic mechanisms, and expand the understanding of the biology of very aggressive tumors. PMID:23698379

  3. Targeting c-Myc-activated genes with a correlation method: Detection of global changes in large gene expression network dynamics

    PubMed Central

    Remondini, D.; O'Connell, B.; Intrator, N.; Sedivy, J. M.; Neretti, N.; Castellani, G. C.; Cooper, L. N.

    2005-01-01

    This work studies the dynamics of a gene expression time series network. The network, which is obtained from the correlation of gene expressions, exhibits global dynamic properties that emerge after a cell state perturbation. The main features of this network appear to be more robust when compared with those obtained with a network obtained from a linear Markov model. In particular, the network properties strongly depend on the exact time sequence relationships between genes and are destroyed by random temporal data shuffling. We discuss in detail the problem of finding targets of the c-myc protooncogene, which encodes a transcriptional regulator whose inappropriate expression has been correlated with a wide array of malignancies. The data used for network construction are a time series of gene expression, collected by microarray analysis of a rat fibroblast cell line expressing a conditional Myc-estrogen receptor oncoprotein. We show that the correlation-based model can establish a clear relationship between network structure and the cascade of c-myc-activated genes. PMID:15867157

  4. A transcribed ultraconserved noncoding RNA, Uc.173, is a key molecule for the inhibition of lead-induced neuronal apoptosis.

    PubMed

    Nan, Aruo; Zhou, Xinke; Chen, Lijian; Liu, Meiling; Zhang, Nan; Zhang, Li; Luo, Yuanwei; Liu, Zhenzhong; Dai, Lijun; Jiang, Yiguo

    2016-01-05

    As a common toxic metal, lead has significant neurotoxicity to brain development. Long non-coding RNAs (lncRNAs) function in multiple biological processes. However, whether lncRNAs are involved in lead-induced neurotoxicity remains unclear. Uc.173 is a lncRNA from a transcribed ultra-conservative region (T-UCR) of human, mouse and rat genomes. We established a lead-induced nerve injury mouse model. It showed the levels of Uc.173 decreased significantly in hippocampus tissue and serum of the model. We further tested the expression of Uc.173 in serum of lead-exposed children, which also showed a tendency to decrease. To explore the effects of Uc.173 on lead-induced nerve injury, we overexpressed Uc.173 in an N2a mouse nerve cell line and found Uc.173 had an inhibitory effect on lead-induced apoptosis of N2a. To investigate the molecular mechanisms of Uc.173 in apoptosis associated with lead-induced nerve injury, we predicted the target microRNAs of Uc.173 by using miRanda, TargetScan and RegRNA. After performing quantitative real-time PCR and bioinformatics analysis, we showed Uc.173 might inter-regulate with miR-291a-3p in lead-induced apoptosis and regulate apoptosis-associated genes. Our study suggests Uc.173 significantly inhibits the apoptosis of nerve cells, which may be mediated by inter-regulation with miRNAs in lead-induced nerve injury.

  5. Nuclear internal transcribed spacer-1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio.

    PubMed

    Minamoto, Toshifumi; Uchii, Kimiko; Takahara, Teruhiko; Kitayoshi, Takumi; Tsuji, Satsuki; Yamanaka, Hiroki; Doi, Hideyuki

    2017-03-01

    The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.

  6. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

    PubMed

    Perera, Omaththage P; Allen, Kerry C; Jain, Devendra; Purcell, Matthew; Little, Nathan S; Luttrell, Randall G

    2015-01-01

    Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

  7. Genetic diversity of Ephedra plants in mongolia inferred from internal transcribed spacer sequence of nuclear ribosomal DNA.

    PubMed

    Kitani, Yuki; Zhu, Shu; Batkhuu, Javzan; Sanchir, Chinbat; Komatsu, Katsuko

    2011-01-01

    Ephedrae herba has been used for treating colds, relieving coughs and asthma from ancient times. We previously reported the distribution of Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and Ephedra sp. in Mongolia, and among them E. sinica and E. equisetina were potential new resources of Ephedrae herba of Japanese pharmacopoeia grade, based on our field survey and subsequent molecular and chemical assessments. However, the Ephedra population in southwestern areas showed a high possibility of having hybrid origins. Further field surveys in southwestern areas, and sequence analysis of the partial nuclear internal transcribed spacer 1 (ITS1) region, besides trnK and 18S ribosomal RNA (rRNA) gene regions, were conducted in order to obtain detailed evidence of hybridization status. As a result, the distribution of E. glauca in western area and E. lomatolepis in western-most area was confirmed. The ITS sequences from all 8 Ephedra species collected in Mongolia were roughly divided into 5 types (types I-V). Type II sequence, having several additive nucleotides, was found in Ephedra sp., E. glauca, E. regeliana and E. sinica, which provided useful information for tracing hybrid origins. Morphological, genetic and distribution evidence suggested that the hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii and E. intermedia were involved in these events. Integrated with our previous report, trnK-, 18S- and ITS-types from pure lines of each species are proposed. In addition, we propose a practicable method for detecting additive peaks on a direct sequencing electropherogram.

  8. Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus: implications for molecular diagnosis in clinical microbiology laboratories.

    PubMed

    Woo, Patrick C Y; Leung, Shui-Yee; To, Kelvin K W; Chan, Jasper F W; Ngan, Antonio H Y; Cheng, Vincent C C; Lau, Susanna K P; Yuen, Kwok-Yung

    2010-01-01

    Although internal transcribed spacer region (ITS) sequence heterogeneity has been reported in a few fungal species, it has very rarely been reported in pathogenic fungi and has never been described in Mucorales, causes of the highly fatal mucormycosis. In a recent outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus infection in patients with hematological malignancies, PCR of the ITS of four of the 28 R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp. Direct sequencing of the purified bands showed frequent double peaks along all of the sequence traces and occasional triple peaks for P12, D3-1, and D4-1. The thick bands of the four R. microsporus strains were purified and cloned. Sequencing of 10 clones for each strain revealed two different ITS sequences for P11 and three different ITS sequences for P12, D3-1, and D4-1. Variations in ITS sequence among the different ribosomal DNA (rDNA) operons in the same strain were observed in only ITS1 and ITS2 and not the 5.8S rDNA region. One copy of P11, P12, and D4-1, respectively, and one copy of P11, P12, D3-1, and D4-1, respectively, showed identical sequences. This represents the first evidence of ITS sequence heterogeneity in Mucorales. ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of fungi in clinical microbiology laboratories. When thick bands and double peaks are observed during PCR sequencing of a gene target, such a strain should be sent to reference laboratories proficient in molecular technologies for further identification and/or genotyping.

  9. Activity-dependent regulation of genes implicated in X-linked non-specific mental retardation.

    PubMed

    Boda, B; Mas, C; Muller, D

    2002-01-01

    X-linked forms of non-specific mental retardation are complex disorders, for which mutations in several genes have recently been identified. These include OPHN1, GDI1, PAK3, IL1RAPL, TM4SF2, FMR2 and RSK2. To investigate the mechanisms through which alterations of these gene products could result in cognitive impairment, we analyzed their expression using quantitative PCR technique in two in vitro models of activity-dependent gene regulation: kainate-induced seizures and long-term synaptic potentiation (LTP). We found that the level of expression of four genes, PAK3, IL1RAPL, RSK2 and TM4SF2, was significantly up-regulated following kainate treatment. Furthermore we observed a significant increase in mRNA levels of PAK3 and IL1RAPL following LTP induction. These results suggest a possible role for these four genes in activity-dependent brain plasticity.

  10. NF-Y activates genes of metabolic pathways altered in cancer cells.

    PubMed

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-12

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

  11. 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.

  12. Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

    PubMed Central

    Pan, Yi; Comiskey, Daniel F.; Kelly, Lisa E.; Chandler, Dawn S.

    2016-01-01

    Purpose The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. Methods X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. Results We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. Conclusions vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system. PMID:28003732

  13. Intricate regulation of tyrosine hydroxylase activity and gene expression.

    PubMed

    Kumer, S C; Vrana, K E

    1996-08-01

    Tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of tyrosine hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of tyrosine hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady-state levels of active tyrosine hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation-dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate tyrosine hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.

  14. Screening for novel human genes associated with CRE pathway activation with cell microarray.

    PubMed

    Tian, Linjie; Wang, Pingzhang; Guo, Jinhai; Wang, Xinyu; Deng, Weiwei; Zhang, Chenying; Fu, Dongxu; Gao, Xia; Shi, Taiping; Ma, Dalong

    2007-07-01

    In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format.

  15. Genome-wide analysis of short interspersed nuclear elements SINES revealed high sequence conservation, gene association and retrotranspositional activity in wheat

    PubMed Central

    Ben-David, Smadar; Yaakov, Beery; Kashkush, Khalil

    2013-01-01

    Short interspersed nuclear elements (SINEs) are non-autonomous non-LTR retroelements that are present in most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, they are poorly studied in plants, especially in wheat (Triticum aestivum). We used quantitative PCR of various wheat species to determine the copy number of a wheat SINE family, termed Au SINE, combined with computer-assisted analyses of the publicly available 454 pyrosequencing database of T. aestivum. In addition, we utilized site-specific PCR on 57 Au SINE insertions, transposon methylation display and transposon display on newly formed wheat polyploids to assess retrotranspositional activity, epigenetic status and genetic rearrangements in Au SINE, respectively. We retrieved 3706 different insertions of Au SINE from the 454 pyrosequencing database of T. aestivum, and found that most of the elements are inserted in A/T-rich regions, while approximately 38% of the insertions are associated with transcribed regions, including known wheat genes. We observed typical retrotransposition of Au SINE in the second generation of a newly formed wheat allohexaploid, and massive hypermethylation in CCGG sites surrounding Au SINE in the third generation. Finally, we observed huge differences in the copy numbers in diploid Triticum and Aegilops species, and a significant increase in the copy numbers in natural wheat polyploids, but no significant increase in the copy number of Au SINE in the first four generations for two of three newly formed allopolyploid species used in this study. Our data indicate that SINEs may play a prominent role in the genomic evolution of wheat through stress-induced activation. PMID:23855320

  16. The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals

    PubMed Central

    Handschin, Christoph; Blättler, Sharon; Roth, Adrian; Looser, Renate; Oscarson, Mikael; Kaufmann, Michel R; Podvinec, Michael; Gnerre, Carmela; Meyer, Urs A

    2004-01-01

    Background Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. Results To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. Conclusion Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species. PMID:15479477

  17. A new PCR primer for the identification of Paracoccidioides brasiliensis based on rRNA sequences coding the internal transcribed spacers (ITS) and 5 x 8S regions.

    PubMed

    Imai, T; Sano, A; Mikami, Y; Watanabe, K; Aoki, F H; Branchini, M L; Negroni, R; Nishimura, K; Miyaji, M

    2000-08-01

    Internal transcribed spacer (ITS) genes including the 5.8S ribosomal (r)RNA of Paracoccidioides brasiliensis were amplified and the DNA sequences were determined. Based on a comparison of the sequence information, a new polymerase chain reaction (PCR) primer pair was designed for specific amplification of DNA for P. brasiliensis. This primer pair amplified a 418-bp DNA sequence and was 100% successful in identifying 29 strains of P. brasiliensis (including the reference strains) isolated from the regions of Brazil, Costa Rica, Japan, Argentina or from different sources. The results of specificity tests of these primers to compare the fungus with those of Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei are also reported.

  18. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions.

    PubMed

    Scorzetti, Gloria; Fell, J W; Fonseca, A; Statzell-Tallman, Adele

    2002-12-01

    Basidiomycetous yeasts in the Urediniomycetes and Hymenomycetes were examined by sequence analysis in two ribosomal DNA regions: the D1/D2 variable domains at the 5' end of the large subunit rRNA gene (D1/D2) and the internal transcribed spacers (ITS) 1 and 2. Four major lineages were recognized in each class: Microbotryum, Sporidiobolus, Erythrobasidium and Agaricostilbum in the Urediniomycetes; Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales in the Hymenomycetes. Bootstrap support for many of the clades within those lineages is weak; however, phylogenetic analysis provides a focal point for in-depth study of biological relationships. Combined sequence analysis of the D1/D2 and ITS regions is recommended for species identification, while species definition requires classical biological information such as life cycles and phenotypic characterization.

  19. Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

    SciTech Connect

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-07-08

    Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

  20. The occ1 gene is preferentially expressed in the primary visual cortex in an activity-dependent manner: a pattern of gene expression related to the cytoarchitectonic area in adult macaque neocortex.

    PubMed

    Tochitani, S; Liang, F; Watakabe, A; Hashikawa, T; Yamamori, T

    2001-01-01

    Marker molecules to visualize specific subsets of neurons are useful for studying the functional organization of the neocortex. One approach to identify such molecular markers is to examine the differences in molecular properties among morphologically and physiologically distinct neuronal cell types. We used differential display to compare mRNA expression in the anatomically and functionally distinct areas of the adult macaque neocortex. We found that a gene, designated occ1, was preferentially transcribed in the posterior region of the neocortex, especially in area 17. Complete sequence analysis revealed that occ1 encodes a macaque homolog of a secretable protein, TSC-36/follistatin-related protein (FRP). In situ hybridization histochemistry confirmed the characteristic neocortical expression pattern of occ1 and showed that occ1 transcription is high in layers II, III, IVA and IVC of area 17. In addition, occ1 transcription was observed selectively in cells of the magnocellular layers in the lateral geniculate nucleus (LGN). Dual labeling immunohistochemistry showed that the occ1-positive neurons in area 17 include both gamma-aminobutyric acid (GABA)-positive aspiny inhibitory cells and the alpha-subunit of type II calcium/calmodulin-dependent protein kinase (CaMKII alpha)-positive spiny excitatory cells. With brief periods of monocular deprivation, the occ1 mRNA level decreased markedly in deprived ocular dominance columns of area 17. From this we conclude that the expression of occ1 mRNA is present in a subset of neurons that are preferentially localized in particular laminae of area 17 and consist of various morphological and physiological neuronal types, and, furthermore, occ1 transcription is subject to visually driven activity-dependent regulation.

  1. Transcriptionally active genome regions are preferred targets for retrovirus integration.

    PubMed Central

    Scherdin, U; Rhodes, K; Breindl, M

    1990-01-01

    We have analyzed the transcriptional activity of cellular target sequences for Moloney murine leukemia virus integration in mouse fibroblasts. At least five of the nine random, unselected integration target sequences studied showed direct evidence for transcriptional activity by hybridization to nuclear run-on transcripts prepared from uninfected cells. At least four of the sequences contained multiple recognition sites for several restriction enzymes that cut preferentially in CpG-rich islands, indicating integration into 5' or 3' ends or flanking regions of genes. Assuming that only a minor fraction (less than 20%) of the genome is transcribed in mammalian cells, we calculated the probability that this association of retroviral integration sites with transcribed sequences is due to chance to be very low (1.6 x 10(-2]. Thus, our results strongly suggest that transcriptionally active genome regions are preferred targets for retrovirus integration. Images PMID:2296087

  2. Gene expression analysis during acute hepatitis C virus infection associates dendritic cell activation with viral clearance.

    PubMed

    Zabaleta, Aintzane; Riezu-Boj, Jose-Ignacio; Larrea, Esther; Villanueva, Lorea; Lasarte, Juan Jose; Guruceaga, Elizabeth; Fisicaro, Paola; Ezzikouri, Sayeh; Missale, Gabriele; Ferrari, Carlo; Benjelloun, Soumaya; Prieto, Jesús; Sarobe, Pablo

    2016-05-01

    Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection.

  3. Modulation of activation-associated host cell gene expression by the apicomplexan parasite Theileria annulata

    PubMed Central

    Durrani, Zeeshan; Weir, William; Pillai, Sreerekha; Kinnaird, Jane; Shiels, Brian

    2012-01-01

    Summary Infection of bovine leucocytes by Theileria annulata results in establishment of transformed, infected cells. Infection of the host cell is known to promote constitutive activation of pro-inflammatory transcription factors that have the potential to be beneficial or detrimental. In this study we have compared the effect of LPS activation on uninfected bovine leucocytes (BL20 cells) and their Theileria-infected counterpart (TBL20). Gene expression profiles representing activated uninfected BL20 relative to TBL20 cells were also compared. The results show that while prolonged stimulation with LPS induces cell death and activation of NF-κB in BL20 cells, the viability of Theileria-infected cells was unaffected. Analysis of gene expression networks provided evidence that the parasite establishes tight control over pathways associated with cellular activation by modulating reception of extrinsic stimuli and by significantly altering the expression outcome of genes targeted by infection-activated transcription factors. Pathway analysis of the data set identified novel candidate genes involved in manipulation of cellular functions associated with the infected transformed cell. The data indicate that the T. annulata parasite can irreversibly reconfigure host cell gene expression networks associated with development of inflammatory disease and cancer to generate an outcome thatis beneficial to survival and propagation of the infected leucocyte. PMID:22533473

  4. Physical activity-associated gene expression signature in nonhuman primate motor cortex.

    PubMed

    Mitchell, Amanda C; Leak, Rehana K; Garbett, Krassimira; Zigmond, Michael J; Cameron, Judy L; Mirnics, Károly

    2012-03-01

    It has been established that weight gain and weight loss are heavily influenced by activity level. In this study, we hypothesized that the motor cortex exhibits a distinct physical activity-associated gene expression profile, which may underlie changes in weight associated with movement. Using DNA microarrays we profiled gene expression in the motor cortex of a group of 14 female rhesus monkeys (Macaca mulatta) with a wide range of stable physical activity levels. We found that neuronal growth factor signaling and nutrient sensing transcripts in the brain were highly correlated with physical activity. A follow-up of AKT3 expression changes (a gene at the apex of neuronal survival and nutrient sensing) revealed increased protein levels of total AKT, phosphorylated AKT, and forkhead box O3 (FOXO3), one of AKT's main downstream effectors. In addition, we successfully validated three other genes via quantitative polymerase chain reaction (qPCR) (cereblon (CRBN), origin recognition complex subunit 4-like, and pyruvate dehydrogenase 4 (PDK4)). We conclude that these genes are important in the physical activity-associated pathway in the motor cortex, and may be critical for physical activity-associated changes in body weight and neuroprotection.

  5. Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.

    PubMed

    Warren, Curtis R; Kassir, Elias; Spurlin, James; Martinez, Jerahme; Putnam, Nicholas H; Farach-Carson, Mary C

    2015-01-01

    The heparan sulfate proteoglycan 2 (HSPG2)/perlecan gene is ancient and conserved in all triploblastic species. Its presence maintains critical cell boundaries in tissue and its large (up to ~900 kDa) modular structure has prompted speculation about the evolutionary origin of the gene. The gene's conservation amongst basal metazoans is unclear. After the recent sequencing of their genomes, the cnidarian Nematostella vectensis and the placozoan Trichoplax adhaerens have become favorite models for studying tissue regeneration and the evolution of multicellularity. More ancient basal metazoan phyla include the poriferan and ctenophore, whose evolutionary relationship has been clarified recently. Our in silico and PCR-based methods indicate that the HSPG2 gene is conserved in both the placozoan and cnidarian genomes, but not in those of the ctenophores and only partly in poriferan genomes. HSPG2 also is absent from published ctenophore and Capsaspora owczarzaki genomes. The gene in T. adhaerens is encoded as two separate but genetically juxtaposed genes that house all of the constituent pieces of the mammalian HSPG2 gene in tandem. These genetic constituents are found in isolated genes of various poriferan species, indicating a possible intronic recombinatory mechanism for assembly of the HSPG2 gene. Perlecan's expression during wound healing and boundary formation is conserved, as expression of the gene was activated during tissue regeneration and reformation of the basement membrane of N. vectensis. These data indicate that the complex HSPG2 gene evolved concurrently in a common ancestor of placozoans, cnidarians and bilaterians, likely along with the development of differentiated cell types separated by acellular matrices, and is activated to reestablish these tissue borders during wound healing.

  6. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  7. The role of EKLF in human beta-globin gene competition.

    PubMed

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  8. Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression.

    PubMed

    Kauffmann, Hans Martin; Pfannschmidt, Sylvia; Zöller, Heike; Benz, Anke; Vorderstemann, Birgit; Webster, Jeanette I; Schrenk, Dieter

    2002-02-28

    In the present study, we investigated the inducibility of the drug conjugate transporter genes MRP1 and MRP2 by redox-active compounds such as tertiary butylated hydroquinone (tBHQ) and quercetin and by chemicals known to activate the pregnane X receptor (PXR) such as rifampicin and clotrimazol and by the metalloid compound arsenite. The human MRP2 gene was found to be inducible in HepG2 cells by rifampicin, clotrimazol, arsenite and tBHQ. As MRP1 expression is extremely low in HepG2 cells, its inducibility was studied in MCF-7 cells. However, only tBHQ and quercetin acted as inducers, but not the other compounds investigated. Reporter gene assays demonstrated that proximal promoter regions of the genes contribute to the induction by tBHQ, quercetin (MRP1) and clotrimazol (MRP2). However, the deletion of binding sites supposed to mediate the induction process (a PXR-binding element-like sequence for the clotrimazol effect and an ARE (antioxidative response element) for the tBHQ/quercetin effect) did not result in a significant decrease in the induction factor indicating that other parts of the promoter are probably involved in the induction process. In summary, expression of both genes can be up-regulated by redox-active compounds, while the other compounds tested induced only MRP2 but not MRP1 expression.

  9. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    PubMed Central

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  10. Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

    NASA Astrophysics Data System (ADS)

    Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

    2016-07-01

    The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

  11. Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

    PubMed Central

    Xie, W Q; Whitton, B A; Simon, J W; Jäger, K; Reed, D; Potts, M

    1989-01-01

    A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants. Images PMID:2536677

  12. Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxoplasma gondii

    PubMed Central

    Gissot, Mathieu; Kelly, Krystyna A; Ajioka, James W; Greally, John M; Kim, Kami

    2007-01-01

    Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation “activation” marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code. PMID:17559302

  13. Regulation of nitrogen metabolism, starch utilisation and the beta-hbd-adh1 gene cluster in Clostridium acetobutylicum.

    PubMed

    Woods, D R; Reid, S J

    1995-10-01

    The successful genetic manipulation of Clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. The glutamine synthetase (glnA) gene is regulated by antisense RNA, transcribed from a downstream promoter, in the opposite direction to the glnA gene. An open reading frame (ORF) was detected downstream of the glnA gene, which has sequence homology to response regulators with anti-termination activity and may be involved in sensing nitrogen conditions. The expression of the linked beta-hbd, adh1 and fixB genes was investigated throughout the bacterial growth cycle by RNA hybridisation techniques. The adh1 gene was independently expressed as a 2.4-kb transcript which peaked at 12 h, immediately prior to the solventogenic phase. The beta-hbd and fixB genes were transcribed throughout the acidogenic and solventogenic phases. A regulator gene, regA, which complements a Bacillus subtilis ccpA mutant, has been identified and sequenced from C. acetobutylicum P262. The regA gene repressed the degradation of starch by an uncharacterised C. acetobutylicum gene, and may therefore play a role in the utilisation of carbohydrate substrates in this organism.

  14. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

    PubMed Central

    McGrath, J P; Jentsch, S; Varshavsky, A

    1991-01-01

    All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability. Images PMID:1989885

  15. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M.; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ≤ 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  16. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium.

    PubMed

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R; Aleksunes, Lauren M; Thomas, Russell S; Applegate, Dawn; Klaassen, Curtis D; Corton, J Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher's algorithm (p-value ≤ 10(-4))) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  17. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

    PubMed

    Green, Benjamin B; Houseman, E Andres; Johnson, Kevin C; Guerin, Dylan J; Armstrong, David A; Christensen, Brock C; Marsit, Carmen J

    2016-08-01

    The conversion of cytosine to 5-methylcystosine (5mC) is an important regulator of gene expression. 5mC may be enzymatically converted to 5-hydroxymethylcytosine (5hmC), with a potentially distinct regulatory function. We sought to investigate these cytosine modifications and their effect on gene expression by parallel processing of genomic DNA using bisulfite and oxidative bisulfite conversion in conjunction with RNA sequencing. Although values of 5hmC across the placental genome were generally low, we identified ∼21,000 loci with consistently elevated levels of 5-hydroxymethycytosine. Absence of 5hmC was observed in CpG islands and, to a greater extent, in non-CpG island-associated regions. 5hmC was enriched within poised enhancers, and depleted within active enhancers, as defined by H3K27ac and H3K4me1 measurements. 5hmC and 5mC were significantly elevated in transcriptionally silent genes when compared with actively transcribed genes. 5hmC was positively associated with transcription in actively transcribed genes only. Our data suggest that dynamic cytosine regulation, associated with transcription, provides the most complete epigenomic landscape of the human placenta, and will be useful for future studies of the placental epigenome.-Green, B. B., Houseman, E. A., Johnson, K. C., Guerin, D. J., Armstrong, D. A., Christensen, B. C., Marsit, C. J. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

  18. Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.

    PubMed Central

    Hanson, R D; Ley, T J

    1990-01-01

    The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined. Images PMID:2233710

  19. Hypermethylation of Wnt antagonist gene promoters and activation of Wnt pathway in myelodysplastic marrow cells.

    PubMed

    Masala, Erico; Valencia, Ana; Buchi, Francesca; Nosi, Daniele; Spinelli, Elena; Gozzini, Antonella; Sassolini, Francesca; Sanna, Alessandro; Zecchi, Sandra; Bosi, Alberto; Santini, Valeria

    2012-10-01

    We observed aberrant gene methylation of Wnt antagonists: sFRP1, sFRP2, sFRP4, sFRP5 and DKK1 in marrow cells of 55 MDS cases. Methylation of Wnt antagonist genes was associated with activation of the Wnt signaling pathway, consistent with the up-regulation of the Wnt downstream genes TCF1 and LEF1. Azacitidine exposure induced demethylation of Wnt-antagonist gene promoters and reduction of the non-phosphorylated β-catenin (NPBC) which is prevalent during Wnt pathway inactivation. Presence of ≥5% of bone marrow blasts was associated with methylation of sFRP1 and DKK1 and with methylation of more than two of the five Wnt antagonist genes.

  20. The New State of the Art: Cas9 for Gene Activation and Repression

    PubMed Central

    La Russa, Marie F.

    2015-01-01

    CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies. PMID:26370509

  1. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    PubMed

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  2. The circadian Clock gene regulates acrosin activity of sperm through serine protease inhibitor A3K

    PubMed Central

    Cheng, Shuting; Liang, Xin; Wang, Yuhui; Jiang, Zhou; Liu, Yanyou; Hou, Wang; Li, Shiping; Zhang, Jing

    2015-01-01

    Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements. PMID:26264441

  3. High intensity focused ultrasound-induced gene activation in sublethally injured tumor cells in vitro

    NASA Astrophysics Data System (ADS)

    Liu, Yunbo; Kon, Takashi; Li, Chuanyuan; Zhong, Pei

    2005-11-01

    Cultured human cervical cancer (HeLa) and rat mammary carcinoma (R3230Ac) cells were transfected with vectors encoding green fluorescent protein (GFP) under the control of hsp70B promoter. Aliquots of 10-μl transfected cells (5×107 cells/ml) were placed in 0.2-ml thin-wall polymerase chain reaction tubes and exposed to 1.1-MHz high intensity focused ultrasound (HIFU) at a peak negative pressure P-=2.68 MPa. By adjusting the duty cycle of the HIFU transducer, the cell suspensions were heated to a peak temperature from 50 to 70 °C in 1-10 s. Exposure dependent cell viability and gene activation were evaluated. For a 5-s HIFU exposure, cell viability dropped from 95% at 50 °C to 13% at 70 °C. Concomitantly, gene activation in sublethally injured tumor cells increased from 4% at 50 °C to 41% at 70 °C. A similar trend was observed at 60 °C peak temperature as the exposure time increased from 1 to 5 s. Further increase of exposure duration to 10 s led to significantly reduced cell viability and lower overall gene activation in exposed cells. Altogether, maximum HIFU-induced gene activation was achieved at 60 °C in 5 s. Under these experimental conditions, HIFU-induced gene activation was found to be produced primarily by thermal rather than mechanical stresses.

  4. Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia.

    PubMed Central

    Chang, K S; Stass, S A; Chu, D T; Deaven, L L; Trujillo, J M; Freireich, E J

    1992-01-01

    A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the

  5. SATB1 packages densely-looped, transciptionally-active chromatinfor coordinated expression of cytokine genes

    SciTech Connect

    Cai, Shutao; Lee, Charles C.; Kohwi-Shigematsu, Terumi

    2006-05-23

    SATB1 is an important regulator of nuclear architecture that anchors specialized DNA sequences onto its cage-like network and recruits chromatin remodeling/modifying factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4, and Il13 from the 200kb cytokine gene cluster region of mouse chromosome 11 during T-helper 2 (Th2)-cell activation. We show that upon cell activation, SATB1 is rapidly induced to form a unique transcriptionally-active chromatin structure that includes the cytokine gene region. Chromatin is folded into numerous small loops all anchored by SATB1, is histone H3 acetylated at lysine 9/14, and associated with Th2-specific factors, GATA3, STAT6, c-Maf, the chromatin-remodeling enzyme Brg-1, and RNA polymerase II across the 200kb region. Before activation, the chromatin displays some of these features, such as association with GATA3 and STAT6, but these were insufficient for cytokine gene expression. Using RNA interference (RNAi), we show that upon cell activation, SATB1 is not only required for chromatin folding into dense loops, but also for c-Maf induction and subsequently for Il4, Il5, and Il13 transcription. Our results show that SATB1 is an important determinant for chromatin architecture that constitutes a novel higher-order, transcriptionally-active chromatin structure upon Th2-cell activation.

  6. Trithorax group proteins: switching genes on and keeping them active.

    PubMed

    Schuettengruber, Bernd; Martinez, Anne-Marie; Iovino, Nicola; Cavalli, Giacomo

    2011-11-23

    Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.

  7. Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation.

    PubMed Central

    Birren, B W; Taplitz, S J; Herschman, H R

    1987-01-01

    We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation. Images PMID:3431545

  8. 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.

  9. Genes Involved in Interleukin-1 Receptor Type II Activities Are Associated With Asthmatic Phenotypes

    PubMed Central

    Madore, Anne-Marie; Vaillancourt, Vanessa T.; Bouzigon, Emmanuelle; Sarnowski, Chloé; Monier, Florent; Dizier, Marie-Hélène; Demenais, Florence

    2016-01-01

    Purpose Interleukin-1 (IL-1) plays a key role in inflammation and immunity and its decoy receptor, IL-1R2, has been implicated in transcriptomic and genetic studies of asthma. Methods Two large asthma family collections, the French-Canadian Saguenay—Lac-St-Jean (SLSJ) study and the French Epidemiological Study on the Genetics and Environment of Asthma (EGEA), were used to investigate the association of SNPs in 10 genes that modulate IL-1R2 activities with asthma, allergic asthma, and atopy. Gene-gene interactions were also tested. Results One SNP in BACE2 was associated with allergic asthma in the SLSJ study and replicated in the EGEA study before statistical correction for multiple testing. Additionally, two SNPs in the MMP2 gene were replicated in both studies prior to statistical correction and reached significance in the combined analysis. Moreover, three gene-gene interactions also survived statistical correction in the combined analyses (BACE1-IL1RAP in asthma and allergic asthma and IL1R1-IL1RAP in atopy). Conclusions Our results highlight the relevance of genes involved in the IL-1R2 activity in the context of asthma and asthma-related traits. PMID:27334786

  10. Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster.

    PubMed

    Thanapipatsiri, Anyarat; Gomez-Escribano, Juan Pablo; Song, Lijiang; Bibb, Maureen J; Al-Bassam, Mahmoud; Chandra, Govind; Thamchaipenet, Arinthip; Challis, Gregory L; Bibb, Mervyn J

    2016-11-17

    Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one-vemR-that encodes a transcriptional activator of the large ATP-binding LuxR-like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co-expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin-producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.

  11. Organization of human ACAT-2 gene and its cell-type-specific promoter activity.

    PubMed

    Song, B L; Qi, W; Yang, X Y; Chang, C C; Zhu, J Q; Chang, T Y; Li, B L

    2001-03-30

    Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Two ACAT genes exist in mammals. We report here the genomic organization of human ACAT-2 gene and analysis of its promoter activity in various cell lines. The human ACAT-2 gene spans over 18 kb and contains 15 exons. Three transcription start sites and one poly(A) site are identified by the 5'/3'-RACE. In addition, the human ACAT-2 gene is linked to the insulin-like growth factor binding protein 6 (IGFBP-6) gene in a head-to-tail manner with a small intergenic region of about 1.2 kb. The 5'-flanking region of human ACAT-2 gene contains many potential cis-acting elements for multiple transcriptional regulatory factors but lacks TATA and CCAAT boxes. Using promoter-luciferase reporter assays, we demonstrate the transcriptional activity of ACAT-2 gene promoter is high in Caco-2 cells, especially after these cells become postconfluent and behave as intestinal enterocytes.

  12. Genome-wide distribution of Auts2 binding localizes with active neurodevelopmental genes.

    PubMed

    Oksenberg, N; Haliburton, G D E; Eckalbar, W L; Oren, I; Nishizaki, S; Murphy, K; Pollard, K S; Birnbaum, R Y; Ahituv, N

    2014-09-02

    The autism susceptibility candidate 2 gene (AUTS2) has been associated with multiple neurological diseases including autism spectrum disorders (ASDs). Previous studies showed that AUTS2 has an important neurodevelopmental function and is a suspected master regulator of genes implicated in ASD-related pathways. However, the regulatory role and targets of Auts2 are not well known. Here, by using ChIP-seq (chromatin immunoprecipitation followed by deep sequencing) and RNA-seq on mouse embryonic day 16.5 forebrains, we elucidated the gene regulatory networks of Auts2. We find that the majority of promoters bound by Auts2 belong to genes highly expressed in the developing forebrain, suggesting that Auts2 is involved in transcriptional activation. Auts2 non-promoter-bound regions significantly overlap developing brain-associated enhancer marks and are located near genes involved in neurodevelopment. Auts2-marked sequences are enriched for binding site motifs of neurodevelopmental transcription factors, including Pitx3 and TCF3. In addition, we characterized two functional brain enhancers marked by Auts2 near NRXN1 and ATP2B2, both ASD-implicated genes. Our results implicate Auts2 as an active regulator of important neurodevelopmental genes and pathways and identify novel genomic regions that could be associated with ASD and other neurodevelopmental diseases.

  13. Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster

    PubMed Central

    Thanapipatsiri, Anyarat; Gomez‐Escribano, Juan Pablo; Song, Lijiang; Bibb, Maureen J.; Al‐Bassam, Mahmoud; Chandra, Govind

    2016-01-01

    Abstract Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one—vemR—that encodes a transcriptional activator of the large ATP‐binding LuxR‐like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co‐expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin‐producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases. PMID:27605017

  14. [A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture].

    PubMed

    Vardevanian, P O; Davtian, A M; Tiratsuian, S G; Vardevanian, A O

    1990-01-01

    A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.

  15. Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD

    PubMed Central

    Colton, Carol A; Mott, Ryan T; Sharpe, Hayley; Xu, Qing; Van Nostrand, William E; Vitek, Michael P

    2006-01-01

    Background Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state. Methods To study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1). Results Our findings confirmed that treatment of microglia with anti-inflammatory cytokines such as IL-4 and IL-13 induces a gene profile typical of alternative activation similar to that previously observed in peripheral macrophages. We then used this gene expression profile to examine two mouse models of AD, the APPsw (Tg-2576) and Tg-SwDI, models for amyloid deposition and for cerebral amyloid angiopathy (CAA) respectively. AGI, MRC1 and YM1 mRNA levels were significantly increased in the Tg-2576 mouse brains compared to age-matched controls while TNFα and NOS2 mRNA levels, genes commonly associated with classical activation, increased or did not change, respectively. Only TNFα mRNA increased in the Tg-SwDI mouse brain. Alternative activation genes were also identified in brain samples from individuals with AD and were compared to age-matched control individuals. In AD brain, mRNAs for TNFα, AGI, MRC1 and the chitinase-3 like 1 and 2 genes (CHI3L1; CHI3L2) were significantly increased

  16. ERK signaling pathway regulates sleep duration through activity-induced gene expression during wakefulness.

    PubMed

    Mikhail, Cyril; Vaucher, Angélique; Jimenez, Sonia; Tafti, Mehdi

    2017-01-24

    Wakefulness is accompanied by experience-dependent synaptic plasticity and an increase in activity-regulated gene transcription. Wake-induced genes are certainly markers of neuronal activity and may also directly regulate the duration of and need for sleep. We stimulated murine cortical cultures with the neuromodulatory signals that are known to control wakefulness in the brain and found that norepinephrine alone or a mixture of these neuromodulators induced activity-regulated gene transcription. Pharmacological inhibition of the various signaling pathways involved in the regulation of gene expression indicated that the extracellular signal-regulated kinase (ERK) pathway is the principal one mediating the effects of waking neuromodulators on gene expression. In mice, ERK phosphorylation in the cortex increased and decreased with wakefulness and sleep. Whole-body or cortical neuron-specific deletion of Erk1 or Erk2 significantly increased the duration of wakefulness in mice, and pharmacological inhibition of ERK phosphorylation decreased sleep duration and increased the duration of wakefulness bouts. Thus, this signaling pathway, which is highly conserved from Drosophila to mammals, is a key pathway that links waking experience-induced neuronal gene expression to sleep duration and quality.

  17. Identification of the nik Gene Cluster of Brucella suis: Regulation and Contribution to Urease Activity

    PubMed Central

    Jubier-Maurin, Véronique; Rodrigue, Agnès; Ouahrani-Bettache, Safia; Layssac, Marion; Mandrand-Berthelot, Marie-Andrée; Köhler, Stephan; Liautard, Jean-Pierre

    2001-01-01

    Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl2 excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host. PMID:11133934

  18. Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae.

    PubMed Central

    Cabrera-Juárez, E; Setlow, J K

    1985-01-01

    Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude (J. K. Setlow, E. Cabrera-Juárez, W. L. Albritton, D. Spikes, and A. Mutschler, J. Bacteriol. 164:525-534, 1985). Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The gyrase and copy number were considerably lower in plasmid-bearing strains carrying the prophage HP1c1. Two mutations affecting gyrase that are apparently regulatory caused an increase in gyrase without a concomitant increase in copy number. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. We conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell. PMID:2997116

  19. Involvement of Trichoderma Trichothecenes in the Biocontrol Activity and Induction of Plant Defense-Related Genes

    PubMed Central

    Malmierca, M. G.; Cardoza, R. E.; Alexander, N. J.; McCormick, S. P.; Hermosa, R.; Monte, E.

    2012-01-01

    Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied. PMID:22562989

  20. Inhibiting AP-1 activity alters cocaine induced gene expression and potentiates sensitization

    PubMed Central

    Paletzki, Ronald F.; Myakishev, Max V.; Polesskaya, Oksana; Orosz, Andras; Hyman, Steven E.; Vinson, Charles

    2008-01-01

    We have expressed A-FOS, an inhibitor of AP-1 DNA binding, in adult mouse striatal neurons. We observe normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral responses to cocaine. We analyzed mRNA from the striatum before and 4 and 24 hours after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 hours following cocaine treatment relative to controls. However, 24 hours after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. 56 gene are down regulated while 28 genes are up regulated including previously identified candidates for addiction including BDNF and Per1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared to human genome scans of addiction to identify potential genes in humans that are involved in addiction. PMID:18355967

  1. Child dopamine active transporter 1 genotype and parenting: evidence for evocative gene-environment correlations.

    PubMed

    Hayden, Elizabeth P; Hanna, Brigitte; Sheikh, Haroon I; Laptook, Rebecca S; Kim, Jiyon; Singh, Shiva M; Klein, Daniel N

    2013-02-01

    The dopamine active transporter 1 (DAT1) gene is implicated in psychopathology risk. Although the processes by which this gene exerts its effects on risk are poorly understood, a small body of research suggests that the DAT1 gene influences early emerging negative emotionality, a marker of children's psychopathology risk. As child negative emotionality evokes negative parenting practices, the DAT1 gene may also play a role in gene-environment correlations. To test this model, children (N = 365) were genotyped for the DAT1 gene and participated in standardized parent-child interaction tasks with their primary caregiver. The DAT1 gene 9-repeat variant was associated with child negative affect expressed toward the parent during parent-child interactions, and parents of children with a 9-repeat allele exhibited more hostility and lower guidance/engagement than parents of children without a 9-repeat allele. These gene-environment associations were partially mediated by child negative affect toward the parent. The findings implicate a specific polymorphism in eliciting negative parenting, suggesting that evocative associations play a role in elevating children's risk for emotional trajectories toward psychopathology risk.

  2. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

    PubMed

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

    2015-10-22

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

  3. Internal transcribed spacer sequence phylogeny of Crambe L. (Brassicaceae): molecular data reveal two Old World disjunctions.

    PubMed

    Francisco-Ortega, J; Fuertes-Aguilar, J; Gómez-Campo, C; Santos-Guerra, A; Jansen, R K

    1999-04-01

    Crambe L. (Brassicaceae) is an Old World genus with a disjunct distribution among four major centers of species diversity. A phylogenetic analysis of nucleotide sequences of the internal transcribed spacers (ITS) of the nuclear ribosomal repeat was conducted with 27 species of Crambe and 18 related genera. Cladistic analyses using weighted and unweighted parsimony support Crambe as a monophyletic genus with three major lineages. The first comprises those taxa endemic to the Macaronesian archipelagos. Taxa with a predominant Mediterranean distribution form the second assemblage, and a disjunction between east Africa (C. abyssinica) and the Mediterranean (C. hispanica) occurs in this clade. The third lineage includes all Eurosiberian-Asian taxa and C. kilimandscharica, a species from the highlands of east Africa. A basal biogeographic split between east Africa and Eurasia is present in the third clade. The patterns of relationships in the ITS tree are concordant with known climatic events in northern Africa and southwestern Asia since the middle Miocene. The ITS trees are congruent with the current sectional classification except for a few members of sections Crambe, Leptocrambe, and Orientecrambe (C. cordifolia, C. endentula, C. kilimandscharica, and C. kotschyana). Low levels of support in the basal branches do not allow resolution of which genera of the subtribes Raphaniae or Brassicinae are sister to Crambe. Both subtribes appear to be highly polyphyletic in the ITS trees.

  4. Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga.

    PubMed

    Estrada-Gómez, Sebastian; Vargas-Muñoz, Leidy Johana; Saldarriaga-Córdoba, Mónica; Cifuentes, Yeimy; Perafan, Carlos

    2017-04-01

    Theraphosidae spider venoms are well known for possess a complex mixture of protein and non-protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity.

  5. A Phylogenetic Analysis of Heterorhabditis (Nemata: Rhabditidae) Based on Internal Transcribed Spacer 1 DNA Sequence Data

    PubMed Central

    Adams, B. J.; Burnell, A. M.; Powers, T. O.

    1998-01-01

    Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of homologous characters for resolving relationships between closely related taxa but provided more tenuous resolution among more divergent lineages. A high degree of sequence identity and lack of autapomorphic characters suggest that sister species pairs within three distinct lineages may be mutually conspecific. Application of these molecular data and current morphological knowledge to the delimitation of species is hindered by an incomplete understanding of their variability in natural populations. PMID:19274196

  6. Energy efficiency trade-offs drive nucleotide usage in transcribed regions.

    PubMed

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J

    2016-04-21

    Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of 'A' versus 'T' and 'G' versus 'C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides 'U' and 'C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides 'A' and 'G' at non-synonymous coding sites.

  7. CpSAT-1, a transcribed satellite sequence from the codling moth, Cydia pomonella.

    PubMed

    Věchtová, Pavlína; Dalíková, Martina; Sýkorová, Miroslava; Žurovcová, Martina; Füssy, Zoltán; Zrzavá, Magda

    2016-08-01

    Satellite DNA (satDNA) is a non-coding component of eukaryotic genomes, located mainly in heterochromatic regions. Relevance of satDNA began to emerge with accumulating evidence of its potential yet hardly comprehensible role that it can play in the genome of many organisms. We isolated the first satDNA of the codling moth (Cydia pomonella, Tortricidae, Lepidoptera), a species with holokinetic chromosomes and a single large heterochromatic element, the W chromosome in females. The satDNA, called CpSAT-1, is located on all chromosomes of the complement, although in different amounts. Surprisingly, the satellite is almost missing in the heterochromatic W chromosome. Additionally, we isolated mRNA from all developmental stages (1st-5th instar larva, pupa, adult), both sexes (adult male and female) and several tissues (Malpighian tubules, gut, heart, testes, and ovaries) of the codling moth and showed the CpSAT-1 sequence was transcribed in all tested samples. Using CpSAT-1 specific primers we amplified, cloned and sequenced 40 monomers from cDNA and gDNA, respectively. The sequence analysis revealed a high mutation rate and the presence of potentially functional motifs, mainly in non-conserved regions of the monomers. Both the chromosomal distribution and the sequence analysis suggest that CPSAT-1 has no function in the C. pomonella genome.

  8. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

    PubMed

    Robideau, Gregg P; De Cock, Arthur W A M; Coffey, Michael D; Voglmayr, Hermann; Brouwer, Henk; Bala, Kanak; Chitty, David W; Désaulniers, Nicole; Eggertson, Quinn A; Gachon, Claire M M; Hu, Chia-Hui; Küpper, Frithjof C; Rintoul, Tara L; Sarhan, Ehab; Verstappen, Els C P; Zhang, Yonghong; Bonants, Peter J M; Ristaino, Jean B; Lévesque, C André

    2011-11-01

    Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

  9. High-resolution profiling of novel transcribed regions during rat spermatogenesis.

    PubMed

    Chalmel, Frédéric; Lardenois, Aurélie; Evrard, Bertrand; Rolland, Antoine D; Sallou, Olivier; Dumargne, Marie-Charlotte; Coiffec, Isabelle; Collin, Olivier; Primig, Michael; Jégou, Bernard

    2014-07-01

    Mammalian spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-encoding transcriptome, little effort has yet been devoted to the systematic identification of novel unannotated transcribed regions expressed during mammalian spermatogenesis. We report high-resolution expression profiling of male germ cells in rat, using next-generation sequencing technology and highly enriched testicular cell populations. Among 20 424 high-confidence transcripts reconstructed, we defined a stringent set of 1419 long multi-exonic unannotated transcripts expressed in the testis (testis-expressed unannotated transcripts [TUTs]). TUTs were divided into 7 groups with different expression patterns. Most TUTs share many of the characteristics of vertebrate long noncoding RNAs (lncRNAs). We also markedly reinforced the finding that TUTs and known lncRNAs accumulate during the meiotic and postmeiotic stages of spermatogenesis in mammals and that X-linked meiotic TUTs do not escape the silencing effects of meiotic sex chromosome inactivation. Importantly, we discovered that TUTs and known lncRNAs with a peak expression during meiosis define a distinct class of noncoding transcripts that exhibit exons twice as long as those of other transcripts. Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource for novel loci expressed during spermatogenesis that significantly contributes to rat genome annotation.

  10. Molecular phylogenetic analysis of Indonesia Solanaceae based on DNA sequences of internal transcribed spacer region

    NASA Astrophysics Data System (ADS)

    Hidayat, Topik; Priyandoko, Didik; Islami, Dina Karina; Wardiny, Putri Yunitha

    2016-02-01

    Solanaceae is one of largest family in Angiosperm group with highly diverse in morphological character. In Indonesia, this group of plant is very popular due to its usefulness as food, ornamental and medicinal plants. However, investigation on phylogenetic relationship among the member of this family in Indonesia remains less attention. The purpose of this study was to evaluate the phylogenetics relationship of the family especially distributed in Indonesia. DNA sequences of Internal Transcribed Spacer (ITS) region of 19 species of Solanaceae and three species of outgroup, which belongs to family Convolvulaceae, Apocynaceae, and Plantaginaceae, were isolated, amplified, and sequenced. Phylogenetic tree analysis based on parsimony method was conducted with using data derived from the ITS-1, 5.8S, and ITS-2, separately, and the combination of all. Results indicated that the phylogenetic tree derived from the combined data established better pattern of relationship than separate data. Thus, three major groups were revealed. Group 1 consists of tribe Datureae, Cestreae, and Petunieae, whereas group 2 is member of tribe Physaleae. Group 3 belongs to tribe Solaneae. The use of the ITS region as a molecular markers, in general, support the global Solanaceae relationship that has been previously reported.

  11. Identification of dendrobium species used for herbal medicines based on ribosomal DNA internal transcribed spacer sequence.

    PubMed

    Takamiya, Tomoko; Wongsawad, Pheravut; Tajima, Natsuko; Shioda, Nao; Lu, Jun Feng; Wen, Chi Luan; Wu, Jin Bin; Handa, Takashi; Iijima, Hiroshi; Kitanaka, Susumu; Yukawa, Tomohisa

    2011-01-01

    Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).

  12. Internal Transcribed Spacer 1 (ITS1) based sequence typing reveals phylogenetically distinct Ascaris population.

    PubMed

    Das, Koushik; Chowdhury, Punam; Ganguly, Sandipan

    2015-01-01

    Taxonomic differentiation among morphologically identical Ascaris species is a debatable scientific issue in the context of Ascariasis epidemiology. To explain the disease epidemiology and also the taxonomic position of different Ascaris species, genome information of infecting strains from endemic areas throughout the world is certainly crucial. Ascaris population from human has been genetically characterized based on the widely used genetic marker, internal transcribed spacer1 (ITS1). Along with previously reported and prevalent genotype G1, 8 new sequence variants of ITS1 have been identified. Genotype G1 was significantly present among female patients aged between 10 to 15 years. Intragenic linkage disequilibrium (LD) analysis at target locus within our study population has identified an incomplete LD value with potential recombination events. A separate cluster of Indian isolates with high bootstrap value indicate their distinct phylogenetic position in comparison to the global Ascaris population. Genetic shuffling through recombination could be a possible reason for high population diversity and frequent emergence of new sequence variants, identified in present and other previous studies. This study explores the genetic organization of Indian Ascaris population for the first time which certainly includes some fundamental information on the molecular epidemiology of Ascariasis.

  13. Definition of Eight Mulberry Species in the Genus Morus by Internal Transcribed Spacer-Based Phylogeny.

    PubMed

    Zeng, Qiwei; Chen, Hongyu; Zhang, Chao; Han, Minjing; Li, Tian; Qi, Xiwu; Xiang, Zhonghuai; He, Ningjia

    2015-01-01

    Mulberry, belonging to the order Rosales, family Moraceae, and genus Morus, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus Morus has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify Morus species, resulting in varied numbers of designated Morus spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus Morus. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. M. alba and M. notabilis showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the M. notabilis genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the Morus genus should be classified into eight species, including M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus Morus.

  14. Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas.

    PubMed

    Taylor, D Lee; McCormick, Melissa K

    2008-01-01

    Despite advances owing to molecular approaches, several hurdles still obstruct the identification of fungi forming orchid mycorrhizas. The Tulasnellaceae exhibit accelerated evolution of the nuclear ribosomal operon, causing most standard primers to fail in polymerase chain reaction (PCR) trials. Insufficient sequences are available from well characterized isolates and fruitbodies. Lastly, taxon-specific PCR primers are needed in order to explore the ecology of the fungi outside of the orchid root. Here, progress in overcoming these hurdles is reported. Broad-spectrum basidiomycete internal transcribed spacer (ITS) primers that do not exclude most known Tulasnellaceae are presented. blast searches and empirical PCR tests support their wide utility within the Basidiomycota. Taxon-specific ITS primers are presented targeted to orchid-associated Tulasnella, and a core component of the Thelephora-Tomentella complex. The efficiency and selectivity of these primer sets are again supported by blast searches and empirical tests. Lastly, ITS DNA sequences are presented from several strains of Epulorhiza, Ceratorhiza, Ceratobasidium, Sistotrema, Thanatephorus and Tulasnella that were originally described in the landmark mycorrhizal studies of Currah and Warcup. Detailed phylogenetic analyses reveal some inconsistencies in species concepts in these taxonomically challenging resupinate basidiomycetes, but also help to place several sequences from environmental samples.

  15. Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides

    PubMed Central

    Sharma, Sonal; Shrivastava, Neeta

    2016-01-01

    Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides (L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer (ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy. PMID:27175337

  16. Endangered Uyghur Medicinal Plant Ferula Identification through the Second Internal Transcribed Spacer

    PubMed Central

    Fan, Congzhao; Li, Xiaojin; Zhu, Jun; Song, Jingyuan; Yao, Hui

    2015-01-01

    The medicinal plant Ferula has been widely used in Asian medicine, especially in Uyghur medicine in Xinjiang, China. Given that various substitutes and closely related species have similar morphological characteristics, Ferula is difficult to distinguish based on morphology alone, thereby causing confusion and threatening the safe use of Ferula. In this study, internal transcribed spacer 2 (ITS2) sequences were analyzed and assessed for the accurate identification of two salable Ferula species (Ferula sinkiangensis and Ferula fukangensis) and eight substitutes or closely related species. Results showed that the sequence length of ITS2 ranged from 451 bp to 45 bp, whereas guanine and cytosine contents (GC) were from 53.6% to 56.2%. A total of 77 variation sites were detected, including 63 base mutations and 14 insertion/deletion mutations. The ITS2 sequence correctly identified 100% of the samples at the species level using the basic local alignment search tool 1 and nearest-distance method. Furthermore, neighbor-joining tree successfully identified the genuine plants F. sinkiangensis and F. fukangensis from their succedaneum and closely related species. These results indicated that ITS2 sequence could be used as a valuable barcode to distinguish Uyghur medicine Ferula from counterfeits and closely related species. This study may broaden DNA barcoding application in the Uyghur medicinal plant field. PMID:26120347

  17. Internal Transcribed Spacer Sequencing versus Biochemical Profiling for Identification of Medically Important Yeasts

    PubMed Central

    Ciardo, D. E.; Schär, G.; Böttger, E. C.; Altwegg, M.; Bosshard, P. P.

    2006-01-01

    In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region. PMID:16390952

  18. Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran.

    PubMed

    Ghatee, Mohammad Amin; Sharifi, Iraj; Kuhls, Katrin; Kanannejad, Zahra; Harandi, Majid Fasihi; de Almeida, Marcos E; Hatam, Gholamreza; Mirhendi, Hossein

    2014-09-01

    Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.

  19. Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR

    PubMed Central

    Couto, Isabel; Pereira, Sandro; Miragaia, Maria; Sanches, Ilda Santos; de Lencastre, Hermínia

    2001-01-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems. PMID:11526135

  20. Internal transcribed spacer 2 barcode: a good tool for identifying Acanthopanacis cortex

    PubMed Central

    Zhao, Sha; Chen, Xiaochen; Song, Jingyuan; Pang, Xiaohui; Chen, Shilin

    2015-01-01

    Acanthopanacis cortex has been used in clinical applications for a long time. Considering some historical and geographical factors, Acanthopanacis cortex is easily confused with other herbs in medicine markets, thereby causing potential safety issues. In this study, we used the internal transcribed spacer 2 (ITS2) barcode to identify 69 samples belonging to six species, including Acanthopanacis cortex and its adulterants. The nearest distance, single-nucleotide polymorphisms (SNPs), and neighbor-joining (NJ) tree methods were used to evaluate the identification ability of the ITS2 barcode. According to the kimura-2-parameter model, the intraspecific distance of Eleutherococcus nodiflorus ITS2 sequences ranged from 0 to 0.0132. The minimum interspecific distance between E. nodiflorus and E. giraldii was 0.0221, which was larger than the maximum intraspecific distance of E. nodiflorus. Three stable SNPs in ITS2 can be used to distinguish Acanthopanacis cortex and its closely related species. The NJ tree indicated that the Acanthopanacis cortex samples clustered into one clade, which can be distinguished clearly from the adulterants of this herb. A secondary structure of ITS2 provided another dimensionality to identify species. In conclusion, the ITS2 barcode effectively identifies Acanthopanacis cortex, and DNA barcoding is a convenient tool for medicine market supervision. PMID:26500674

  1. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    PubMed

    Marathe, Himangi G; Mehta, Gaurav; Zhang, Xiaolu; Datar, Ila; Mehrotra, Aanchal; Yeung, Kam C; de la Serna, Ivana L

    2013-01-01

    SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to

  2. Customized Regulation of Diverse Stress Response Genes by the Multiple Antibiotic Resistance Activator MarA

    PubMed Central

    2017-01-01

    Stress response networks frequently have a single upstream regulator that controls many downstream genes. However, the downstream targets are often diverse, therefore it remains unclear how their expression is specialized when under the command of a common regulator. To address this, we focused on a stress response network where the multiple antibiotic resistance activator MarA from Escherichia coli regulates diverse targets ranging from small RNAs to efflux pumps. Using single-cell experiments and computational modeling, we showed that each downstream gene studied has distinct activation, noise, and information transmission properties. Critically, our results demonstrate that understanding biological context is essential; we found examples where strong activation only occurs outside physiologically relevant ranges of MarA and others where noise is high at wild type MarA levels and decreases as MarA reaches its physiological limit. These results demonstrate how a single regulatory protein can maintain specificity while orchestrating the response of many downstream genes. PMID:28060821

  3. Customized Regulation of Diverse Stress Response Genes by the Multiple Antibiotic Resistance Activator MarA.

    PubMed

    Rossi, Nicholas A; Dunlop, Mary J

    2017-01-01

    Stress response networks frequently have a single upstream regulator that controls many downstream genes. However, the downstream targets are often diverse, therefore it remains unclear how their expression is specialized when under the command of a common regulator. To address this, we focused on a stress response network where the multiple antibiotic resistance activator MarA from Escherichia coli regulates diverse targets ranging from small RNAs to efflux pumps. Using single-cell experiments and computational modeling, we showed that each downstream gene studied has distinct activation, noise, and information transmission properties. Critically, our results demonstrate that understanding biological context is essential; we found examples where strong activation only occurs outside physiologically relevant ranges of MarA and others where noise is high at wild type MarA levels and decreases as MarA reaches its physiological limit. These results demonstrate how a single regulatory protein can maintain specificity while orchestrating the response of many downstream genes.

  4. Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

    PubMed

    Ramirez, Kevin; Chandler, Katherine J; Spaulding, Christina; Zandi, Sasan; Sigvardsson, Mikael; Graves, Barbara J; Kee, Barbara L

    2012-06-29

    Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

  5. Gene activation properties of a mouse DNA sequence isolated by expression selection.

    PubMed Central

    von Hoyningen-Huene, V; Norbury, C; Griffiths, M; Fried, M

    1986-01-01

    The MES-1 element was previously isolated from restricted total mouse cellular DNA by "expression selection"--the ability to reactivate expression of a test gene devoid of its 5' enhancer sequences. Mes-1 has been tested in long-term transformation and short-term CAT expression assays. In both assays MES-1 is active independent of orientation and at a distance when placed 5' to the test gene. The element is active with heterologous promoters and functions efficiently in both rat and mouse cells. MES-1 activates expression by increasing transcription from the test gene's own start (cap) site. Thus the expression selection technique can be used for the isolation of DNA sequences with enhancer-like properties from total cellular DNA. Images PMID:3016657

  6. Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity.

    PubMed

    Venturi, V; Wolfs, K; Leong, J; Weisbeek, P J

    1994-10-17

    Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting conditions. The genes encoding pseudobactin 358 biosynthesis are iron-regulated at the level of transcription. In this study, the molecular characterization is reported of a cosmid clone of WCS358 DNA that can stimulate, in an iron-dependent manner, the activity of a WCS358 siderophore gene promoter in the heterologous Pseudomonas strain A225. The functional region in the clone was identified by subcloning, transposon mutagenesis and DNA sequencing as the groESL operon of strain WCS358. This increase in promoter activity was not observed when the groESL genes of strain WCS358 were integrated via a transposon vector into the genome of Pseudomonas A225, indicating that multiple copies of the operon are necessary for the increase in siderophore gene promoter activity. Amplification of the Escherichia coli and WCS358 groESL genes also increased iron-regulated promoter activity in the parent strain WCS358. The groESL operon codes for the chaperone proteins GroES and GroEL, which are responsible for mediating the folding and assembly of many proteins.

  7. Macro optical projection tomography for large scale 3D imaging of plant structures and gene activity.

    PubMed

    Lee, Karen J I; Calder, Grant M; Hindle, Christopher R; Newman, Jacob L; Robinson, Simon N; Avondo, Jerome J H Y; Coen, Enrico S

    2016-12-26

    Optical projection tomography (OPT) is a well-established method for visualising gene activity in plants and animals. However, a limitation of conventional OPT is that the specimen upper size limit precludes its application to larger structures. To address this problem we constructed a macro version called Macro OPT (M-OPT). We apply M-OPT to 3D live imaging of gene activity in growing whole plants and to visualise structural morphology in large optically cleared plant and insect specimens up to 60 mm tall and 45 mm deep. We also show how M-OPT can be used to image gene expression domains in 3D within fixed tissue and to visualise gene activity in 3D in clones of growing young whole Arabidopsis plants. A further application of M-OPT is to visualise plant-insect interactions. Thus M-OPT provides an effective 3D imaging platform that allows the study of gene activity, internal plant structures and plant-insect interactions at a macroscopic scale.

  8. Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis.

    PubMed

    Carnevali, O; Cionna, C; Tosti, L; Cerdà, J; Gioacchini, G

    2008-01-01

    The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.

  9. CG gene body DNA methylation changes and evolution of duplicated genes in cassava.

    PubMed

    Wang, Haifeng; Beyene, Getu; Zhai, Jixian; Feng, Suhua; Fahlgren, Noah; Taylor, Nigel J; Bart, Rebecca; Carrington, James C; Jacobsen, Steven E; Ausin, Israel

    2015-11-03

    DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

  10. CG gene body DNA methylation changes and evolution of duplicated genes in cassava

    PubMed Central

    Wang, Haifeng; Beyene, Getu; Zhai, Jixian; Feng, Suhua; Fahlgren, Noah; Taylor, Nigel J.; Bart, Rebecca; Carrington, James C.; Jacobsen, Steven E.; Ausin, Israel

    2015-01-01

    DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits. PMID:26483493

  11. Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

    PubMed Central

    McGregor, W G; Chen, R H; Lukash, L; Maher, V M; McCormick, J J

    1991-01-01

    To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene. PMID:2005888

  12. Characterization of human TCR Vbeta gene promoter. Role of the dodecamer motif in promoter activity.

    PubMed

    Deng, X; Sun, G R; Zheng, Q; Li, Y

    1998-09-11

    During T-lymphocyte development, the T-cell antigen receptor (TCR) gene expression is controlled by its promoter and enhancer elements and regulated in tissue- and development stage-specific manner. To uncover the promoter function and to define positive and negative regulatory elements in TCR gene promoters, the promoter activities from 13 human TCR Vbeta genes were determined by the transient transfection system and luciferase reporter assay. Although most of the TCR Vbeta gene promoters that we tested are inactive by themselves, some promoters were found to be constitutively strong. Among them, Vbeta6.7 is the strongest. 5'-Deletion and fragmentation experiments have narrowed the full promoter activity of Vbeta6.7 to a fragment of 147 base pairs immediately 5' to the transcription initiation site. A decanucleotide motif with the consensus sequence AGTGAYRTCA has been found to be conserved in most TCR Vbeta gene promoters. There are three such decamer motifs in the promoter region of Vbeta6.7, and the contribution of each such motif to the promoter activity has been examined. Further site-directed mutagenesis analyses showed that: 1) when two Ts in the decamer were mutated, the promoter activity was totally abolished; 2) when two additional nucleotides 3' to the end of decamer were mutated, the promoter activity was decreased to two-thirds of the full level; and 3) when the element with the sequence AGTGATGTCACT was inserted into other promoters, the original weak promoters become very strong. Taken together, our data suggest that the positive regulatory element in Vbeta6.7 should be considered a dodecamer rather than a decamer and that it confers strong basal transcriptional activity on TCR Vbeta genes.

  13. Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

    PubMed Central

    Lock, L F; Melton, D W; Caskey, C T; Martin, G R

    1986-01-01

    It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt

  14. Unified English Braille in the United Kingdom: Part 2--Examination by Literary Braille Users, Braille Teachers, and Transcribers

    ERIC Educational Resources Information Center

    Cryer, Heather; Home, Sarah; Morley Wilkins, Sarah

    2013-01-01

    To inform decision-making around the adoption of the Unified English Braille (UEB) code in the United Kingdom, a suite of research was carried out. This study involved a variety of braille stakeholders--student braille readers (in full time education), adult braille readers, braille teachers, and braille transcribers. Participants were sent…

  15. Regulation of spblimp1/krox1a, an alternatively transcribed isoform expressed in midgut and hindgut of the sea urchin gastrula.

    PubMed

    Livi, Carolina B; Davidson, Eric H

    2007-01-01

    The sea urchin regulatory gene Spblimp1/krox produces alternatively transcribed and spliced isoforms, 1a and 1b, which have different temporal and spatial patterns of expression. Here we describe a cis-regulatory module that controls the expression of the 1a splice form in the midgut and hindgut at the beginning of gastrulation. Conserved sequence patches revealed by a comparison of the blimp1/krox locus in Strongylocentrotus purpuratus and Lytechinus variegatus genomes were tested by gene transfer, in association with GFP or CAT reporter genes. An expression construct containing a conserved sequence patch immediately 5' of exon 1a included the transcription initiation site for blimp1/krox1a. This construct displays specific mid and hindgut expression, indicating that these are the locations of endogenous blimp1/krox1a transcription during the gastrula stage. Its sequence contains binding sites for Brn1/2/4, Otx, and Blimp1/Krox itself, as predicted in a prior regulatory network analysis.

  16. Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    PubMed Central

    Nitulescu, Ioana I.; Tangpeerachaikul, Anupong; Poss, Zachary C.; Da Silva, Diogo H.; Caruso, Brittany T.; Arefolov, Alexander; Fadeyi, Olugbeminiyi; Christie, Amanda L.; Du, Karrie; Banka, Deepti; Schneider, Elisabeth V.; Jestel, Anja; Zou, Ge; Si, Chong; Ebmeier, Christopher C.; Bronson, Roderick T.; Krivtsov, Andrei V.; Myers, Andrew G.; Kohl, Nancy E.; Kung, Andrew L.; Armstrong, Scott A.; Lemieux, Madeleine E.; Taatjes, Dylan J.; Shair, Matthew D.

    2015-01-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML. PMID:26416749

  17. Identification and characterization of genes related to cellulolytic activity in basidiomycetes.

    PubMed

    Volpini, A F N; Thomazine, T; Umeo, S H; Pereira, G A; Linde, G A; Valle, J S; Colauto, N B; Barcellos, F G; Souza, S G H

    2016-09-16

    Enzymes produced by basidiomycetes that are involved in the cellulose degradation process, and their respective codifying genes, must be identified to facilitate the development of novel biotechnological strategies and applications in the agro-industry. The objective of this study was to identify prospective cellulase-producing genes and characterize their cellulolytic activity, in order to elucidate the potential biotechnological applications (with respect to vegetal residues) of basidiomycetes. The basidiomycete strains Lentinula edodes U8-1, Lentinus crinitus U9-1, and Schizophyllum commune U6-7 were analyzed in this study. The cellulolytic activities of these fungi were evaluated based on the halo formation in carboxymethyl cellulose culture medium after dyeing with Congo red. The presence of cellulase-codifying genes (cel7A, cel6B, cel3A, and egl) in these fungal strains was also evaluated. L. edodes and S. commune presented the highest cellulolytic halo to mycelial growth radius ratio, followed by L. crinitus. Four genes were amplified in the L. edodes strain, whereas three and one genes were isolated from L. crinitus and S. commune, respectively. The cel6B gene (L. edodes) presented the conserved domain glyco_hydro_6 and characterized as cellobiohydrolase gene. The results of this study contribute to the existing knowledge on cellulases in basidiomycetes, and serve as a basis for future studies on the expression of these genes and the characterization of the catalytic activity of these enzymes. This allows for better utilization of these fungi in degrading vegetal fibers from agro-industrial residues and in other biotechnological applications.

  18. VSG gene expression site control in insect form Trypanosoma brucei.

    PubMed

    Rudenko, G; Blundell, P A; Taylor, M C; Kieft, R; Borst, P

    1994-11-15

    When the African trypanosome Trypanosoma brucei is taken up from mammals by a tse-tse fly, it replaces its variant surface glycoprotein (VSG) coat by a procyclin coat. Transcription of VSG genes stops in the fly, but transcription of sequences derived from the promoter area of the VSG expression site(s) remains high. Whether this is due to continuing high activity of one promoter or to low activity of many promoters was unclear. We have used the small differences between the sequences of different expression sites to show that multiple expression site promoters are active in insect form trypanosomes. This is confirmed by the low expression of single copy marker genes introduced into the transcribed area. However, if the expression site promoter is removed from the genomic location of the expression site and inserted in the non-transcribed spacer of the ribosomal DNA (rDNA), it is derepressed. Derepression of transcription can also be accomplished by replacing the promoter of an expression site by an rDNA promoter. We conclude that the down-regulation of VSG gene expression site promoters in insect form trypanosomes is affected by both the DNA sequence of the promoter and the genomic context in which it resides.

  19. Heterogeneous activation of a slow myosin gene in proliferating myoblasts and differentiated single myofibers

    PubMed Central

    Wang, Jing-hua; Wang, Qiao-jing; Wang, Chao; Reinholt, Brad; Grant, Alan L; Gerrard, David E; Kuang, Shihuan

    2015-01-01

    Each skeletal muscle contains a fixed ratio of fast and slow myofibers that are distributed in a stereotyped pattern to achieve a specific motor function. How myofibers are specified during development and regeneration is poorly understood. Here we address this question using transgenic reporter mice that indelibly mark the myofiber lineages based on activation of fast or slow myosin. Lineage tracing indicates that during development all muscles have activated the fast myosin gene Myl1, but not the slow myosin gene Myh7, which is activated in all slow but a subset of fast myofibers. Similarly, most nascent myofibers do not activate Myh7 during fast muscle regeneration, but the ratio and pattern of fast and slow myofibers are restored at the completion of regeneration. At the single myofiber level, most mature fast myofibers are heterogeneous in nuclear composition, manifested by mosaic activation of Myh7. Strikingly, Myh7 is activated in a subpopulation of proliferating myoblasts that co-express the myogenic progenitor marker Pax7. When induced to differentiate, the Myh7-activated myoblasts differentiate more readily than the non-activated myoblasts, and have a higher tendency, but not restricted, to become slow myotubes. Together, our data reveal significant nuclear heterogeneity within a single myofiber, and challenge the conventional view that myosin genes are only expressed after myogenic differentiation. These results provide novel insights into the regulation of muscle fiber type specification. PMID:25794679

  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. Repression of telomere-associated genes by microglia activation in neuropsychiatric disease.

    PubMed

    Kronenberg, Golo; Uhlemann, Ria; Schöner, Johanna; Wegner, Stephanie; Boujon, Valérie; Deigendesch, Nikolas; Endres, Matthias; Gertz, Karen

    2016-11-28

    Microglia senescence may promote neuropsychiatric disease. This prompted us to examine the relationship between microglia activation states and telomere biology. A panel of candidate genes associated with telomere maintenance, mitochondrial biogenesis, and cell-cycle regulation were investigated in M1- and M2-polarized microglia in vitro as well as in MACS-purified CD11b+ microglia/brain macrophages from models of stroke, Alzheimer's disease, and chronic stress. M1 polarization, ischemia, and Alzheimer pathology elicited a strikingly similar transcriptomic profile with, in particular, reduced expression of murine Tert. Our results link classical microglia activation with repression of telomere-associated genes, suggesting a new mechanism underlying microglia dysfunction.

  2. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    SciTech Connect

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  3. A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

    PubMed Central

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

  4. Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

    PubMed

    Ochi, Kozo; Tanaka, Yukinori; Tojo, Shigeo

    2014-02-01

    Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

  5. Genetic Characterization of the Homeodomain-Independent Activity of the Drosophila Fushi Tarazu Gene Product

    PubMed Central

    Hyduk, D.; Percival-Smith, A.

    1996-01-01

    The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results. PMID:8852847

  6. Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

    PubMed

    Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

    2005-03-01

    We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

  7. Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells

    SciTech Connect

    McGregor, W.G.; Chen, Ruey-Hwa; Lukash, L.; Maher, V.M.; McCormick, J.J. )

    1991-04-01

    To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), the authors synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G, phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, they also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G[sub 1] and the repair-proficient mutants from S, [approximately]62% were G [center dot] C[r arrow]A [center dot] T. In the repair-proficient mutants from G[sub 1], 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G[sub 1], only 20% (3 of 15) were. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.

  8. Preferential repair of DNA double-strand break at the active gene in vivo.

    PubMed

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

  9. Timing and Variability of Galactose Metabolic Gene Activation Depend on the Rate of Environmental Change.

    PubMed

    Nguyen-Huu, Truong D; Gupta, Chinmaya; Ma, Bo; Ott, William; Josić, Krešimir; Bennett, Matthew R

    2015-07-01

    Modulation of gene network activity allows cells to respond to changes in environmental conditions. For example, the galactose utilization network in Saccharomyces cerevisiae is activated by the presence of galactose but repressed by glucose. If both sugars are present, the yeast will first metabolize glucose, depleting it from the extracellular environment. Upon depletion of glucose, the genes encoding galactose metabolic proteins will activate. Here, we show that the rate at which glucose levels are depleted determines the timing and variability of galactose gene activation. Paradoxically, we find that Gal1p, an enzyme needed for galactose metabolism, accumulates more quickly if glucose is depleted slowly rather than taken away quickly. Furthermore, the variability of induction times in individual cells depends non-monotonically on the rate of glucose depletion and exhibits a minimum at intermediate depletion rates. Our mathematical modeling suggests that the dynamics of the metabolic transition from glucose to galactose are responsible for the variability in galactose gene activation. These findings demonstrate that environmental dynamics can determine the phenotypic outcome at both the single-cell and population levels.

  10. Computer-aided design of modular protein devices: Boolean AND gene activation

    NASA Astrophysics Data System (ADS)

    Salis, H.; Kaznessis, Y. N.

    2006-12-01

    Many potentially useful synthetic gene networks require the expression of an engineered gene if and only if two different DNA-binding proteins exist in sufficient concentration. While some natural and engineered systems activate gene expression according to a logical AND-like behavior, they often utilize allosteric or cooperative protein-protein interactions, rendering their components unsuitable for a toolbox of modular parts for use in multiple applications. Here, we develop a quantitative model to demonstrate that a small system of interacting fusion proteins, called a protein device, can activate an engineered gene according to the Boolean AND behavior while using only modular protein domains and DNA sites. The fusion proteins are created from transactivating, DNA-binding, non-DNA binding, and protein-protein interaction domains along with the corresponding peptide ligands. Using a combined kinetic and thermodynamic model, we identify the characteristics of the molecular components and their rates of constitutive production that maximize the fidelity of AND behavior. These AND protein devices facilitate the creation of complex genetic programs and may be used to create gene therapies, biosensors and other biomedical and biotechnological applications that turn on gene expression only when multiple DNA-binding proteins are simultaneously present.

  11. Structural requirements for the functional activity of a U1 snRNA gene enhancer.

    PubMed Central

    Cheung, C H; Fan, Q N; Stumph, W E

    1993-01-01

    The transcriptional enhancer of a chicken U1 small nuclear RNA (snRNA) gene contains a GC-box, an octamer motif, and an SPH motif that are recognized by the transcription factors Sp1, Oct-1, and SBF respectively. Previous work indicated that the octamer and the SPH motifs were both required for U1 gene enhancer activity in frog oocytes when the U1 gene was coinjected with a competing snRNA gene template. Here we show that neither two copies of the octamer motif, nor two copies of the SPH motif, can effectively substitute for the natural combination of octamer and SPH. Furthermore, neither the octamer nor the SPH motif (in the absence of the other) functioned efficiently in combination with a GC-box. Alteration of the spacing between the octamer and SPH motifs also reduced U1 template activity. Several potential cis-acting elements other than the SPH motif, with one possible exception among those tested, were unable to cooperate with the octamer motif to effectively enhance U1 gene expression. These results indicate that rather stringent structural requirements exist with respect to the essential cis-acting motifs present in the U1 enhancer, possibly reflecting the unique properties of the transcription complexes assembled on snRNA gene promoters. Images PMID:8441636

  12. The phn Genes of Burkholderia sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism

    PubMed Central

    Laurie, Andrew D.; Lloyd-Jones, Gareth

    1999-01-01

    Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. This phn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenanthrene-degrading Burkholderia sp. strain RP007. The phn genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in Escherichia coli the recombinant phn enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein α and β subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the phn catabolic genes are two putative regulatory genes, phnR and phnS. Sequence homology suggests that phnS is a LysR-type transcriptional activator and that phnR, which is divergently transcribed with respect to phnSFECDAcAdB, is a member of the ς54-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve

  13. Structure and ANR-dependent transcription of the nir genes for denitrification from Pseudomonas aeruginosa.

    PubMed

    Arai, H; Igarashi, Y; Kodama, T

    1994-07-01

    In the denitrification gene cluster from Pseudomonas aeruginosa, an operon encoding three open reading frames (nirQ, ORF2, ORF3) was upstream of the structural gene for nitrite reductase (nirS) as a divergent transcriptional organization. A nucleotide-binding protein encoded by nirQ was 76% identical to the Pseudomonas stutzeri nirQ gene product, which was shown to be necessary for activating nitrite and nitric oxide reductases. The gene product of ORF2 was homologous to subunit III of cytochrome oxidases. The nirQ gene was transcribed under denitrifying conditions. The intergenic region of nirS and nirQ has only one binding motif for ANR, a regulatory protein for anaerobic gene expression correspond to FNR in E. coli. Complementation analyses showed that the transcription of both nirS and nirQ completely depended on ANR.

  14. Diversity of laccase-like multicopper oxidase genes in Morchellaceae: identification of genes potentially involved in extracellular activities related to plant litter decay.

    PubMed

    Kellner, Harald; Luis, Patricia; Buscot, François

    2007-07-01

    Despite the important role played by soil-inhabiting ascomycetes in plant litter decay processes, studies on the diversity and function of their laccase-like multicopper oxidase (LMCO) genes are scarce. In the present work, the LMCO gene diversity in 15 strains representing nine Morchellaceae and one Discinaceae species was evaluated by PCR. One to six different genes were found within the species, representing 26 different sequence types. Cluster analysis revealed LMCO genes belonging to four main gene families encoding different protein classes (Class I-IV). To identify the genes related to extracellular activities and potentially involved in litter decay processes, liquid cultures were induced by different aromatic compounds. Morchella conica and Verpa conica showed the strongest LMCO activity enhancement in the presence of the naturally occurring phenolic compound guaiacol, and their expressed LMCO genes were identified by sequencing. Only genes belonging to the gene families encoding the Class II and III proteins were expressed. Both genes (Class II and III) of the mycorrhizal-like strain M. conica were exclusively expressed in the presence of guaiacol. In contrast to the saprotrophic strain V. conica, the gene encoding the Class III protein was constitutively expressed as it was also found in control cultures without guaiacol.

  15. Tissue plasminogen activator and plasminogen activator inhibitor type 1 gene polymorphism in patients with gastric ulcer complicated with bleeding.

    PubMed Central

    Kim, Hong-Soo; Hwang, Kyu-Yoon; Chung, Il-Kwon; Park, Sang-Heum; Lee, Moon-Ho; Kim, Sun-Joo; Hong, Sae-Yong

    2003-01-01

    Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) may be involved in the pathogenesis of peptic ulcers through suppression of fibrinolysis. This study was designed to investigate associations of t-PA and PAI-1 genes with clinical features of the patients with bleeding gastric ulcers. Eighty-four patients with peptic ulcers and 100 controls were studied between January 1998 and April 2000. We used polymerase chain reaction and endonuclease digestion to genotype for 4G/5G polymorphism in the promoter region of the PAI-1 gene and the Alurepeat insertion/deletion (I/D) polymorphism in intron h of the t-PA gene. Various clinical features, including lesion site, bleeding event, recurrence of ulcer, and rebleeding, were assessed using a multiple logistic regression model. The genotype distributions of both the t-PA and PAI-1 genes did not differ between the patient and control groups. The occurrence of the I/D or D/D genotype of t-PA was significantly higher in cases of duodenal ulcer (adjusted OR=4.39, 95% CI=1.12-17.21). When a dominant effect (i.e., 4G/4G or 4G/5G versus 5G/5G) of the 4G allele was assumed, the PAI-1 4G/4G genotype was independently associated with rebleeding after hemostasis (adjusted OR=5.07, 95% CI=1.03-24.87). Our data suggest that t-PA gene polymorphism is associated with duodenal ulcers, and that the PAI-1 gene may be a risk factor leading to recurrent bleeding after initial hemostasis. PMID:12589088

  16. Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes.

    PubMed

    Renier, Nicolas; Adams, Eliza L; Kirst, Christoph; Wu, Zhuhao; Azevedo, Ricardo; Kohl, Johannes; Autry, Anita E; Kadiri, Lolahon; Umadevi Venkataraju, Kannan; Zhou, Yu; Wang, Victoria X; Tang, Cheuk Y; Olsen, Olav; Dulac, Catherine; Osten, Pavel; Tessier-Lavigne, Marc

    2016-06-16

    Understanding how neural information is processed in physiological and pathological states would benefit from precise detection, localization, and quantification of the activity of all neurons across the entire brain, which has not, to date, been achieved in the mammalian brain. We introduce a pipeline for high-speed acquisition of brain activity at cellular resolution through profiling immediate early gene expression using immunostaining and light-sheet fluorescence imaging, followed by automated mapping and analysis of activity by an open-source software program we term ClearMap. We validate the pipeline first by analysis of brain regions activated in response to haloperidol. Next, we report new cortical regions downstream of whisker-evoked sensory processing during active exploration. Last, we combine activity mapping with axon tracing to uncover new brain regions differentially activated during parenting behavior. This pipeline is widely applicable to different experimental paradigms, including animal species for which transgenic activity reporters are not readily available.

  17. Controlling Hox gene expression and activity to build the vertebrate axial skeleton.

    PubMed

    Casaca, Ana; Santos, Ana Cristina; Mallo, Moisés

    2014-01-01

    It has long been known that Hox genes are central players in patterning the vertebrate axial skeleton. Extensive genetic studies in the mouse have revealed that the combinatorial activity of Hox genes along the anterior-posterior body axis specifies different vertebral identities. In addition, Hox genes were instrumental for the evolutionary diversification of the vertebrate body plan. In this review, we focus on fundamental questions regarding the intricate mechanisms controlling Hox gene activity. In particular, we discuss the functional relevance of the precise timing of Hox gene activation in the embryo. Moreover, we provide insight into the epigenetic regulatory mechanisms that are likely to control this process and are responsible for the maintenance of spatially restricted Hox expression domains throughout embryonic development. We also analyze how specific features of each Hox protein may contribute to the functional diversity of Hox family. Altogether, the work reviewed here further supports the notion that the Hox program is far more complex than initially assumed. Exciting new findings will surely emerge in the years ahead.

  18. Stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer.

    PubMed

    Yin, Perry T; Shah, Shreyas; Pasquale, Nicholas J; Garbuzenko, Olga B; Minko, Tamara; Lee, Ki-Bum

    2016-03-01

    Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo.

  19. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

    DOE PAGES

    Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; ...

    2016-06-08

    In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less

  20. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    PubMed

    Book, Adam J; Lewin, Gina R; McDonald, Bradon R; Takasuka, Taichi E; Wendt-Pienkowski, Evelyn; Doering, Drew T; Suh, Steven; Raffa, Kenneth F; Fox, Brian G; Currie, Cameron R

    2016-06-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  1. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

    PubMed Central

    McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-01-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

  2. Keeping the blood flowing-plasminogen activator genes and feeding behavior in vampire bats.

    PubMed

    Tellgren-Roth, Asa; Dittmar, Katharina; Massey, Steven E; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A; Liberles, David A

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  3. Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

    NASA Astrophysics Data System (ADS)

    Tellgren-Roth, Åsa; Dittmar, Katharina; Massey, Steven E.; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A.; Liberles, David A.

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  4. Gene targeting in rats using transcription activator-like effector nucleases.

    PubMed

    Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Thépenier, Virginie; Thinard, Reynald; Ouisse, Laure-Hélène; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-15

    The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

  5. Structure-activity investigation on the gene transfection properties of cardiolipin mimicking gemini lipid analogues.

    PubMed

    Bajaj, Avinash; Paul, Bishwajit; Kondaiah, Paturu; Bhattacharya, Santanu

    2008-06-01

    A structure-activity relationship has been explored on the gene transfection efficiencies of cardiolipin mimicking gemini lipid analogues upon variation of length and hydrophilicity of the spacer between the cationic ammonium headgroups and lipid hydrocarbon chain lengths. All the gemini lipids were found to be highly superior in gene transfer abilities as compared to their monomeric lipid and a related commercially available formulation. Pseudoglyceryl gemini lipids bearing an oxyethylene (-CH2-(CH2-O-CH2)m-CH2-) spacer were found to be superior gene transfecting agents as compared to those bearing polymethylene (-CH2)m-) spacers. The major characteristic feature of the present set of gemini lipids is their serum compatibility, which is most often the major hurdle in liposome-mediated gene delivery.

  6. Molecular analysis of fungal populations in patients with oral candidiasis using internal transcribed spacer region.

    PubMed

    Ieda, Shinsuke; Moriyama, Masafumi; Takeshita, Toru; Takashita, Toru; Maehara, Takashi; Imabayashi, Yumi; Shinozaki, Shoichi; Tanaka, Akihiko; Hayashida, Jun-Nosuke; Furukawa, Sachiko; Ohta, Miho; Yamashita, Yoshihisa; Nakamura, Seiji

    2014-01-01

    Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.

  7. Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region

    PubMed Central

    Ieda, Shinsuke; Moriyama, Masafumi; Takashita, Toru; Maehara, Takashi; Imabayashi, Yumi; Shinozaki, Shoichi; Tanaka, Akihiko; Hayashida, Jun-Nosuke; Furukawa, Sachiko; Ohta, Miho; Yamashita, Yoshihisa; Nakamura, Seiji

    2014-01-01

    Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection. PMID:24979710

  8. Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes

    PubMed Central

    Šmajs, David; Weinstock, George M.

    2001-01-01

    The 5.2-kb ColJs plasmid of a colicinogenic strain of Shigella sonnei (colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the rep, mob, and cer regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (cja), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (cji) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (cjl) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the cjl gene was found upstream from cja. Moreover, the promoter upstream from cjl was similar to promoters described upstream from several colicin activity genes. The cji gene was found to be located downstream from cja with a transcription polarity opposite to that of the cjl and cja genes. The cja, cji, and cjl genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus. PMID:11395458

  9. Gene expression profiling of pituitary melanotrope cells during their physiological activation.

    PubMed

    Kuribara, Miyuki; van Bakel, Nick H M; Ramekers, Dyan; de Gouw, Daan; Neijts, Roel; Roubos, Eric W; Scheenen, Wim J J M; Martens, Gerard J M; Jenks, Bruce G

    2012-01-01

    The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

  10. Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

    PubMed

    Kremmydas, Gerasimos F; Tampakaki, Anastasia P; Georgakopoulos, Dimitrios G

    2013-01-01

    Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocont