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Sample records for essential gene products

  1. Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin

    PubMed Central

    Dairi, Tohru; Hamano, Yoshimitsu; Kuzuyama, Tomohisa; Itoh, Nobuya; Furihata, Kazuo; Seto, Haruo

    2001-01-01

    A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:16271635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC. PMID:11567009

  2. The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production

    PubMed Central

    Lysøe, Erik; Klemsdal, Sonja S.; Bone, Karen R.; Frandsen, Rasmus J. N.; Johansen, Thomas; Thrane, Ulf; Giese, Henriette

    2006-01-01

    Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others. PMID:16751498

  3. The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity.

    PubMed Central

    Wuenscher, M D; Köhler, S; Bubert, A; Gerike, U; Goebel, W

    1993-01-01

    Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtilis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikticus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division. Images PMID:8099071

  4. A highly conserved baculovirus gene p48 (ac103) is essential for BV production and ODV envelopment

    SciTech Connect

    Yuan Meijin; Wu Wenbi; Liu Chao; Wang Yanjie; Hu Zhaoyang; Yang Kai Pang Yi

    2008-09-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc{sup P48-KO-PH-GFP} was unable to propagate in cell culture, while a 'repair' Bacmid vAc{sup P48-REP-PH-GFP} was able to replicate in a manner similar to a wild-type Bacmid vAc{sup PH-GFP}. Titration assays and Western blotting confirmed that vAc{sup P48-KO-PH-GFP} was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

  5. Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture.

    PubMed Central

    Schmitt, J; Keil, G M

    1996-01-01

    The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture. PMID:8551568

  6. The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

    PubMed Central

    Brosh, Robert M.; Balajee, Adayabalam S.; Selzer, Rebecca R.; Sunesen, Morten; De Santis, Luca Proietti; Bohr, Vilhelm A.

    1999-01-01

    Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. PMID:10564257

  7. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    PubMed Central

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas O.

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature. PMID:22344643

  8. The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential.

    PubMed

    Blinkova, A; Hervas, C; Stukenberg, P T; Onrust, R; O'Donnell, M E; Walker, J R

    1993-09-01

    The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded. PMID:8376347

  9. Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment

    SciTech Connect

    McCarthy, Christina B.; Da, Xiaojiang; Donly, Cam; Theilmann, David A.

    2008-03-15

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC{sup ac142KO-PH-GFP}). Fluorescence and light microscopy revealed that AcBAC{sup ac142KO-PH-GFP} exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC{sup ac142KO-PH-GFP} is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC{sup ac142KO-PH-GFP}-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.

  10. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

    SciTech Connect

    McCarthy, Christina B.; Theilmann, David A.

    2008-05-25

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC{sup ac142REP-ac143KO}). Fluorescence and light microscopy showed that infection by AcBAC{sup ac142REP-ac143KO} is limited to a single cell and titration assays confirmed that AcBAC{sup ac142REP-ac143KO} was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC{sup ac142REP-ac143KO} transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

  11. Genetic Interactions between an Essential 3′ cis-Acting RNA Pseudoknot, Replicase Gene Products, and the Extreme 3′ End of the Mouse Coronavirus Genome▿

    PubMed Central

    Züst, Roland; Miller, Timothy B.; Goebel, Scott J.; Thiel, Volker; Masters, Paul S.

    2008-01-01

    The upstream end of the 3′ untranslated region (UTR) of the mouse hepatitis virus genome contains two essential and overlapping RNA secondary structures, a bulged stem-loop and a pseudoknot, which have been proposed to be elements of a molecular switch that is critical for viral RNA synthesis. It has previously been shown that a particular six-base insertion in loop 1 of the pseudoknot is extremely deleterious to the virus. We have now isolated multiple independent second-site revertants of the loop 1 insertion mutant, and we used reverse-genetics methods to confirm the identities of suppressor mutations that could compensate for the original insertion. The suppressors were localized to two separate regions of the genome. Members of one class of suppressor were mapped to the portions of gene 1 that encode nsp8 and nsp9, thereby providing the first evidence for specific interactions between coronavirus replicase gene products and a cis-acting genomic RNA element. The second class of suppressor was mapped to the extreme 3′ end of the genome, a result which pointed to the existence of a direct base-pairing interaction between loop 1 of the pseudoknot and the genomic terminus. The latter finding was strongly supported by phylogenetic evidence and by the construction of a deletion mutant that reduced the 3′ UTR to its minimal essential elements. Taken together, the interactions revealed by the two classes of suppressors suggest a model for the initiation of coronavirus negative-strand RNA synthesis. PMID:18032506

  12. Network rewiring is an important mechanism of gene essentiality change

    PubMed Central

    Kim, Jinho; Kim, Inhae; Han, Seong Kyu; Bowie, James U.; Kim, Sanguk

    2012-01-01

    Gene essentiality changes are crucial for organismal evolution. However, it is unclear how essentiality of orthologs varies across species. We investigated the underlying mechanism of gene essentiality changes between yeast and mouse based on the framework of network evolution and comparative genomic analysis. We found that yeast nonessential genes become essential in mouse when their network connections rapidly increase through engagement in protein complexes. The increased interactions allowed the previously nonessential genes to become members of vital pathways. By accounting for changes in gene essentiality, we firmly reestablished the centrality-lethality rule, which proposed the relationship of essential genes and network hubs. Furthermore, we discovered that the number of connections associated with essential and non-essential genes depends on whether they were essential in ancestral species. Our study describes for the first time how network evolution occurs to change gene essentiality. PMID:23198090

  13. The essential gene set of a photosynthetic organism.

    PubMed

    Rubin, Benjamin E; Wetmore, Kelly M; Price, Morgan N; Diamond, Spencer; Shultzaberger, Ryan K; Lowe, Laura C; Curtin, Genevieve; Arkin, Adam P; Deutschbauer, Adam; Golden, Susan S

    2015-12-01

    Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼ 250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism's 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA(Leu), which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism's physiology and defines the essential gene set required for the growth of a photosynthetic organism. PMID:26508635

  14. Identification and characterization of essential genes in the human genome

    PubMed Central

    Wang, Tim; Birsoy, K?van; Hughes, Nicholas W.; Krupczak, Kevin M.; Post, Yorick; Wei, Jenny J.; Lander, Eric S.; Sabatini, David M.

    2015-01-01

    Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA (sgRNA) library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated by an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Lastly, screens in additional cell lines showed a high degree of overlap in gene essentiality, but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells. PMID:26472758

  15. Identification and characterization of essential genes in the human genome.

    PubMed

    Wang, Tim; Birsoy, Kıvanç; Hughes, Nicholas W; Krupczak, Kevin M; Post, Yorick; Wei, Jenny J; Lander, Eric S; Sabatini, David M

    2015-11-27

    Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells. PMID:26472758

  16. Exploring the Optimal Strategy to Predict Essential Genes in Microbes

    PubMed Central

    Deng, Jingyuan; Tan, Lirong; Lin, Xiaodong; Lu, Yao; Lu, Long J.

    2011-01-01

    Accurately predicting essential genes is important in many aspects of biology, medicine and bioengineering. In previous research, we have developed a machine learning based integrative algorithm to predict essential genes in bacterial species. This algorithm lends itself to two approaches for predicting essential genes: learning the traits from known essential genes in the target organism, or transferring essential gene annotations from a closely related model organism. However, for an understudied microbe, each approach has its potential limitations. The first is constricted by the often small number of known essential genes. The second is limited by the availability of model organisms and by evolutionary distance. In this study, we aim to determine the optimal strategy for predicting essential genes by examining four microbes with well-characterized essential genes. Our results suggest that, unless the known essential genes are few, learning from the known essential genes in the target organism usually outperforms transferring essential gene annotations from a related model organism. In fact, the required number of known essential genes is surprisingly small to make accurate predictions. In prokaryotes, when the number of known essential genes is greater than 2% of total genes, this approach already comes close to its optimal performance. In eukaryotes, achieving the same best performance requires over 4% of total genes, reflecting the increased complexity of eukaryotic organisms. Combining the two approaches resulted in an increased performance when the known essential genes are few. Our investigation thus provides key information on accurately predicting essential genes and will greatly facilitate annotations of microbial genomes. PMID:24970124

  17. Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media.

    PubMed Central

    Karow, M; Fayet, O; Cegielska, A; Ziegelhoffer, T; Georgopoulos, C

    1991-01-01

    We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33 degrees C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB+ gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates. Images PMID:1846149

  18. Network analysis of gene essentiality in functional genomics experiments.

    PubMed

    Jiang, Peng; Wang, Hongfang; Li, Wei; Zang, Chongzhi; Li, Bo; Wong, Yinling J; Meyer, Cliff; Liu, Jun S; Aster, Jon C; Liu, X Shirley

    2015-01-01

    Many genomic techniques have been developed to study gene essentiality genome-wide, such as CRISPR and shRNA screens. Our analyses of public CRISPR screens suggest protein interaction networks, when integrated with gene expression or histone marks, are highly predictive of gene essentiality. Meanwhile, the quality of CRISPR and shRNA screen results can be significantly enhanced through network neighbor information. We also found network neighbor information to be very informative on prioritizing ChIP-seq target genes and survival indicator genes from tumor profiling. Thus, our study provides a general method for gene essentiality analysis in functional genomic experiments ( http://nest.dfci.harvard.edu ). PMID:26518695

  19. A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit.

    PubMed

    Barad, Shiri; Horowitz, Sigal Brown; Moscovitz, Oren; Lichter, Amnon; Sherman, Amir; Prusky, Dov

    2012-06-01

    Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum. PMID:22352719

  20. The ha72 Core Gene of Baculovirus Is Essential for Budded Virus Production and Occlusion-Derived Virus Embedding, and Amino Acid K22 Plays an Important Role in Its Function

    PubMed Central

    Huang, Huachao; Wang, Manli; Deng, Fei; Hou, Dianhai; Arif, Basil M.; Wang, Hualin

    2014-01-01

    ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33. PMID:24089571

  1. Mutualistic Polydnaviruses Share Essential Replication Gene Functions with Pathogenic Ancestors

    PubMed Central

    Burke, Gaelen R.; Thomas, Sarah A.; Eum, Jai H.; Strand, Michael R.

    2013-01-01

    Viruses are usually thought to form parasitic associations with hosts, but all members of the family Polydnaviridae are obligate mutualists of insects called parasitoid wasps. Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses. However, pronounced differences in the biology of BVs and baculoviruses together with high divergence of many shared genes make it unclear whether BV homologs still retain baculovirus-like functions. Here we report that virions from Microplitis demolitor bracovirus (MdBV) contain multiple baculovirus-like and nudivirus-like conserved gene products. We further show that RNA interference effectively and specifically knocks down MdBV gene expression. Coupling RNAi knockdown methods with functional assays, we examined the activity of six genes in the MdBV conserved gene set that are known to have essential roles in transcription (lef-4, lef-9), capsid assembly (vp39, vlf-1), and envelope formation (p74, pif-1) during baculovirus replication. Our results indicated that MdBV produces a baculovirus-like RNA polymerase that transcribes virus structural genes. Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions. Additional experiments suggested that vlf-1 together with the nudivirus-like gene int-1 also have novel functions in regulating excision of MdBV proviral DNAs for packaging into virions. Overall, these data provide the first experimental insights into the function of BV genes in virion formation. PMID:23671417

  2. Conditional promoters for analysis of essential genes in Zymoseptoria tritici.

    PubMed

    Kilaru, S; Ma, W; Schuster, M; Courbot, M; Steinberg, G

    2015-06-01

    Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase (Pnar1; induced by nitrogen and repressed by ammonium), 1,4-β-endoxylanase A (Pex1A; induced by xylose and repressed by maltodextrin), l-arabinofuranosidase B (PlaraB; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (Pgal7; induced by galactose and repressed by glucose) and isocitrate lyase (Picl1; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici. PMID:26092803

  3. Conditional promoters for analysis of essential genes in Zymoseptoria tritici☆

    PubMed Central

    Kilaru, S.; Ma, W.; Schuster, M.; Courbot, M.; Steinberg, G.

    2015-01-01

    Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase (Pnar1; induced by nitrogen and repressed by ammonium), 1,4-β-endoxylanase A (Pex1A; induced by xylose and repressed by maltodextrin), l-arabinofuranosidase B (PlaraB; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (Pgal7; induced by galactose and repressed by glucose) and isocitrate lyase (Picl1; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici. PMID:26092803

  4. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    SciTech Connect

    Salem, Tamer Z.; Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619; Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 ; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  5. A new computational strategy for predicting essential genes

    PubMed Central

    2013-01-01

    Background Determination of the minimum gene set for cellular life is one of the central goals in biology. Genome-wide essential gene identification has progressed rapidly in certain bacterial species; however, it remains difficult to achieve in most eukaryotic species. Several computational models have recently been developed to integrate gene features and used as alternatives to transfer gene essentiality annotations between organisms. Results We first collected features that were widely used by previous predictive models and assessed the relationships between gene features and gene essentiality using a stepwise regression model. We found two issues that could significantly reduce model accuracy: (i) the effect of multicollinearity among gene features and (ii) the diverse and even contrasting correlations between gene features and gene essentiality existing within and among different species. To address these issues, we developed a novel model called feature-based weighted Naïve Bayes model (FWM), which is based on Naïve Bayes classifiers, logistic regression, and genetic algorithm. The proposed model assesses features and filters out the effects of multicollinearity and diversity. The performance of FWM was compared with other popular models, such as support vector machine, Naïve Bayes model, and logistic regression model, by applying FWM to reciprocally predict essential genes among and within 21 species. Our results showed that FWM significantly improves the accuracy and robustness of essential gene prediction. Conclusions FWM can remarkably improve the accuracy of essential gene prediction and may be used as an alternative method for other classification work. This method can contribute substantially to the knowledge of the minimum gene sets required for living organisms and the discovery of new drug targets. PMID:24359534

  6. Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

    PubMed

    Liu, Gaowen; Yong, Mei Yun Jacy; Yurieva, Marina; Srinivasan, Kandhadayar Gopalan; Liu, Jaron; Lim, John Soon Yew; Poidinger, Michael; Wright, Graham Daniel; Zolezzi, Francesca; Choi, Hyungwon; Pavelka, Norman; Rancati, Giulia

    2015-12-01

    Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance. PMID:26627736

  7. Analysis of pan-genome to identify the core genes and essential genes of Brucella spp.

    PubMed

    Yang, Xiaowen; Li, Yajie; Zang, Juan; Li, Yexia; Bie, Pengfei; Lu, Yanli; Wu, Qingmin

    2016-04-01

    Brucella spp. are facultative intracellular pathogens, that cause a contagious zoonotic disease, that can result in such outcomes as abortion or sterility in susceptible animal hosts and grave, debilitating illness in humans. For deciphering the survival mechanism of Brucella spp. in vivo, 42 Brucella complete genomes from NCBI were analyzed for the pan-genome and core genome by identification of their composition and function of Brucella genomes. The results showed that the total 132,143 protein-coding genes in these genomes were divided into 5369 clusters. Among these, 1710 clusters were associated with the core genome, 1182 clusters with strain-specific genes and 2477 clusters with dispensable genomes. COG analysis indicated that 44 % of the core genes were devoted to metabolism, which were mainly responsible for energy production and conversion (COG category C), and amino acid transport and metabolism (COG category E). Meanwhile, approximately 35 % of the core genes were in positive selection. In addition, 1252 potential essential genes were predicted in the core genome by comparison with a prokaryote database of essential genes. The results suggested that the core genes in Brucella genomes are relatively conservation, and the energy and amino acid metabolism play a more important role in the process of growth and reproduction in Brucella spp. This study might help us to better understand the mechanisms of Brucella persistent infection and provide some clues for further exploring the gene modules of the intracellular survival in Brucella spp. PMID:26724943

  8. Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis

    PubMed Central

    2012-01-01

    Background Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. Results In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions. Conclusions A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. Understanding the putative essential genes of P. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies. PMID:23114059

  9. Identification of essential and non-essential genes in Ambystoma tigrinum virus.

    PubMed

    Aron, Mariah M; Allen, Alexander G; Kromer, Mathew; Galvez, Hector; Vigil, Brianna; Jancovich, James K

    2016-06-01

    Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155kbp in length and they are predicted to encode around 90-125 genes. Currently, our knowledge of the function of ∼50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV. PMID:27025572

  10. Methods for identifying an essential gene in a prokaryotic microorganism

    DOEpatents

    Shizuya, Hiroaki

    2006-01-31

    Methods are provided for the rapid identification of essential or conditionally essential DNA segments in any species of haploid cell (one copy chromosome per cell) that is capable of being transformed by artificial means and is capable of undergoing DNA recombination. This system offers an enhanced means of identifying essential function genes in diploid pathogens, such as gram-negative and gram-positive bacteria.

  11. Training Set Selection for the Prediction of Essential Genes

    PubMed Central

    Zhao, Li; Li, Xiangchen; Liu, Yanlin; Tao, Shiheng

    2014-01-01

    Various computational models have been developed to transfer annotations of gene essentiality between organisms. However, despite the increasing number of microorganisms with well-characterized sets of essential genes, selection of appropriate training sets for predicting the essential genes of poorly-studied or newly sequenced organisms remains challenging. In this study, a machine learning approach was applied reciprocally to predict the essential genes in 21 microorganisms. Results showed that training set selection greatly influenced predictive accuracy. We determined four criteria for training set selection: (1) essential genes in the selected training set should be reliable; (2) the growth conditions in which essential genes are defined should be consistent in training and prediction sets; (3) species used as training set should be closely related to the target organism; and (4) organisms used as training and prediction sets should exhibit similar phenotypes or lifestyles. We then analyzed the performance of an incomplete training set and an integrated training set with multiple organisms. We found that the size of the training set should be at least 10% of the total genes to yield accurate predictions. Additionally, the integrated training sets exhibited remarkable increase in stability and accuracy compared with single sets. Finally, we compared the performance of the integrated training sets with the four criteria and with random selection. The results revealed that a rational selection of training sets based on our criteria yields better performance than random selection. Thus, our results provide empirical guidance on training set selection for the identification of essential genes on a genome-wide scale. PMID:24466248

  12. Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures.

    PubMed Central

    Lipinska, B; Fayet, O; Baird, L; Georgopoulos, C

    1989-01-01

    We identified and cloned an Escherichia coli gene called htrA (high temperature requirement). The htrA gene was originally discovered because mini-Tn10 transposon insertions in it allowed E. coli growth at 30 degrees C but prevented growth at elevated temperatures (above 42 degrees C). The htrA insertion mutants underwent a block in macromolecular synthesis and eventually lysed at the nonpermissive temperature. The htrA gene was located at approximately 3.7 min (between the fhuA and dapD loci) on the genetic map of E. coli and between 180 and 187.5 kilobases on the physical map. It coded for an unstable, 51-kilodalton protein which was processed by removal of an amino-terminal fragment, resulting in a stable, 48-kilodalton protein. Images PMID:2537822

  13. Gene trap mice reveal an essential function of dual specificity phosphatase Dusp16/MKP-7 in perinatal survival and regulation of Toll-like receptor (TLR)-induced cytokine production.

    PubMed

    Niedzielska, Magdalena; Bodendorfer, Barbara; Münch, Sandra; Eichner, Alexander; Derigs, Marcus; da Costa, Olivia; Schweizer, Astrid; Neff, Frauke; Nitschke, Lars; Sparwasser, Tim; Keyse, Stephen M; Lang, Roland

    2014-01-24

    MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16. PMID:24311790

  14. Essential gene identification and drug target prioritization in Aspergillus fumigatus.

    PubMed

    Hu, Wenqi; Sillaots, Susan; Lemieux, Sebastien; Davison, John; Kauffman, Sarah; Breton, Anouk; Linteau, Annie; Xin, Chunlin; Bowman, Joel; Becker, Jeff; Jiang, Bo; Roemer, Terry

    2007-03-01

    Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BDelta, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors. PMID:17352532

  15. Improvement of the viability of cultured rat neurons by the non-essential amino acids L-serine and glycine that upregulates expression of the anti-apoptotic gene product Bcl-w.

    PubMed

    Yang, L; Zhang, B; Toku, K; Maeda, N; Sakanaka, M; Tanaka, J

    2000-12-01

    The non-essential amino acids L-serine (Ser) and glycine (Gly) have recently been shown to exhibit specific actions in the nervous system. In the present study, L-Ser and Gly promoted the survival of cultured rat cerebrocortical neurons in a concentration-dependent manner as revealed by Alamar blue assay and microtubule-associated protein-2 (MAP2) immunoblotting. The maximum effects of the amino acids were detected at the concentrations of 30-100 microM. L-Ser was more effective than Gly. D-Ser failed to promote neuronal survival. L-Ser and Gly upregulated expression of the anti-apoptotic gene product Bcl-w, while they did not affect the expression of Bcl-xL. The promotion of neuronal survival by L-Ser and Gly may be, at least in part, attributable to the upregulated Bcl-w. PMID:11090983

  16. Transcriptional landscape and essential genes of Neisseria gonorrhoeae

    PubMed Central

    Remmele, Christian W.; Xian, Yibo; Albrecht, Marco; Faulstich, Michaela; Fraunholz, Martin; Heinrichs, Elisabeth; Dittrich, Marcus T.; Müller, Tobias; Reinhardt, Richard; Rudel, Thomas

    2014-01-01

    The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. PMID:25143534

  17. Antibiotics: natural products essential to human health.

    PubMed

    Demain, Arnold L

    2009-11-01

    For more than 50 years, natural products have served us well in combating infectious bacteria and fungi. Microbial and plant secondary metabolites helped to double our life span during the 20th century, reduced pain and suffering, and revolutionized medicine. Most antibiotics are either (i) natural products of microorganisms, (ii) semi-synthetically produced from natural products, or (iii) chemically synthesized based on the structure of the natural products. Production of antibiotics began with penicillin in the late 1940s and proceeded with great success until the 1970-1980s when it became harder and harder to discover new and useful products. Furthermore, resistance development in pathogens became a major problem, which is still with us today. In addition, new pathogens are continually emerging and there are still bacteria that are not eliminated by any antibiotic, e.g., Pseudomonas aeruginosa. In addition to these problems, many of the major pharmaceutical companies have abandoned the antibiotic field, leaving much of the discovery efforts to small companies, new companies, and the biotechnology industries. Despite these problems, development of new antibiotics has continued, albeit at a much lower pace than in the last century. We have seen the (i) appearance of newly discovered antibiotics (e.g., candins), (ii) development of old but unutilized antibiotics (e.g., daptomycin), (iii) production of new semi-synthetic versions of old antibiotics (e.g., glycylcyclines, streptogrammins), as well as the (iv) very useful application of old but underutilized antibiotics (e.g., teicoplanin). PMID:19291695

  18. The evolutionary study of susceptibility genes for essential hypertension.

    PubMed

    Ji, Lindan; Qian, Haixia; Xu, Jin

    2014-12-01

    Genetic studies, including familial linkage analysis, candidate gene approach and genome-wide association study, to some extent, have failed in detecting confirmative susceptibility genes/loci for essential hypertension (EH) in the general population. Previous genetic studies have suggested that differential susceptibility to many metabolic diseases is due to different environmental adaptation patterns during the out-of-Africa expansion, which provides a new strategy for the genetic study of EH. In this review, we introduce the principle and the latest progress of evolutionary study of susceptibility genes for EH. Furthermore, our recent evolutionary screening for EH susceptible genes/loci in Chinese Han population is also summarized. This review is expected to provide useful information for future genetic studies of EH and many other diseases. PMID:25487263

  19. DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis

    PubMed Central

    Chamberlain, Alyssa A.; Lin, Mingyan; Lister, Rolanda L.; Maslov, Alex A.; Wang, Yidong; Suzuki, Masako; Wu, Bingruo; Greally, John M.; Zheng, Deyou; Zhou, Bin

    2014-01-01

    Background DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development. Methods and Results We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function. Conclusions DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease. PMID:24947998

  20. Human factors and pathways essential for mediating epigenetic gene silencing

    PubMed Central

    Poleshko, Andrey; Kossenkov, Andrew V; Shalginskikh, Natalia; Pecherskaya, Anna; Pecherskaya, Anna; Einarson, Margret B; Einarson, Margret B; Marie Skalka, Anna; Marie Skalka, Anna; Katz, Richard A; Katz, Richard A

    2014-01-01

    Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming. PMID:25147916

  1. Conditional lethal amber mutations in essential Escherichia coli genes.

    PubMed

    Herring, Christopher D; Blattner, Frederick R

    2004-05-01

    The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development. PMID:15090508

  2. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology. PMID:26472760

  3. A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination.

    PubMed Central

    Ramakrishnan, L; Tran, H T; Federspiel, N A; Falkow, S

    1997-01-01

    A gene essential for light-induced pigment production was isolated from the photochromogen Mycobacterium marinum by heterologous complementation of an M. marinum cosmid library in the nonchromogen Mycobacterium smegmatis. This gene is part of an operon and homologous to the Streptomyces griseus and Myxococcus xanthus crtB genes encoding phytoene synthase. Gene replacement at this locus was achieved via homologous recombination, demonstrating that its expression is essential for photochromogenicity. The ease of targeted gene disruption in this pathogenic Mycobacterium allows for the dissection of the molecular basis of mycobacterial pathogenesis. PMID:9294446

  4. Essentiality drives the orientation bias of bacterial genes in a continuous manner

    PubMed Central

    Zheng, Wen-Xin; Luo, Cheng-Si; Deng, Yan-Yan; Guo, Feng-Biao

    2015-01-01

    Studies had found that bacterial genes are preferentially located on the leading strands. Subsequently, the preferences of essential genes and highly expressed genes were compared by classifying all genes into four groups, which showed that the former has an exclusive influence on orientation. However, only some functional classes of essential genes have this orientation bias. Nevertheless, previous studies only performed comparative analyzes by differentiating the orientation bias extent of two types of genes. Thus, it is unclear whether the influence of essentiality on strand bias works continuously. Herein, we found a significant correlation between essentiality and orientation bias extent in 19 of 21 analyzed bacterial genomes, based on quantitative measurement of gene essentiality (or fitness). The correlation coefficient was much higher than that derived from binary essentiality measures (essential or non-essential). This suggested that genes with relatively lower essentiality, i.e., conditionally essential genes, also have some orientation bias, although it is weaker than that of absolutely essential genes. The results demonstrated the continuous influence of essentiality on orientation bias and provided details on this visible structural feature of bacterial genomes. It also proved that Geptop and IFIM could serve as useful resources of bacterial gene essentiality, particularly for quantitative analysis. PMID:26560889

  5. The tobacco plastid accD gene is essential and is required for leaf development.

    PubMed

    Kode, Vasumathi; Mudd, Elisabeth A; Iamtham, Siriluck; Day, Anil

    2005-10-01

    Angiosperm plastid genomes typically encode approximately 80 polypeptides, mainly specifying plastid-localized functions such as photosynthesis and gene expression. Plastid protein synthesis and expression of the plastid clpP1 gene are essential for development in tobacco, indicating the presence of one or more plastid genes whose influence extends beyond the plastid compartment. The plastid accD gene encodes the beta-carboxyl transferase subunit of acetyl-CoA carboxylase and is present in the plastids of most flowering plants, including non-photosynthetic parasitic plants. We replaced the wild-type accD gene with an aadA-disrupted mutant allele using homologous recombination. Persistent heteroplasmy in the presence of antibiotics indicated that the wild-type accD allele was essential. The phenotype of the accD knockout was revealed in plastid transformants grown in the absence of antibiotics. Leaves contained pale green sectors and lacked part or all of the leaf lamina due to arrested division or loss of cells. Abnormal structures were present in plastids found in mutant plants, indicating that accD might be required to maintain the plastid compartment. Loss of the plastid compartment would be expected to be lethal. These results provide genetic evidence showing the essential role of plastid ACCase in the pathway leading to the synthesis of products required for the extra-plastidic processes needed for leaf development. PMID:16212603

  6. Characterization of the angR Gene of Vibrio anguillarum: Essential Role in Virulence

    PubMed Central

    Wertheimer, A. M.; Verweij, W.; Chen, Q.; Crosa, L. M.; Nagasawa, M.; Tolmasky, M. E.; Actis, L. A.; Crosa, J. H.

    1999-01-01

    The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5′-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression. PMID:10569768

  7. Uncovering major genomic features of essential genes in Bacteria and a methanogenic Archaea.

    PubMed

    Grazziotin, Ana Laura; Vidal, Newton M; Venancio, Thiago M

    2015-09-01

    Identification of essential genes is critical to understanding the physiology of a species, proposing novel drug targets and uncovering minimal gene sets required for life. Although essential gene sets of several organisms have been determined using large-scale mutagenesis techniques, systematic studies addressing their conservation, genomic context and functions remain scant. Here we integrate 17 essential gene sets from genome-wide in vitro screenings and three gene collections required for growth in vivo, encompassing 15 Bacteria and one Archaea. We refine and generalize important theories proposed using Escherichia coli. Essential genes are typically monogenic and more conserved than nonessential genes. Genes required in vivo are less conserved than those essential in vitro, suggesting that more divergent strategies are deployed when the organism is stressed by the host immune system and unstable nutrient availability. We identified essential analogous pathways that would probably be missed by orthology-based essentiality prediction strategies. For example, Streptococcus sanguinis carries horizontally transferred isoprenoid biosynthesis genes that are widespread in Archaea. Genes specifically essential in Mycobacterium tuberculosis and Burkholderia pseudomallei are reported as potential drug targets. Moreover, essential genes are not only preferentially located in operons, but also occupy the first position therein, supporting the influence of their regulatory regions in driving transcription of whole operons. Finally, these important genomic features are shared between Bacteria and at least one Archaea, suggesting that high order properties of gene essentiality and genome architecture were probably present in the last universal common ancestor or evolved independently in the prokaryotic domains. PMID:26084810

  8. Identifying Essential Streptococcus sanguinis Genes Using Genome-Wide Deletion Mutation

    PubMed Central

    Chen, Lei; Ge, Xiuchun; Xu, Ping

    2016-01-01

    Essential genes in pathogens are important for the development of antibacterial drugs. In this report, we described a protocol to identify essential genes in the Streptococcus sanguinis SK36 strain using genome-wide deletion mutation. A fusion PCR-based method is used to construct gene deletion fragments, which contain kanamycin resistance cassettes with two flanking arms of DNA upstream and downstream of the target gene. The linear fused PCR amplicons were transformed into S. sanguinis SK36 cells. No kanamycin-resistant transformants suggested the gene essentiality because the deletion of the essential gene renders a lethal phenotype of the transformants. The putative essential genes were further confirmed by independent transformations up to five attempts. The false nonessential genes were also identified by removing double-band mutants. PMID:25636610

  9. The Essential Gene EMB1611 Maintains Shoot Apical Meristem Function During Arabidopsis Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis thaliana genome contains hundreds of genes essential for seed development. Because null mutations in these genes cause embryo lethality, their specific molecular and developmental functions are largely unknown. Here, we identify a role for EMB1611/MEE22, an essential gene in Arabidop...

  10. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    PubMed Central

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  11. Towards a compendium of essential genes – From model organisms to synthetic lethality in cancer cells

    PubMed Central

    Zhan, Tianzuo; Boutros, Michael

    2016-01-01

    Abstract Essential genes are defined by their requirement to sustain life in cells or whole organisms. The systematic identification of essential gene sets not only allows insights into the fundamental building blocks of life, but may also provide novel therapeutic targets in oncology. The discovery of essential genes has been tightly linked to the development and deployment of various screening technologies. Here, we describe how gene essentiality was addressed in different eukaryotic model organisms, covering a range of organisms from yeast to mouse. We describe how increasing knowledge of evolutionarily divergent genomes facilitate identification of gene essentiality across species. Finally, the impact of gene essentiality and synthetic lethality on cancer research and the clinical translation of screening results are highlighted. PMID:26627871

  12. Towards a compendium of essential genes - From model organisms to synthetic lethality in cancer cells.

    PubMed

    Zhan, Tianzuo; Boutros, Michael

    2016-01-01

    Essential genes are defined by their requirement to sustain life in cells or whole organisms. The systematic identification of essential gene sets not only allows insights into the fundamental building blocks of life, but may also provide novel therapeutic targets in oncology. The discovery of essential genes has been tightly linked to the development and deployment of various screening technologies. Here, we describe how gene essentiality was addressed in different eukaryotic model organisms, covering a range of organisms from yeast to mouse. We describe how increasing knowledge of evolutionarily divergent genomes facilitate identification of gene essentiality across species. Finally, the impact of gene essentiality and synthetic lethality on cancer research and the clinical translation of screening results are highlighted. PMID:26627871

  13. Human lupus anti-DNA autoantibodies undergo essentially primary V kappa gene rearrangements.

    PubMed Central

    Bensimon, C; Chastagner, P; Zouali, M

    1994-01-01

    We have recently characterized the heavy chain variable region (VH) genes expressed by a panel of human anti-DNA antibodies derived from four patients with systemic lupus erythematosus and expressing an idiotypic marker representative of a subset of pathogenic autoantibodies. Here, we have cloned and sequenced the kappa chain variable region genes (V kappa) of the clones whose VH genes had been previously analysed. All the V kappa genes utilized map to the 280 kb portion of the 3' end of the locus, suggesting that they represent essentially the products of primary rearrangements. This proximal clustering of the V kappa genes used contrasts with the broad distribution of immunization-induced human antibody V kappa genes over 1400 kb of the locus. In addition, lupus autoantibodies show no tendency to express the downstream junctional (J kappa) exons--another indication of infrequent secondary variable gene assembly. Since successive rearrangements may extinguish high-affinity recognition of self antigens, we propose that this bias in V kappa and J kappa expression reflects a low rate of secondary light chain rearrangements among lupus autoantibodies. We also postulate that the corrective mechanism capable of editing potentially aggressive, self-reactive antibodies in these patients may be deficient--a deficit that could be genetically determined and/or somatically acquired. Images PMID:8039491

  14. Statistical Analysis of Hurst Exponents of Essential/Nonessential Genes in 33 Bacterial Genomes

    PubMed Central

    Liu, Xiao; Wang, Baojin; Xu, Luo

    2015-01-01

    Methods for identifying essential genes currently depend predominantly on biochemical experiments. However, there is demand for improved computational methods for determining gene essentiality. In this study, we used the Hurst exponent, a characteristic parameter to describe long-range correlation in DNA, and analyzed its distribution in 33 bacterial genomes. In most genomes (31 out of 33) the significance levels of the Hurst exponents of the essential genes were significantly higher than for the corresponding full-gene-set, whereas the significance levels of the Hurst exponents of the nonessential genes remained unchanged or increased only slightly. All of the Hurst exponents of essential genes followed a normal distribution, with one exception. We therefore propose that the distribution feature of Hurst exponents of essential genes can be used as a classification index for essential gene prediction in bacteria. For computer-aided design in the field of synthetic biology, this feature can build a restraint for pre- or post-design checking of bacterial essential genes. Moreover, considering the relationship between gene essentiality and evolution, the Hurst exponents could be used as a descriptive parameter related to evolutionary level, or be added to the annotation of each gene. PMID:26067107

  15. Expression of essential B cell genes and immunoglobulin isotypes suggests active development and gene recombination during equine gestation.

    PubMed

    Tallmadge, Rebecca L; McLaughlin, Kristin; Secor, Erica; Ruano, Diana; Matychak, Mary Beth; Flaminio, M Julia B F

    2009-09-01

    Many features of the equine immune system develop during fetal life, yet the naïve or immature immune state of the neonate renders the foal uniquely susceptible to particular pathogens. RT-PCR and immunohistochemical experiments investigated the progressive expression of developmental B cell markers and immunoglobulins in lymphoid tissues from equine fetus, pre-suckle neonate, foal, and adult horses. Serum IgM, IgG isotype, and IgA concentrations were also quantified in pre-suckle foals and adult horses. The expression of essential B cell genes suggests active development and gene recombination during equine gestation, including immunoglobulin isotype switching. The corresponding production of IgM and IgG proteins is detectable in a limited scale at birth. Although the equine neonate humoral response seems competent, B cell activation factors derived from antigen presenting cells and T cells may control critical developmental regulation and immunoglobulin production during the initial months of life. PMID:19442687

  16. Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88.

    PubMed Central

    Yamazaki, M; Thorne, L; Mikolajczak, M; Armentrout, R W; Pollock, T J

    1996-01-01

    Several structurally related capsular polysaccharides that are secreted by members of the genus Sphingomonas are being developed as aqueous rheological control agents for diverse industrial and food applications. They include gellan (S-60), welan (S-130), rhamsan (S-194), S-657, S-88, S-198, S-7, and NW-11. We refer to these polysaccharides as sphingans, after the genus name. This paper characterizes the first gene cluster isolated from a Sphingomonas species (S88) that is required for capsule synthesis. Overlapping DNA segments which spanned about 50 kbp of S88 DNA restored the synthesis of sphingan S-88 in capsule-negative mutants. The mutations were mapped into functional complementation groups, and the contiguous nucleotide sequence for the 29-kbp cluster was determined. The genetic complementation map and the DNA sequences were interpreted as an extended multicistronic locus containing genes essential for the assembly and secretion of polysaccharide S-88. Many of the deduced amino acid sequences were similar to gene products from other polysaccharide-secreting bacteria such as Rhizobium meliloti (succinoglycan), Xanthomonas campestris (xanthan gum), and Salmonella enterica (O antigen). The S88 locus contained a four-gene operon for the biosynthesis of dTDP-L-rhamnose, an essential precursor for the sphingans. Unexpectedly, there were also two genes for secretion of a lytic or toxin-like protein nested within the polysaccharide cluster. The conservation and linkage of genes that code for a defensive capsule and genes for secretion of an offensive lysin or toxin suggest a heretofore unknown pathogenic life history for Sphingomonas strain S88. PMID:8626338

  17. Predicting Essential Genes and Proteins Based on Machine Learning and Network Topological Features: A Comprehensive Review

    PubMed Central

    Zhang, Xue; Acencio, Marcio Luis; Lemke, Ney

    2016-01-01

    Essential proteins/genes are indispensable to the survival or reproduction of an organism, and the deletion of such essential proteins will result in lethality or infertility. The identification of essential genes is very important not only for understanding the minimal requirements for survival of an organism, but also for finding human disease genes and new drug targets. Experimental methods for identifying essential genes are costly, time-consuming, and laborious. With the accumulation of sequenced genomes data and high-throughput experimental data, many computational methods for identifying essential proteins are proposed, which are useful complements to experimental methods. In this review, we show the state-of-the-art methods for identifying essential genes and proteins based on machine learning and network topological features, point out the progress and limitations of current methods, and discuss the challenges and directions for further research. PMID:27014079

  18. The functional diversity of essential genes required for mammalian cardiac development

    PubMed Central

    Clowes, Christopher; Boylan, Michael GS; Ridge, Liam A; Barnes, Emma; Wright, Jayne A; Hentges, Kathryn E

    2014-01-01

    Genes required for an organism to develop to maturity (for which no other gene can compensate) are considered essential. The continuing functional annotation of the mouse genome has enabled the identification of many essential genes required for specific developmental processes including cardiac development. Patterns are now emerging regarding the functional nature of genes required at specific points throughout gestation. Essential genes required for development beyond cardiac progenitor cell migration and induction include a small and functionally homogenous group encoding transcription factors, ligands and receptors. Actions of core cardiogenic transcription factors from the Gata, Nkx, Mef, Hand, and Tbx families trigger a marked expansion in the functional diversity of essential genes from midgestation onwards. As the embryo grows in size and complexity, genes required to maintain a functional heartbeat and to provide muscular strength and regulate blood flow are well represented. These essential genes regulate further specialization and polarization of cell types along with proliferative, migratory, adhesive, contractile, and structural processes. The identification of patterns regarding the functional nature of essential genes across numerous developmental systems may aid prediction of further essential genes and those important to development and/or progression of disease. genesis 52:713–737, 2014. PMID:24866031

  19. Identification and functional analysis of essential, conserved, housekeeping and duplicated genes.

    PubMed

    Arun, P V Parvati Sai; Miryala, Sravan Kumar; Chattopadhyay, Subhayan; Thiyyagura, Kranthi; Bawa, Payal; Bhattacharjee, Madhuchhanda; Yellaboina, Sailu

    2016-05-01

    Gene conservation, duplication and constitutive expression are intricately linked and strong predictors of essentiality. Here, we introduce metrics based on diversity indices to measure gene conservation, duplication and constitutive expression and validate them by measuring their performance in prediction of essential genes. Conservation and duplication were measured using the diversity indices on the bit score profile of Escherichia coli K12 orthologues, across the genomes, and paralogues, within the genome respectively. Constitutive expression was measured using expression diversity of E. coli K12 genes across different conditions. In addition, we developed a systematic method for enrichment analysis of gene-sets in a given ranked list of genes. The method was used to identify genome-wide functions of essential, conserved, constitutively expressed and duplicated genes. Furthermore, we also ranked various operons, complexes and pathways according to their essentiality, conservation, constitutive expression and duplication. PMID:27129600

  20. Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems.

    PubMed

    Curtis, Patrick D; Brun, Yves V

    2014-08-01

    The cell cycle of Caulobacter crescentus is controlled by a complex signalling network that co-ordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism. PMID:24975755

  1. Identification of essential Alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems

    PubMed Central

    Curtis, Patrick D.; Brun, Yves V.

    2014-01-01

    Summary The cell cycle of Caulobacter crescentus is controlled by a complex signaling network that coordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism’s different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism’s essential gene pool is specific to that organism. PMID:24975755

  2. small ORFs: A new class of essential genes for development

    PubMed Central

    Albuquerque, João Paulo; Tobias-Santos, Vitória; Rodrigues, Aline Cáceres; Mury, Flávia Borges; da Fonseca, Rodrigo Nunes

    2015-01-01

    Genes that contain small open reading frames (smORFs) constitute a new group of eukaryotic genes and are expected to represent 5% of the Drosophila melanogaster transcribed genes. In this review we provide a historical perspective of their recent discovery, describe their general mechanism and discuss the importance of smORFs for future genomic and transcriptomic studies. Finally, we discuss the biological role of the most studied smORF so far, the Mlpt/Pri/Tal gene in arthropods. The pleiotropic action of Mlpt/Pri/Tal in D. melanogaster suggests a complex evolutionary scenario that can be used to understand the origins, evolution and integration of smORFs into complex gene regulatory networks. PMID:26500431

  3. Systematic identification of novel, essential host genes affecting bromovirus RNA replication.

    PubMed

    Gancarz, Brandi L; Hao, Linhui; He, Qiuling; Newton, Michael A; Ahlquist, Paul

    2011-01-01

    Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ?100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ?900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ?81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control. PMID:21915247

  4. Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli.

    PubMed Central

    Lee, J H; Wendt, J C; Shanmugam, K T

    1990-01-01

    A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase. This phenotype was suppressed by supplementing the media with molybdate. Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport. The molR gene is located at 65.3 min in the E. coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished. The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min). The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media. The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium. In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient. In low-sulfur medium, this value decreased to about 50 nM. The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity. These data suggest that in the absence of the high-affinity molybdate transport system, E. coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system. PMID:2156810

  5. Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans.

    PubMed

    Chen, Xiangyang; Li, Mu; Feng, Xuezhu; Guang, Shouhong

    2015-12-01

    Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss-of-function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we have developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology and have applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual small guide RNA targeting genes on different chromosomes resulted in reciprocal translocation between nonhomologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss-of-function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss-of-function alleles of essential genes with detailed genetic background information. PMID:26482793

  6. Construction of an inducible system for the analysis of essential genes in Yersinia pestis.

    PubMed

    Ford, D C; Ireland, P M; Bullifent, H L; Saint, R J; McAlister, E V; Sarkar-Tyson, M; Oyston, P C F

    2014-05-01

    Yersinia pestis, a Gram negative bacterium, causes bubonic and pneumonic plague. Emerging antibiotic resistance in clinical isolates is driving a need to develop novel antibiotics to treat infection by this transmissible and highly virulent pathogen. Proteins required for viability, so called essential genes, are attractive potential therapeutic targets, however, confirmation of essentiality is problematic. For the first time, we report the development of a system that allows the rapid determination of Y. pestis gene essentiality through mutagenesis and inducible expression of a plasmid borne copy of the target gene. Using this approach, we have confirmed the uridine monophosphate kinase PyrH as an essential protein in Y. pestis. This methodology and the tools we have developed will allow the confirmation of other putative essential genes in this dangerous pathogen, and facilitate the identification of novel targets for antimicrobial development. PMID:24524852

  7. Bayesian analysis of gene essentiality based on sequencing of transposon insertion libraries

    PubMed Central

    DeJesus, Michael A.; Zhang, Yanjia J.; Sassetti, Christopher M.; Rubin, Eric J.; Sacchettini, James C.; Ioerger, Thomas R.

    2013-01-01

    Motivation: Next-generation sequencing affords an efficient analysis of transposon insertion libraries, which can be used to identify essential genes in bacteria. To analyse this high-resolution data, we present a formal Bayesian framework for estimating the posterior probability of essentiality for each gene, using the extreme-value distribution to characterize the statistical significance of the longest region lacking insertions within a gene. We describe a sampling procedure based on the Metropolis–Hastings algorithm to calculate posterior probabilities of essentiality while simultaneously integrating over unknown internal parameters. Results: Using a sequence dataset from a transposon library for Mycobacterium tuberculosis, we show that this Bayesian approach predicts essential genes that correspond well with genes shown to be essential in previous studies. Furthermore, we show that by using the extreme-value distribution to characterize genomic regions lacking transposon insertions, this method is capable of identifying essential domains within genes. This approach can be used for analysing transposon libraries in other organisms and augmenting essentiality predictions with statistical confidence scores. Availability: A python script implementing the method described is available for download from http://saclab.tamu.edu/essentiality/. Contact: michael.dejesus@tamu.edu or ioerger@cs.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23361328

  8. Evolutionary Conservation of Bacterial Essential Metabolic Genes across All Bacterial Culture Media

    PubMed Central

    Ish-Am, Oren; Kristensen, David M.; Ruppin, Eytan

    2015-01-01

    One of the basic postulates of molecular evolution is that functionally important genes should evolve slower than genes of lesser significance. Essential genes, whose knockout leads to a lethal phenotype are considered of high functional importance, yet whether they are truly more conserved than nonessential genes has been the topic of much debate, fuelled by a host of contradictory findings. Here we conduct the first large-scale study utilizing genome-scale metabolic modeling and spanning many bacterial species, which aims to answer this question. Using the novel Media Variation Analysis, we examine the range of conservation of essential vs. nonessential metabolic genes in a given species across all possible media. We are thus able to obtain for the first time, exact upper and lower bounds on the levels of differential conservation of essential genes for each of the species studied. The results show that bacteria do exhibit an overall tendency for differential conservation of their essential genes vs. their non-essential ones, yet this tendency is highly variable across species. We show that the model bacterium E. coli K12 may or may not exhibit differential conservation of essential genes depending on its growth medium, shedding light on previous experimental studies showing opposite trends. PMID:25894004

  9. Burkholderia cenocepacia conditional growth mutant library created by random promoter replacement of essential genes

    PubMed Central

    Bloodworth, Ruhi A M; Gislason, April S; Cardona, Silvia T

    2013-01-01

    Identification of essential genes by construction of conditional knockouts with inducible promoters allows the identification of essential genes and creation of conditional growth (CG) mutants that are then available as genetic tools for further studies. We used large-scale transposon delivery of the rhamnose-inducible promoter, PrhaB followed by robotic screening of rhamnose-dependent growth to construct a genomic library of 106 Burkholderia cenocepacia CG mutants. Transposon insertions were found where PrhaB was in the same orientation of widely conserved, well-characterized essential genes as well as genes with no previous records of essentiality in other microorganisms. Using previously reported global gene-expression analyses, we demonstrate that PrhaB can achieve the wide dynamic range of expression levels required for essential genes when the promoter is delivered randomly and mutants with rhamnose-dependent growth are selected. We also show specific detection of the target of an antibiotic, novobiocin, by enhanced sensitivity of the corresponding CG mutant (PrhaB controlling gyrB expression) within the library. Modulation of gene expression to achieve 30–60% of wild-type growth created conditions for specific hypersensitivity demonstrating the value of the CG mutant library for chemogenomic experiments. In summary, CG mutants can be obtained on a large scale by random delivery of a tightly regulated inducible promoter into the bacterial chromosome followed by a simple screening for the CG phenotype, without previous information on gene essentiality. PMID:23389959

  10. The role of the 'gearbox' in the transcription of essential genes.

    PubMed

    Vicente, M; Kushner, S R; Garrido, T; Aldea, M

    1991-09-01

    Regulation of transcription occurs at different levels, one being in the presence of sequences specifically recognized by different forms of RNA polymerase, i.e. the promoters. Three different kinds of promoter are defined according, among other things, to their dependence on the growth rate of the cell: the 'house-keeper' promoter of many metabolic genes, the stringent promoter found at several rRNA and ribosomal protein genes, and the 'gearbox' at genes whose products are required at higher relative amounts at lower growth rates. The identified gearbox promoters of Escherichia coli share specific homologies in the -10, -35 and upstream regions. Although there may be different types of gearbox promoters, the -10 sequence of one of these promoters has been found to be essential for functioning as a gearbox. This suggests the existence of specific sigma factors for its transcription. RpoS (KatF) is a likely candidate for being one of these sigma factors. Computer simulation allows us to predict that such sigma factors should, in turn, be expressed following a gearbox mode, which would then imply the existence of self-regulated loops contributing to the expression of some genes of bacterial division. PMID:1766382

  11. The Drosophila Couch Potato Gene: An Essential Gene Required for Normal Adult Behavior

    PubMed Central

    Bellen, H. J.; Vaessin, H.; Bier, E.; Kolodkin, A.; D'Evelyn, D.; Kooyer, S.; Jan, Y. N.

    1992-01-01

    Through enhancer detection screens we have isolated 14 insertions in an essential gene that is expressed in embryonic sensory mother cells (SMC), in most cells of the mature embryonic peripheral nervous system (PNS), and in glial cells of the PNS and the central nervous system (CNS). Embryos homozygote for amorphic alleles die, but show no obvious defects in their cuticle, PNS or CNS. The gene has been named couch potato (cpo) because several insertional alleles alter adult behavior. Homozygous hypomorphic cpo flies recover slowly from ether anaesthesia, show aberrant flight behavior, fail to move toward light and do not exhibit normal negative geotactic behavior. However, the flies are able to groom and walk, and some are able to fly when prodded, indicating that not all processes required for behavior are severely affected. A molecular analysis shows that the 14 insertions are confined to a few hundred nucleotides which probably contain key regulatory sequences of the gene. The orientation of these insertions and their position within this DNA fragment play an important role in the couch potato phenotype. In situ hybridization to whole mount embryos suggest that some insertions affect the levels of transcription of cpo in most cells in which it is expressed. PMID:1644278

  12. A multi-level multi-scale approach to study essential genes in Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background The set of indispensable genes that are required by an organism to grow and sustain life are termed as essential genes. There is a strong interest in identification of the set of essential genes, particularly in pathogens, not only for a better understanding of the pathogen biology, but also for identifying drug targets and the minimal gene set for the organism. Essentiality is inherently a systems property and requires consideration of the system as a whole for their identification. The available experimental approaches capture some aspects but each method comes with its own limitations. Moreover, they do not explain the basis for essentiality in most cases. A powerful prediction method to recognize this gene pool including rationalization of the known essential genes in a given organism would be very useful. Here we describe a multi-level multi-scale approach to identify the essential gene pool in a deadly pathogen, Mycobacterium tuberculosis. Results The multi-level workflow analyses the bacterial cell by studying (a) genome-wide gene expression profiles to identify the set of genes which show consistent and significant levels of expression in multiple samples of the same condition, (b) indispensability for growth by using gene expression integrated flux balance analysis of a genome-scale metabolic model, (c) importance for maintaining the integrity and flow in a protein-protein interaction network and (d) evolutionary conservation in a set of genomes of the same ecological niche. In the gene pool identified, the functional basis for essentiality has been addressed by studying residue level conservation and the sub-structure at the ligand binding pockets, from which essential amino acid residues in that pocket have also been identified. 283 genes were identified as essential genes with high-confidence. An agreement of about 73.5% is observed with that obtained from the experimental transposon mutagenesis technique. A large proportion of the identified genes belong to the class of intermediary metabolism and respiration. Conclusions The multi-scale, multi-level approach described can be generally applied to other pathogens as well. The essential gene pool identified form a basis for designing experiments to probe their finer functional roles and also serve as a ready shortlist for identifying drug targets. PMID:24308365

  13. Correlating Traits of Gene Retention, Sequence Divergence, Duplicability and Essentiality in Vertebrates, Arthropods, and Fungi

    PubMed Central

    Waterhouse, Robert M.; Zdobnov, Evgeny M.; Kriventseva, Evgenia V.

    2011-01-01

    Delineating ancestral gene relations among a large set of sequenced eukaryotic genomes allowed us to rigorously examine links between evolutionary and functional traits. We classified 86% of over 1.36 million protein-coding genes from 40 vertebrates, 23 arthropods, and 32 fungi into orthologous groups and linked over 90% of them to Gene Ontology or InterPro annotations. Quantifying properties of ortholog phyletic retention, copy-number variation, and sequence conservation, we examined correlations with gene essentiality and functional traits. More than half of vertebrate, arthropod, and fungal orthologs are universally present across each lineage. These universal orthologs are preferentially distributed in groups with almost all single-copy or all multicopy genes, and sequence evolution of the predominantly single-copy orthologous groups is markedly more constrained. Essential genes from representative model organisms, Mus musculus, Drosophila melanogaster, and Saccharomyces cerevisiae, are significantly enriched in universal orthologs within each lineage, and essential-gene-containing groups consistently exhibit greater sequence conservation than those without. This study of eukaryotic gene repertoire evolution identifies shared fundamental principles and highlights lineage-specific features, it also confirms that essential genes are highly retained and conclusively supports the “knockout-rate prediction” of stronger constraints on essential gene sequence evolution. However, the distinction between sequence conservation of single- versus multicopy orthologs is quantitatively more prominent than between orthologous groups with and without essential genes. The previously underappreciated difference in the tolerance of gene duplications and contrasting evolutionary modes of “single-copy control” versus “multicopy license” may reflect a major evolutionary mechanism that allows extended exploration of gene sequence space. PMID:21148284

  14. The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control.

    PubMed Central

    Kinoshita, N; Goebl, M; Yanagida, M

    1991-01-01

    The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis. Images PMID:1944266

  15. Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products

    PubMed Central

    Houël, Emeline; Gonzalez, German; Bessière, Jean-Marie; Odonne, Guillaume; Eparvier, Véronique; Deharo, Eric; Stien, Didier

    2015-01-01

    This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis. PMID:25742270

  16. Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products.

    PubMed

    Houël, Emeline; Gonzalez, German; Bessière, Jean-Marie; Odonne, Guillaume; Eparvier, Véronique; Deharo, Eric; Stien, Didier

    2015-02-01

    This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis. PMID:25742270

  17. Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli.

    PubMed

    Goh, Shan; Hohmeier, Angela; Stone, Timothy C; Offord, Victoria; Sarabia, Francisco; Garcia-Ruiz, Cristina; Good, Liam

    2015-08-15

    Essential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, the Escherichia coli ribF-ileS-lspA-fkpB-ispH operon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlapping ileS-lspA genes. We found upstream and downstream polar silencing effects when either ileS or lspA was silenced, indicating coupled expression. Weighted MTL50 values (means and standard deviations) of ribF, ileS, and lspA were 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P = 0.71 by weighted one-way analysis of variance). The gene requirement for ispH could not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated that ileS-lspA expression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials. PMID:26070674

  18. An ABC transporter from Bacillus thuringiensis is essential for beta-exotoxin I production.

    PubMed

    Espinasse, Sylvain; Gohar, Michel; Lereclus, Didier; Sanchis, Vincent

    2002-11-01

    beta-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce beta-exotoxin I have suggested that there is a strong correlation between high levels of beta-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry(-))(Pig(+)), with no crystal gene, produced considerable amounts of beta-exotoxin I together with a soluble brown melanin pigment. Therefore, beta-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of beta-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry(-))(Pig(+)) mutants. We screened for nonpigmented mutants with impaired beta-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for beta-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for beta-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers beta-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule. PMID:12374817

  19. An ABC Transporter from Bacillus thuringiensis Is Essential for β-Exotoxin I Production

    PubMed Central

    Espinasse, Sylvain; Gohar, Michel; Lereclus, Didier; Sanchis, Vincent

    2002-01-01

    β-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce β-exotoxin I have suggested that there is a strong correlation between high levels of β-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry−)(Pig+), with no crystal gene, produced considerable amounts of β-exotoxin I together with a soluble brown melanin pigment. Therefore, β-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of β-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry−)(Pig+) mutants. We screened for nonpigmented mutants with impaired β-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for β-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for β-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers β-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule. PMID:12374817

  20. Predicting essential genes in prokaryotic genomes using a linear method: ZUPLS.

    PubMed

    Song, Kai; Tong, Tuopong; Wu, Fang

    2014-04-01

    An effective linear method, ZUPLS, was developed to improve the accuracy and speed of prokaryotic essential gene identification. ZUPLS only uses the Z-curve and other sequence-based features. Such features can be calculated readily from the DNA/amino acid sequences. Therefore, no well-studied biological network knowledge is required for using ZUPLS. This significantly simplifies essential gene identification, especially for newly sequenced species. ZUPLS can also select necessary features automatically by embedding the uninformative variable elimination tool into the partial least squares classifier. No optimized modelling parameters are needed. ZUPLS has been used, herein, to predict essential genes of 12 remotely related prokaryotes to test its performance. The cross-organism predictions yielded AUC (Area Under the Curve) scores between 0.8042 and 0.9319 by using E. coli genes as the training samples. Similarly, ZUPLS achieved AUC scores between 0.8111 and 0.9371 by using B. subtilis genes as the training samples. We also compared it with the best available results of the existing approaches for further testing. The improvement of the AUC score in predicting B. subtilis essential genes using E. coli genes was 0.13. Additionally, in predicting E. coli essential genes using P. aeruginosa genes, the significant improvement was 0.10. Similarly, the exceptional improvement of the average accuracy of M. pulmonis using M. genitalium and M. pulmonis genes was 14.7%. The combined superior feature extraction and selection power of ZUPLS enable it to give reliable prediction of essential genes for both Gram-positive/negative organisms and rich/poor culture media. PMID:24603751

  1. A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: Application to Mycobacterium tuberculosis

    PubMed Central

    Lamichhane, Gyanu; Zignol, Matteo; Blades, Natalie J.; Geiman, Deborah E.; Dougherty, Annette; Grosset, Jacques; Broman, Karl W.; Bishai, William R.

    2003-01-01

    We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35% (95% confidence interval, 28%–41%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide/nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes. PMID:12775759

  2. Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis

    PubMed Central

    1995-01-01

    Schizosaccharomyces pombe cells divide by medial fission. One class of cell division mutants (cdc), the late septation mutants, defines four genes: cdc3, cdc4, cdc8, and cdc12 (Nurse, P., P. Thuriaux, and K. Nasmyth. 1976. Mol. & Gen. Genet. 146:167-178). We have cloned and characterized the cdc4 gene and show that the predicted gene product. Cdc4p, is a 141-amino acid polypeptide that is similar in sequence to EF-hand proteins including myosin light chains, calmodulin, and troponin C. Two temperature-sensitive lethal alleles, cdc4-8 and cdc4- 31, accumulate multiple nuclei and multiple improper F-actin rings and septa but fail to complete cytokinesis. Deletion of cdc4 also results in a lethal terminal phenotype characterized by multinucleate, elongated cells that fail to complete cytokinesis. Sequence comparisons suggest that Cdc4p may be a member of a new class of EF-hand proteins. Cdc4p localizes to a ringlike structure in the medial region of cells undergoing cytokinesis. Thus, Cdc4p appears to be an essential component of the F-actin contractile ring. We find that Cdc4 protein forms a complex with a 200-kD protein which can be cross-linked to UTP, a property common to myosin heavy chains. Together these results suggest that Cdc4p may be a novel myosin light chain. PMID:7622565

  3. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    SciTech Connect

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  4. A Caenorhabditis Elegans RNA Polymerase II Gene, Ama-1 Iv, and Nearby Essential Genes

    PubMed Central

    Rogalski, T. M.; Riddle, D. L.

    1988-01-01

    The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20° but are arrested as larvae at 25°, and two others are fertile at 20° and sterile at 25°. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25° is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four γ-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development. PMID:8608933

  5. The Dpy-30 Gene Encodes an Essential Component of the Caenorhabditis Elegans Dosage Compensation Machinery

    PubMed Central

    Hsu, D. R.; Meyer, B. J.

    1994-01-01

    The need to regulate X chromosome expression in Caenorhabditis elegans arises as a consequence of the primary sex-determining signal, the X/A ratio (the ratio of X chromosomes to sets of autosomes), which directs 1X/2A animals to develop as males and 2X/2A animals to develop as hermaphrodites. C. elegans possesses a dosage compensation mechanism that equalizes X chromosome expression between the two sexes despite their disparity in X chromosome dosage. Previous genetic analysis led to the identification of four autosomal genes, dpy-21, dpy-26, dpy-27 and dpy-28, whose products are essential in XX animals for proper dosage compensation, but not for sex determination. We report the identification and characterization of dpy-30, an essential component of the dosage compensation machinery. Putative null mutations in dpy-30 disrupt dosage compensation and cause a severe maternal-effect, XX-specific lethality. Rare survivors of the dpy-30 lethality are dumpy and express their X-linked genes at higher than wild-type levels. These dpy-30 mutant phenotypes superficially resemble those caused by mutations in dpy-26, dpy-27 and dpy-28; however, detailed phenotypic analysis reveals important differences that distinguish dpy-30 from these genes. In contrast to the XX-specific lethality caused by mutations in the other dpy genes, the XX-specific lethality caused by dpy-30 mutations is completely penetrant and temperature sensitive. In addition, unlike the other genes, dpy-30 is required for the normal development of XO animals. Although dpy-30 mutations do not significantly affect the viability of XO animals, they do cause them to be developmentally delayed and to possess numerous morphological and behavioral abnormalities. Finally, dpy-30 mutations can dramatically influence the choice of sexual fate in animals with an ambiguous sexual identity, despite having no apparent effect on the sexual phenotype of otherwise wild-type animals. Paradoxically, depending on the genetic background, dpy-30 mutations cause either masculinization or feminization, thus revealing the complex regulatory relationship between the sex determination and dosage compensation processes. The novel phenotypes caused by dpy-30 mutations suggest that in addition to acting in the dosage compensation process, dpy-30 may play a more general role in the development of both XX and XO animals. PMID:7982580

  6. Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH)

    PubMed Central

    Chaudhuri, Roy R; Allen, Andrew G; Owen, Paul J; Shalom, Gil; Stone, Karl; Harrison, Marcus; Burgis, Timothy A; Lockyer, Michael; Garcia-Lara, Jorge; Foster, Simon J; Pleasance, Stephen J; Peters, Sarah E; Maskell, Duncan J; Charles, Ian G

    2009-01-01

    Background In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. Results We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. Conclusion We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens. PMID:19570206

  7. Role of Azotobacter vinelandii mucA and mucC gene products in alginate production.

    PubMed

    Núñez, C; León, R; Guzmán, J; Espín, G; Soberón-Chávez, G

    2000-12-01

    Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production. PMID:11073894

  8. Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.

    PubMed Central

    Sankar, P; Lee, J H; Shanmugam, K T

    1985-01-01

    Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome. PMID:3884595

  9. High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile

    PubMed Central

    Dembek, Marcin; Barquist, Lars; Boinett, Christine J.; Cain, Amy K.; Mayho, Matthew; Lawley, Trevor D.; Fagan, Robert P.

    2015-01-01

    ABSTRACT Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. PMID:25714712

  10. MEGA (Multiple Essential Genes Assembling) deletion and replacement method for genome reduction in Escherichia coli.

    PubMed

    Xue, Xiaoli; Wang, Tao; Jiang, Peng; Shao, Yangyang; Zhou, Min; Zhong, Li; Wu, Ronghai; Zhou, Jianting; Xia, Haiyang; Zhao, Guoping; Qin, Zhongjun

    2015-06-19

    Top-down reduction of the bacterial genome to construct desired chassis cells is important for synthetic biology. However, the current progress in the field of genome reduction is greatly hindered by indispensable life-essential genes that are interspersed throughout the chromosomal loci. Here, we described a new method designated as "MEGA (Multiple Essential Genes Assembling) deletion and replacement" that functions by assembling multiple essential genes in an E. coli-S. cerevisiae shuttle vector, removing targeted chromosomal regions containing essential and nonessential genes using a one-round deletion, and then integrating the cloned essential genes into the in situ chromosomal loci via I-SceI endonuclease cleavage. As a proof of concept, we separately generated three large deletions (80-205 kbp) in the E. coli MDS42 chromosome. We believe that the MEGA deletion and replacement method has potential to become widely used in large-scale genome reductions in other sequenced organisms in addition to E. coli. PMID:25494410

  11. Coenzyme Q regulates the expression of essential genes of the pathogen- and xenobiotic-associated defense pathway in C. elegans.

    PubMed

    Fischer, Alexandra; Niklowitz, Petra; Menke, Thomas; Dring, Frank

    2015-11-01

    Coenzyme Q (CoQ) is necessary for mitochondrial energy production and modulates the expression of genes that are important for inflammatory processes, growth and detoxification reactions. A cellular surveillance-activated detoxification and defenses (cSADDs) pathway has been recently identified in C. elegans. The down-regulation of the components of the cSADDs pathway initiates an aversion behavior of the nematode. Here we hypothesized that CoQ regulates genes of the cSADDs pathway. To verify this we generated CoQ-deficient worms ("CoQ-free") and performed whole-genome expression profiling. We found about 30% (120 genes) of the cSADDs pathway genes were differentially regulated under CoQ-deficient condition. Remarkably, 83% of these genes were down-regulated. The majority of the CoQ-sensitive cSADDs pathway genes encode for proteins involved in larval development (enrichment score (ES)=38.0, p=5.0E(-37)), aminoacyl-tRNA biosynthesis, proteasome function (ES 8.2, p=5.9E(-31)) and mitochondria function (ES 3.4, p=1.7E(-5)). 67% (80 genes) of these genes are categorized as lethal. Thus it is shown for the first time that CoQ regulates a substantial number of essential genes that function in the evolutionary conserved cellular surveillance-activated detoxification and defenses pathway in C. elegans. PMID:26566301

  12. Identification of the Physiological Gene Targets of the Essential Lytic Replicative Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein

    PubMed Central

    Verma, Dinesh; Li, Da-Jiang; Krueger, Brian; Renne, Rolf

    2014-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57-null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV-infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, quantitative PCR, Northern blotting, and rapid amplification of cDNA ends methods. Comparison of gene expression from a ΔORF57 KSHV recombinant, a rescued ΔORF57 KSHV recombinant, and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57-dependent genes, ORF57 regulation was promoter and polyadenylation signal independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified to critically require ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly, or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in the causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy. PMID:25410858

  13. Characteristics of Plant Essential Genes Allow for within- and between-Species Prediction of Lethal Mutant Phenotypes[OPEN

    PubMed Central

    Lloyd, John P.; Seddon, Alexander E.; Moghe, Gaurav D.; Simenc, Matthew C.; Shiu, Shin-Han

    2015-01-01

    Essential genes represent critical cellular components whose disruption results in lethality. Characteristics shared among essential genes have been uncovered in fungal and metazoan model systems. However, features associated with plant essential genes are largely unknown and the full set of essential genes remains to be discovered in any plant species. Here, we show that essential genes in Arabidopsis thaliana have distinct features useful for constructing within- and cross-species prediction models. Essential genes in A. thaliana are often single copy or derived from older duplications, highly and broadly expressed, slow evolving, and highly connected within molecular networks compared with genes with nonlethal mutant phenotypes. These gene features allowed the application of machine learning methods that predicted known lethal genes as well as an additional 1970 likely essential genes without documented phenotypes. Prediction models from A. thaliana could also be applied to predict Oryza sativa and Saccharomyces cerevisiae essential genes. Importantly, successful predictions drew upon many features, while any single feature was not sufficient. Our findings show that essential genes can be distinguished from genes with nonlethal phenotypes using features that are similar across kingdoms and indicate the possibility for translational application of our approach to species without extensive functional genomic and phenomic resources. PMID:26286535

  14. Essential gene disruptions reveal complex relationships between phenotypic robustness, pleiotropy, and fitness

    PubMed Central

    Bauer, Christopher R; Li, Shuang; Siegal, Mark L

    2015-01-01

    The concept of robustness in biology has gained much attention recently, but a mechanistic understanding of how genetic networks regulate phenotypic variation has remained elusive. One approach to understand the genetic architecture of variability has been to analyze dispensable gene deletions in model organisms; however, the most important genes cannot be deleted. Here, we have utilized two systems in yeast whereby essential genes have been altered to reduce expression. Using high-throughput microscopy and image analysis, we have characterized a large number of morphological phenotypes, and their associated variation, for the majority of essential genes in yeast. Our results indicate that phenotypic robustness is more highly dependent upon the expression of essential genes than on the presence of dispensable genes. Morphological robustness appears to be a general property of a genotype that is closely related to pleiotropy. While the fitness profile across a range of expression levels is idiosyncratic to each gene, the global pattern indicates that there is a window in which phenotypic variation can be released before fitness effects are observable. PMID:25609648

  15. A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes

    PubMed Central

    Pogoda, Madlen; Bosse, Jens B.; Conzelmann, Karl-Klaus; Koszinowski, Ulrich H.; Ruzsics, Zsolt

    2014-01-01

    Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses. PMID:24733555

  16. The ciliopathy gene Rpgrip1l is essential for hair follicle development.

    PubMed

    Chen, Jiang; Laclef, Christine; Moncayo, Alejandra; Snedecor, Elizabeth R; Yang, Ning; Li, Li; Takemaru, Ken-Ichi; Paus, Ralf; Schneider-Maunoury, Sylvie; Clark, Richard A

    2015-03-01

    The primary cilium is essential for skin morphogenesis through regulating the Notch, Wnt, and hedgehog signaling pathways. Prior studies on the functions of primary cilia in the skin were based on the investigations of genes that are essential for cilium formation. However, none of these ciliogenic genes has been linked to ciliopathy, a group of disorders caused by abnormal formation or function of cilia. To determine whether there is a genetic and molecular link between ciliopathies and skin morphogenesis, we investigated the role of RPGRIP1L, a gene mutated in Joubert (JBTS) and Meckel (MKS) syndromes, two severe forms of ciliopathy, in the context of skin development. We found that RPGRIP1L is essential for hair follicle morphogenesis. Specifically, disrupting the Rpgrip1l gene in mice resulted in reduced proliferation and differentiation of follicular keratinocytes, leading to hair follicle developmental defects. These defects were associated with significantly decreased primary cilium formation and attenuated hedgehog signaling. In contrast, we found that hair follicle induction and polarization and the development of interfollicular epidermis were unaffected. This study indicates that RPGRIP1L, a ciliopathy gene, is essential for hair follicle morphogenesis likely through regulating primary cilia formation and the hedgehog signaling pathway. PMID:25398052

  17. Atrial natriuretic peptide and aldosterone synthase gene in essential hypertension: a case-control study.

    PubMed

    Chandra, Sudhir; Saluja, Daman; Narang, Rajiv; Bhatia, Jagriti; Srivastava, Kamna

    2015-08-01

    The renin-angiotensin-aldosterone system (RAAS) and their candidate genes are principally involved in regulation of blood pressure through salt-water homeostasis. Atrial natriuretic peptide (ANP) and Aldosterone synthase (CYP11B2) are the important RAAS mediators, play a major role in hypertension through regulation of cardiorenal homeostasis and water-electrolytes balance, respectively. Present study reports the expression of ANP and CYP11B2 gene at mRNA and proteins levels in patients with essential hypertension in North Indian subjects. Gene expression at mRNA and protein levels was carried out by Real time PCR and Western blot, respectively. We found a significant down regulation in the ANP gene expression at mRNA (85%) and protein (72.6%) levels and significant increase in the CYP11B2 protein expression in patients as compared to controls. A significant increase in Serum creatinine (14.6%), Sodium (1.15%) and decrease in the Blood urea (8.18%) and Potassium (2.32%) levels were also observed among the patients group having higher expression (based on median delta-CT value) in comparison to the lower expression of CYP11B2 gene. Our results suggest that the down-regulation of ANP gene expression at mRNA and protein levels and up-regulated CYP11B2 protein expression levels may be correlated with the essential hypertension and could serve as circulating prognostic biomarkers for essential hypertension. PMID:25917967

  18. Experimental and Computational Assessment of Conditionally Essential Genes in Escherichia coli▿

    PubMed Central

    Joyce, Andrew R.; Reed, Jennifer L.; White, Aprilfawn; Edwards, Robert; Osterman, Andrei; Baba, Tomoya; Mori, Hirotada; Lesely, Scott A.; Palsson, Bernhard Ø.; Agarwalla, Sanjay

    2006-01-01

    Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium. Out of 3,888 single-deletion mutants tested, 119 mutants were unable to grow on glycerol minimal medium. These conditionally essential genes were then evaluated using a genome scale metabolic and transcriptional-regulatory model of E. coli, and it was found that the model made the correct prediction in ∼91% of the cases. The discrepancies between model predictions and experimental results were analyzed in detail to indicate where model improvements could be made or where the current literature lacks an explanation for the observed phenotypes. The identified set of essential genes and their model-based analysis indicates that our current understanding of the roles these essential genes play is relatively clear and complete. Furthermore, by analyzing the data set in terms of metabolic subsystems across multiple genomes, we can project which metabolic pathways are likely to play equally important roles in other organisms. Overall, this work establishes a paradigm that will drive model enhancement while simultaneously generating hypotheses that will ultimately lead to a better understanding of the organism. PMID:17012394

  19. Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes

    PubMed Central

    Le Breton, Yoann; Belew, Ashton T.; Valdes, Kayla M.; Islam, Emrul; Curry, Patrick; Tettelin, Hervé; Shirtliff, Mark E.; El-Sayed, Najib M.; McIver, Kevin S.

    2015-01-01

    Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci. PMID:25996237

  20. Use of selected essential oils to control aflatoxin contaminated stored cashew and detection of aflatoxin biosynthesis gene.

    PubMed

    Abd El-Aziz, Abeer R M; Mahmoud, Mohamed A; Al-Othman, Monira R; Al-Gahtani, Munirah F

    2015-01-01

    Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method. PMID:25705718

  1. Use of Selected Essential Oils to Control Aflatoxin Contaminated Stored Cashew and Detection of Aflatoxin Biosynthesis Gene

    PubMed Central

    Abd El-Aziz, Abeer R. M.; Mahmoud, Mohamed A.; Al-Othman, Monira R.; Al-Gahtani, Munirah F.

    2015-01-01

    Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method. PMID:25705718

  2. Functional requirements for bacteriophage growth: Gene essentiality and expression in Mycobacteriophage Giles

    PubMed Central

    Dedrick, Rebekah M.; Marinelli, Laura J.; Newton, Gerald L.; Pogliano, Kit; Pogliano, Joseph; Hatfull, Graham F.

    2013-01-01

    Summary Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions – such as virion proteins and repressors – cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny. PMID:23560716

  3. An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

    PubMed

    Kofoed, Megan; Milbury, Karissa L; Chiang, Jennifer H; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C

    2015-09-01

    Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. PMID:26175450

  4. An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes

    PubMed Central

    Kofoed, Megan; Milbury, Karissa L.; Chiang, Jennifer H.; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C.

    2015-01-01

    Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. PMID:26175450

  5. Food production & availability - Essential prerequisites for sustainable food security

    PubMed Central

    Swaminathan, M.S.; Bhavani, R.V.

    2013-01-01

    Food and nutrition security are intimately interconnected, since only a food based approach can help in overcoming malnutrition in an economically and socially sustainable manner. Food production provides the base for food security as it is a key determinant of food availability. This paper deals with different aspects of ensuring high productivity and production without associated ecological harm for ensuring adequate food availability. By mainstreaming ecological considerations in technology development and dissemination, we can enter an era of evergreen revolution and sustainable food and nutrition security. Public policy support is crucial for enabling this. PMID:24135188

  6. The zebrafish maternal-effect gene mission impossible encodes the DEAH-box helicase Dhx16 and is essential for the expression of downstream endodermal genes.

    PubMed

    Putiri, Emily; Pelegri, Francisco

    2011-05-15

    Early animal embryonic development requires maternal products that drive developmental processes prior to the activation of the zygotic genome at the mid-blastula transition. During and after this transition, maternal products may continue to act within incipient zygotic developmental programs. Mechanisms that control maternally-inherited products to spatially and temporally restrict developmental responses remain poorly understood, but necessarily depend on posttranscriptional regulation. We report the functional analysis and molecular identification of the zebrafish maternal-effect gene mission impossible (mis). Our studies suggest requirements for maternally-derived mis function in events that occur during gastrulation, including cell movement and the activation of some endodermal target genes. Cell transplantation experiments show that the cell movement defect is cell autonomous. Within the endoderm induction pathway, mis is not required for the activation of early zygotic genes, but is essential to implement nodal activity downstream of casanova/sox 32 but upstream of sox17 expression. Activation of nodal signaling in blastoderm explants shows that the requirement for mis function in endoderm gene induction is independent of the underlying yolk cell. Positional cloning of mis, including genetic rescue and complementation analysis, shows that it encodes the DEAH-box RNA helicase Dhx16, shown in other systems to act in RNA regulatory processes such as splicing and translational control. Analysis of a previously identified insertional dhx16 mutation shows that the zygotic component of this gene is also essential for embryonic viability. Our studies provide a striking example of the interweaving of maternal and zygotic genetic functions during the egg-to-embryo transition. Maternal RNA helicases have long been known to be involved in the development of the animal germ line, but our findings add to growing evidence that these factors may also control specific gene expression programs in somatic tissues. PMID:21396359

  7. The Zebrafish Maternal-effect Gene mission impossible Encodes the DEAH-box Helicase Dhx16 and is Essential for the Expression of Downstream Endodermal Genes

    PubMed Central

    Putiri, Emily; Pelegri, Francisco

    2011-01-01

    Early animal embryonic development requires maternal products that drive developmental processes prior to the activation of the zygotic genome at the mid-blastula transition. During and after this transition, maternal products may continue to act within incipient zygotic developmental programs. Mechanisms that control maternally-inherited products to spatially and temporally restrict developmental responses remain poorly understood, but necessarily depend on posttranscriptional regulation. We report the functional analysis and molecular identification of the zebrafish maternal-effect gene mission impossible (mis). Our studies suggest requirements for maternally-derived mis function in events that occur during gastrulation, including cell movement and the activation of some endodermal target genes. Cell transplantation experiments show that the cell movement defect is cell autonomous. Within the endoderm induction pathway, mis is not required for the activation of early zygotic genes, but is essential to implement nodal activity downstream of casanova/sox 32 but upstream of sox17 expression. Activation of nodal signaling in blastoderm explants shows that the requirement for mis function in endoderm gene induction is independent of the underlying yolk cell. Positional cloning of mis, including genetic rescue and complementation analysis, shows that it encodes the DEAH-box RNA helicase Dhx16, shown in other systems to act in RNA regulatory processes such as splicing and translational control. Analysis of a previously identified insertional dhx16 mutation shows that the zygotic component of this gene is also essential for embryonic viability. Our studies provide a striking example of the interweaving of maternal and zygotic genetic functions during the egg-to-embryo transition. Maternal RNA helicases have long been known to be involved in the development of the animal germ line, but our findings add to growing evidence that these factors may also control specific gene expression programs in somatic tissues. PMID:21396359

  8. DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements

    PubMed Central

    Luo, Hao; Lin, Yan; Gao, Feng; Zhang, Chun-Ting; Zhang, Ren

    2014-01-01

    The combination of high-density transposon-mediated mutagenesis and high-throughput sequencing has led to significant advancements in research on essential genes, resulting in a dramatic increase in the number of identified prokaryotic essential genes under diverse conditions and a revised essential-gene concept that includes all essential genomic elements, rather than focusing on protein-coding genes only. DEG 10, a new release of the Database of Essential Genes (available at http://www.essentialgene.org), has been developed to accommodate these quantitative and qualitative advancements. In addition to increasing the number of bacterial and archaeal essential genes determined by genome-wide gene essentiality screens, DEG 10 also harbors essential noncoding RNAs, promoters, regulatory sequences and replication origins. These essential genomic elements are determined not only in vitro, but also in vivo, under diverse conditions including those for survival, pathogenesis and antibiotic resistance. We have developed customizable BLAST tools that allow users to perform species- and experiment-specific BLAST searches for a single gene, a list of genes, annotated or unannotated genomes. Therefore, DEG 10 includes essential genomic elements under different conditions in three domains of life, with customizable BLAST tools. PMID:24243843

  9. MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

    PubMed Central

    Luca, Francis C.; Winey, Mark

    1998-01-01

    Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1–1 mutation. Strains containing mob2Δ also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication. PMID:9436989

  10. Cell-specific expression of artificial microRNAs targeting essential genes exhibit potent antitumor effect on hepatocellular carcinoma cells

    PubMed Central

    Mao, Chenyu; Liu, Hao; Chen, Ping; Ye, Jingjia; Teng, Lisong; Jia, Zhenyu; Cao, Jiang

    2015-01-01

    To achieve specific and potent antitumor effect of hepatocyte carcinoma cells, replication defective adenoviral vectors, namely rAd/AFP-amiRG, rAd/AFP-amiRE and rAd/AFP-amiRP, were constructed which were armed with artificial microRNAs (amiRs) targeting essential functional genes glyceraldehyde-3-phosphate dehydrogenase, eukaryotic translation initiation factor 4E and DNA polymerase α respectively under the control of a recombinant promoter comprised of human α-fetoprotein enhancer and basal promoter. The AFP enhancer/promoter showed specific high transcription activity in AFP-positive HCC cells Hep3B, HepG2 and SMMC7721, while low in AFP-negative cell Bcap37. All artificial microRNAs exhibited efficient knockdown of target genes. Decreased ATP production and protein synthesis was observed in rAd/AFP-amiRG and rAd/AFP-amiRE treated HCC cells. All three recombinant adenoviruses showed efficient blockage of cell cycle progression and significant suppression of HCC cells in vitro. In nude mice model bearing Hep3B xenograft, administration of rAd/AFP-amiRG showed potent antitumor effect. The strategy of tumor-specific knockdown of genes essential for cell survival and proliferation may suggest a novel promising approach for HCC gene therapy. PMID:25691059

  11. Identification of genes and gene products necessary for bacterial bioluminescence.

    PubMed Central

    Engebrecht, J; Silverman, M

    1984-01-01

    Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation analysis and the minicell programing experiments. These genes, in the order of their position on a linear map, and the apparent molecular weights of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli containing mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light production and luxR and luxI encode regulatory functions. Images PMID:6377310

  12. Essential Gene Discovery in the Basidiomycete Cryptococcus neoformans for Antifungal Drug Target Prioritization

    PubMed Central

    Ianiri, Giuseppe

    2015-01-01

    ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in both Cryptococcus and ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity. PMID:25827419

  13. Effective identification of essential proteins based on priori knowledge, network topology and gene expressions.

    PubMed

    Li, Min; Zheng, Ruiqing; Zhang, Hanhui; Wang, Jianxin; Pan, Yi

    2014-06-01

    Identification of essential proteins is very important for understanding the minimal requirements for cellular life and also necessary for a series of practical applications, such as drug design. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which makes it possible to detect proteins' essentialities from the network level. Considering that most species already have a number of known essential proteins, we proposed a new priori knowledge-based scheme to discover new essential proteins from protein interaction networks. Based on the new scheme, two essential protein discovery algorithms, CPPK and CEPPK, were developed. CPPK predicts new essential proteins based on network topology and CEPPK detects new essential proteins by integrating network topology and gene expressions. The performances of CPPK and CEPPK were validated based on the protein interaction network of Saccharomyces cerevisiae. The experimental results showed that the priori knowledge of known essential proteins was effective for improving the predicted precision. The predicted precisions of CPPK and CEPPK clearly exceeded that of the other 10 previously proposed essential protein discovery methods: Degree Centrality (DC), Betweenness Centrality (BC), Closeness Centrality (CC), Subgraph Centrality (SC), Eigenvector Centrality (EC), Information Centrality (IC), Bottle Neck (BN), Density of Maximum Neighborhood Component (DMNC), Local Average Connectivity-based method (LAC), and Network Centrality (NC). Especially, CPPK achieved 40% improvement in precision over BC, CC, SC, EC, and BN, and CEPPK performed even better. CEPPK was also compared to four other methods (EPC, ORFL, PeC, and CoEWC) which were not node centralities and CEPPK was showed to achieve the best results. PMID:24565748

  14. Human AZU-1 gene, variants thereof and expressed gene products

    DOEpatents

    Chen, Huei-Mei; Bissell, Mina

    2004-06-22

    A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

  15. The NGATHA Distal Organ Development Genes Are Essential for Style Specification in Arabidopsis[W

    PubMed Central

    Alvarez, John Paul; Goldshmidt, Alexander; Efroni, Idan; Bowman, John L.; Eshed, Yuval

    2009-01-01

    Floral organ identities are specified by a few transcription factors that act as master regulators. Subsequently, specification of organ axes programs the distribution of distinct tissue types within the organs that themselves develop unique identities. The C-class, AGAMOUS-clade MADS box genes are primary promoters of the gynoecium, which is divided into a distal style and a subtending ovary along the apical-basal axis. We show that members of a clade of B3 domain transcription factors, NGATHA1 (NGA1) to NGA4, are expressed distally in all lateral organs, and all four have a redundant and essential role in style development. Loss of all four genes results in gynoecia where style is replaced by valve-like projections and a reduction in style-specific SHATTERPROOF1 (SHP1) expression. In agreement, floral misexpression of NGA1 promotes ectopic style and SHP1 expression. STYLISH1, an auxin biosynthesis inducer, conditionally activated NGA genes, which in turn promoted distal expression of other STY genes in a putative positive feedback loop. Inhibited auxin transport or lack of YABBY1 gene activities resulted in a basally expanded style domain and broader expression of NGA genes. We speculate that early gynoecium factors delimit NGA gene response to an auxin-based signal, elicited by STY gene activity, to restrict the activation of style program to a late and distal carpel domain. PMID:19435933

  16. Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

    PubMed Central

    Budziszewski, G J; Lewis, S P; Glover, L W; Reineke, J; Jones, G; Ziemnik, L S; Lonowski, J; Nyfeler, B; Aux, G; Zhou, Q; McElver, J; Patton, D A; Martienssen, R; Grossniklaus, U; Ma, H; Law, M; Levin, J Z

    2001-01-01

    We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype. PMID:11779813

  17. Endogenous hydrogen sulfide production is essential for dietary restriction benefits.

    PubMed

    Hine, Christopher; Harputlugil, Eylul; Zhang, Yue; Ruckenstuhl, Christoph; Lee, Byung Cheon; Brace, Lear; Longchamp, Alban; Treviño-Villarreal, Jose H; Mejia, Pedro; Ozaki, C Keith; Wang, Rui; Gladyshev, Vadim N; Madeo, Frank; Mair, William B; Mitchell, James R

    2015-01-15

    Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. PAPERFLICK: PMID:25542313

  18. Endogenous Hydrogen Sulfide Production Is Essential for Dietary Restriction Benefits

    PubMed Central

    Hine, Christopher; Harputlugil, Eylul; Zhang, Yue; Ruckenstuhl, Christoph; Lee, Byung Cheon; Brace, Lear; Longchamp, Alban; Trevino-Villarreal, Jose H.; Mejia, Pedro; Ozaki, C. Keith; Wang, Rui; Gladyshev, Vadim N.; Madeo, Frank; Mair, William B.; Mitchell, James R.

    2014-01-01

    Summary Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine ?-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a novel mediator of DR benefits with broad implications for clinical translation. PMID:25542313

  19. Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene

    SciTech Connect

    Bohr, V.A.; Okumoto, D.S.; Hanawalt, P.C.

    1986-06-01

    The survival of UV-irradiated mammalian cells is not necessarily correlated with their overall capacity to carry out DNA repair. Human cells typically remove 80% of the pyrimidine dimers produced by a UV dose of 5 J/m2 within 24 hr. In contrast, a Chinese hamster ovary (CHO) cell line survives UV irradiation equally well while removing only 15% of the dimers. Using a newly developed technique to measure dimer frequencies in single-copy specific sequences, we find that the CHO cells remove 70% of the dimers from the essential dihydrofolate reductase (DHFR) gene but only 20% from sequences located 30 kilobases or more upstream from the 5' end of the gene in a 24-hr period. Repair-deficient human cells from xeroderma pigmentosum complementation group C (XPC) are similar to the CHO cells in overall repair levels, but they are extremely sensitive to killing by UV irradiation. In the XPC cells, we find little or no repair in the DHFR gene; in contrast, in normal human fibroblasts and epidermal keratinocytes, greater than 80% of the dimers induced in the gene by 20 J/m2 are removed in 24 hr. Since the CHO and normal human cells exhibit similar UV resistance, much higher than that of XPC cells, our findings suggest a correlation between efficient repair of essential genes and resistance to DNA-damaging agents such as UV light.

  20. An essential gene of Saccharomyces cerevisiae coding for an actin-related protein.

    PubMed Central

    Harata, M; Karwan, A; Wintersberger, U

    1994-01-01

    Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT1 (coding for the filament actin), and a so-called actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (M(r) = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophilic [corrected] segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3. Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in budding yeast that obviously provide different essential functions for survival. Images PMID:8058791

  1. Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene.

    PubMed Central

    Bohr, V A; Okumoto, D S; Hanawalt, P C

    1986-01-01

    The survival of UV-irradiated mammalian cells is not necessarily correlated with their overall capacity to carry out DNA repair. Human cells typically remove 80% of the pyrimidine dimers produced by a UV dose of 5 J/m2 within 24 hr. In contrast, a Chinese hamster ovary (CHO) cell line survives UV irradiation equally well while removing only 15% of the dimers. Using a newly developed technique to measure dimer frequencies in single-copy specific sequences, we find that the CHO cells remove 70% of the dimers from the essential dihydrofolate reductase (DHFR) gene but only 20% from sequences located 30 kilobases or more upstream from the 5' end of the gene in a 24-hr period. Repair-deficient human cells from xeroderma pigmentosum complementation group C (XPC) are similar to the CHO cells in overall repair levels, but they are extremely sensitive to killing by UV irradiation. In the XPC cells, we find little or no repair in the DHFR gene; in contrast, in normal human fibroblasts and epidermal keratinocytes, greater than 80% of the dimers induced in the gene by 20 J/m2 are removed in 24 hr. Since the CHO and normal human cells exhibit similar UV resistance, much higher than that of XPC cells, our findings suggest a correlation between efficient repair of essential genes and resistance to DNA-damaging agents such as UV light. Images PMID:3459159

  2. Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production

    PubMed Central

    2014-01-01

    Background The soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei. Results We have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes. Conclusions Transcriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression. PMID:24472375

  3. Selection of recombinant MVA by rescue of the essential D4R gene

    PubMed Central

    2011-01-01

    Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant. PMID:22152060

  4. In vivo and in silico determination of essential genes of Campylobacter jejuni.

    TOXLINE Toxicology Bibliographic Information

    Metris A; Reuter M; Gaskin DJ; Baranyi J; van Vliet AH

    2011-01-01

    BACKGROUND: In the United Kingdom, the thermophilic Campylobacter species C. jejuni and C. coli are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barr syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate C. jejuni and C. coli from the food chain.RESULTS: A metabolic model of C. jejuni was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium Helicobacter pylori, and extensive literature mining. Using this model, we have used in silico Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this in silico approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published Campylobacter protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy.CONCLUSIONS: We have constructed the first curated metabolic model for the food-borne pathogen Campylobacter jejuni and have presented the resulting metabolic insights. We have shown that the combination of in silico and in vivo approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to C. jejuni, which are all potential novel Campylobacter intervention targets.

  5. In vivo and in silico determination of essential genes of Campylobacter jejuni

    PubMed Central

    2011-01-01

    Background In the United Kingdom, the thermophilic Campylobacter species C. jejuni and C. coli are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate C. jejuni and C. coli from the food chain. Results A metabolic model of C. jejuni was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium Helicobacter pylori, and extensive literature mining. Using this model, we have used in silico Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this in silico approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published Campylobacter protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy. Conclusions We have constructed the first curated metabolic model for the food-borne pathogen Campylobacter jejuni and have presented the resulting metabolic insights. We have shown that the combination of in silico and in vivo approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to C. jejuni, which are all potential novel Campylobacter intervention targets. PMID:22044676

  6. Association between the MTHFR C677T gene polymorphism and essential hypertension in South West Cameroon.

    PubMed

    Ghogomu, S M; Ngolle, N E; Mouliom, R N; Asa, B F

    2016-01-01

    The association of the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and essential hypertension has been reported but with controversial results in diverse populations in Asia and Europe, thereby suggesting a dependency on ethnicity. The aim of this study was to investigate the association between the MTHFR C677T polymorphism and essential hypertension in a Cameroonian population (Bantu ethnic group) of the South West Region. Analysis of anthropometric and biochemical data in hypertensive and normotensive subjects revealed that age, systolic blood pressure, diastolic blood pressure, low-density lipoprotein cholesterol, serum total cholesterol, and triglycerides are independent risk factors for essential hypertension. Substitution of thymine for cytosine at position 667 of the MTHFR gene was determined by polymerase chain reaction-restriction fragment length polymorphism. Genotype frequencies were found to be 7.3% CC, 58.5% CT, and 34.1% TT for hypertensive subjects compared to 90.0% CC, 10.0% CT, and 0.0% TT for normotensives. Allele frequencies were obtained as 36.6% C and 63.4% T for hypertensive subjects and 95.0% C and 5.0% T for normotensive subjects. These results reveal that the T allele predisposes individuals to hypertension. Therefore, there is an association between variants of the MTHFR gene and hypertension in Cameroonian patients from the South West region. PMID:27051013

  7. Locating essential Escherichia coli genes by using mini-Tn10 transposons: the pdxJ operon.

    PubMed Central

    Takiff, H E; Baker, T; Copeland, T; Chen, S M; Court, D L

    1992-01-01

    The mini-Tn10 transposon (delta 16 delta 17Tn10) confers tetracycline resistance. When inserted between a gene and its promoter, it blocks transcription and prevents expression of that gene. Tetracycline in the medium induces divergent transcription of the tetA and tetR genes within the transposon, and this transcription extends beyond the transposon in both directions into the bacterial genes. If the mini-Tn10 inserts between an essential bacterial gene and its promoter, the insertion mutation can cause conditional growth which is dependent on the presence of tetracycline. Two essential genes in adjacent operons of Escherichia coli have been detected by screening for tetracycline dependence among tetracycline-resistant insertion mutants. These essential genes are the era gene in the rnc operon and the dpj gene in the adjacent pdxJ operon. The pdxJ operon has not been described previously. It consists of two genes, pdxJ and dpj. Whereas the dpj gene is essential for E. coli growth in all media tested, pdxJ is not essential. The pdxJ gene encodes a protein required in the biosynthesis of pyridoxine (vitamin B6). Images PMID:1537799

  8. Identification of a New Gene Essential for Germination of Bacillus subtilis Spores with Ca2+-Dipicolinate

    PubMed Central

    Ragkousi, Katerina; Eichenberger, Patrick; van Ooij, Christiaan; Setlow, Peter

    2003-01-01

    Bacillus subtilis spores can germinate with a 1:1 chelate of Ca2+ and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca2+-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca2+-DPA. The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is σE dependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ. PMID:12644503

  9. Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate.

    PubMed

    Ragkousi, Katerina; Eichenberger, Patrick; van Ooij, Christiaan; Setlow, Peter

    2003-04-01

    Bacillus subtilis spores can germinate with a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca(2+)-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca(2+)-DPA. The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is sigma(E) dependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ. PMID:12644503

  10. Female Inheritance of Malarial lap Genes Is Essential for Mosquito Transmission

    PubMed Central

    Mendoza, Jacqui; Tewari, Rita; Stanway, Rebecca R; Sinden, Robert E

    2007-01-01

    Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite–mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Δpblap sporozoites, but not if the individual Δpblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Δpbs47) and male-deficient (Δpbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Δpblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages. PMID:17335349

  11. Identification of an Essential Gene of Listeria monocytogenes Involved in Teichoic Acid Biogenesis

    PubMed Central

    Dubail, Iharilalao; Bigot, Armelle; Lazarevic, Vladimir; Soldo, Blazenka; Euphrasie, Daniel; Dupuis, Marion; Charbit, Alain

    2006-01-01

    Listeria monocytogenes is a facultative intracellular gram-positive bacterium responsible for severe opportunistic infections in humans and animals. We had previously identified a gene encoding a putative UDP-N-acetylglucosamine 2-epimerase, a precursor of the teichoic acid linkage unit, in the genome of L monocytogenes strain EGD-e. This gene, now designated lmo2537, encodes a protein that shares 62% identity with the cognate epimerase MnaA of Bacillus subtilis and 55% identity with Cap5P of Staphylococcus aureus. Here, we addressed the role of lmo2537 in L. monocytogenes pathogenesis by constructing a conditional knockout mutant. The data presented here demonstrate that lmo2537 is an essential gene of L. monocytogenes that is involved in teichoic acid biogenesis. In vivo, the conditional mutant is very rapidly eliminated from the target organs of infected mice and thus is totally avirulent. PMID:16952950

  12. 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

  13. A CRISPR-based screen identifies genes essential for West Nile virus-induced cell death

    PubMed Central

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N.; Wu, Haoquan

    2015-01-01

    Summary West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. PMID:26190106

  14. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

    PubMed

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

    2015-07-28

    West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. PMID:26190106

  15. Genes, patents, and product development.

    PubMed

    Eisenberg, R S

    1992-08-14

    In the past year, the National Institutes of Health (NIH) has filed patent applications on more than 2750 partial complementary DNA sequences of unknown function. The rationale for the filings--that patent protection may be necessary to ensure that private firms are willing to invest in developing related products--rests on two premises: first, that NIH may obtain patent rights that will offer effective product monopolies to licensee firms, and second, that unless NIH obtains these rights now, firms will be unable to obtain a comparable degree of exclusivity by other means, such as by obtaining patents on their own subsequent innovations. Neither premise is clearly wrong, although both are subject to doubt in view of statements from industry representatives that the NIH patenting strategy will deter rather than promote product development. PMID:1502556

  16. Comparative Genomics Analysis of Mycobacterium ulcerans for the Identification of Putative Essential Genes and Therapeutic Candidates

    PubMed Central

    Tahir, Shifa; Tong, Yigang

    2012-01-01

    Mycobacterium ulcerans, the causative agent of Buruli ulcer, is the third most common mycobacterial disease after tuberculosis and leprosy. The present treatment options are limited and emergence of treatment resistant isolates represents a serious concern and a need for better therapeutics. Conventional drug discovery methods are time consuming and labor-intensive. Unfortunately, the slow growing nature of M. ulcerans in experimental conditions is also a barrier for drug discovery and development. In contrast, recent advancements in complete genome sequencing, in combination with cheminformatics and computational biology, represent an attractive alternative approach for the identification of therapeutic candidates worthy of experimental research. A computational, comparative genomics workflow was defined for the identification of novel therapeutic candidates against M. ulcerans, with the aim that a selected target should be essential to the pathogen, and have no homology in the human host. Initially, a total of 424 genes were predicted as essential from the M. ulcerans genome, via homology searching of essential genome content from 20 different bacteria. Metabolic pathway analysis showed that the most essential genes are associated with carbohydrate and amino acid metabolism. Among these, 236 proteins were identified as non-host and essential, and could serve as potential drug and vaccine candidates. Several drug target prioritization parameters including druggability were also calculated. Enzymes from several pathways are discussed as potential drug targets, including those from cell wall synthesis, thiamine biosynthesis, protein biosynthesis, and histidine biosynthesis. It is expected that our data will facilitate selection of M. ulcerans proteins for successful entry into drug design pipelines. PMID:22912793

  17. Non-essential genes form the hubs of genome scale protein function and environmental gene expression networks in Salmonella enterica serovar Typhimurium

    PubMed Central

    2013-01-01

    Background Salmonella Typhimurium is an important pathogen of human and animals. It shows a broad growth range and survives in harsh conditions. The aim of this study was to analyze transcriptional responses to a number of growth and stress conditions as well as the relationship of metabolic pathways and/or cell functions at the genome-scale-level by network analysis, and further to explore whether highly connected genes (hubs) in these networks were essential for growth, stress adaptation and virulence. Results De novo generated as well as published transcriptional data for 425 selected genes under a number of growth and stress conditions were used to construct a bipartite network connecting culture conditions and significantly regulated genes (transcriptional network). Also, a genome scale network was constructed for strain LT2. The latter connected genes with metabolic pathways and cellular functions. Both networks were shown to belong to the family of scale-free networks characterized by the presence of highly connected nodes or hubs which are genes whose transcription is regulated when responding to many of the assayed culture conditions or genes encoding products involved in a high number of metabolic pathways and cell functions. The five genes with most connections in the transcriptional network (wraB, ygaU, uspA, cbpA and osmC) and in the genome scale network (ychN, siiF (STM4262), yajD, ybeB and dcoC) were selected for mutations, however mutagenesis of ygaU and ybeB proved unsuccessful. No difference between mutants and the wild type strain was observed during growth at unfavorable temperatures, pH values, NaCl concentrations and in the presence of H2O2. Eight mutants were evaluated for virulence in C57/BL6 mice and none differed from the wild type strain. Notably, however, deviations of phenotypes with respect to the wild type were observed when combinations of these genes were deleted. Conclusion Network analysis revealed the presence of hubs in both transcriptional and functional networks of S. Typhimurium. Hubs theoretically confer higher resistance to random mutation but a greater susceptibility to directed attacks, however, we found that genes that formed hubs were dispensable for growth, stress adaptation and virulence, suggesting that evolution favors non-essential genes as main connectors in cellular networks. PMID:24345035

  18. Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori?

    PubMed Central

    Boneca, Ivo G.; Ecobichon, Chantal; Chaput, Catherine; Mathieu, Aurlie; Guadagnini, Stphanie; Prvost, Marie-Christine; Colland, Frdric; Labigne, Agns; de Reuse, Hilde

    2008-01-01

    The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring ?-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-?-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds. PMID:18245237

  19. The Epstein-Barr virus BRRF2 gene product is involved in viral progeny production.

    PubMed

    Watanabe, Takahiro; Tsuruoka, Miyuki; Narita, Yohei; Katsuya, Ryotaro; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

    2015-10-01

    The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, and occasionally switches from the latent state to the lytic cycle. In this report, we identified and examined the role of a lytic gene, BRRF2. We first prepared an antibody against BRRF2 and identified the gene product as a viral lytic protein expressed in B95-8 cells with late kinetics. Immunofluorescence revealed that BRRF2 localized in the cytoplasm of cells during the lytic phase. We also found that BRRF2 protein was phosphorylated in lytic cells, but the only viral protein kinase, BGLF4, was not involved in the phosphorylation. Knockout EBV and a repaired strain were then prepared, and we found that BRRF2 disruption did not affect viral gene expression and DNA replication, but decreased virus production. These results demonstrated that BRRF2 is involved in production of infectious progeny, although it is not essential for lytic replication. PMID:26057150

  20. Expression of Heat Shock Protein 70 Gene and Its Correlation with Inflammatory Markers in Essential Hypertension

    PubMed Central

    Srivastava, Kamna; Narang, Rajiv; Bhatia, Jagriti; Saluja, Daman

    2016-01-01

    Objectives Hypertension is characterized by systemic high blood pressure and is the most common and important risk factor for the development of cardiovascular diseases. Studies have shown that the circulating levels of certain inflammatory markers such as tumor necrosis factor-alpha (TNF-alpha), interlukin-6 (IL-6), c-reactive protein (CRP), and tumor suppressor protein-53 (p53) are upregulated and are independently associated with essential hypertension. However, mechanism of increase in the levels of HSP70 protein is not clear. No such studies are reported in the blood circulation of patients with essential hypertension. In the present study, we investigated the expression of circulating HSP70 at mRNA and protein levels and its relationship with other inflammatory markers in patients with essential hypertension. Materials and Methods We recruited 132 patients with essential hypertension and 132 normal controls from similar socio-economic-geographical background. The expression of HSP70 at mRNA levels was determined by Real Time PCR and at protein levels by indirect Elisa and Western Blot techniques. Results We found a significantly higher expression of HSP70 gene expression (approximately 6.45 fold, P < 0.0001) in hypertensive patients as compared to healthy controls. A significant difference (P < 0.0001) in the protein expression of HSP70 was also observed in plasma of patients as compared to that of controls. Conclusion Higher expression of HSP70 is positively correlated with inflammatory markers in patients with essential hypertension and this correlation could play an important role in essential hypertension. PMID:26989902

  1. Neural stem cell transcriptional networks highlight genes essential for nervous system development

    PubMed Central

    Southall, Tony D; Brand, Andrea H

    2009-01-01

    Neural stem cells must strike a balance between self-renewal and multipotency, and differentiation. Identification of the transcriptional networks regulating stem cell division is an essential step in understanding how this balance is achieved. We have shown that the homeodomain transcription factor, Prospero, acts to repress self-renewal and promote differentiation. Among its targets are three neural stem cell transcription factors, Asense, Deadpan and Snail, of which Asense and Deadpan are repressed by Prospero. Here, we identify the targets of these three factors throughout the genome. We find a large overlap in their target genes, and indeed with the targets of Prospero, with 245 genomic loci bound by all factors. Many of the genes have been implicated in vertebrate stem cell self-renewal, suggesting that this core set of genes is crucial in the switch between self-renewal and differentiation. We also show that multiply bound loci are enriched for genes previously linked to nervous system phenotypes, thereby providing a shortcut to identifying genes important for nervous system development. PMID:19851284

  2. Metazoan Remaining Genes for Essential Amino Acid Biosynthesis: Sequence Conservation and Evolutionary Analyses

    PubMed Central

    Costa, Igor R.; Thompson, Julie D.; Ortega, José Miguel; Prosdocimi, Francisco

    2014-01-01

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals. PMID:25545100

  3. kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila.

    PubMed Central

    Roman, G; He, J; Davis, R L

    2000-01-01

    The kurtz gene encodes a novel nonvisual arrestin. krz is located at the most-distal end of the chromosome 3R, the third gene in from the telomere. krz is expressed throughout development. During early embryogenesis, krz is expressed ubiquitously and later is localized to the central nervous system, maxillary cirri, and antennal sensory organs. In late third instar larvae, krz message is detected in the fat bodies, the ventral portion of the thoracic-abdominal ganglia, the deuterocerebrum, the eye-antennal imaginal disc, and the wing imaginal disc. The krz(1) mutation contains a P-element insertion within the only intron of this gene and results in a severe reduction of function. Mutations in krz have a broad lethal phase extending from late embryogenesis to the third larval instar. The fat bodies of krz(1) larva precociously dissociate during the midthird instar. krz(1) is a type 1 melanotic tumor gene; the fat body is the primary site of melanotic tumor formation during the third instar. We have functionally rescued these phenotypes with both genomic and cDNA transgenes. Importantly, the expression of a full-length krz cDNA within the CNS rescues the krz(1) lethality. These experiments establish the krz nonvisual arrestin as an essential neural gene in Drosophila. PMID:10880488

  4. Essential, Overlapping and Redundant Roles of the Drosophila Protein Phosphatase 1α and 1β Genes

    PubMed Central

    Kirchner, Jasmin; Gross, Sascha; Bennett, Daimark; Alphey, Luke

    2007-01-01

    Protein serine/threonine phosphatase type 1 (PP1) has been found in all eukaryotes examined to date and is involved in the regulation of many cellular functions, including glycogen metabolism, muscle contraction, and mitosis. In Drosophila, four genes code for the catalytic subunit of PP1 (PP1c), three of which belong to the PP1α subtype. PP1β9C (flapwing) encodes the fourth PP1c gene and has a specific and nonredundant function as a nonmuscle myosin phosphatase. PP1α87B is the major form and contributes ∼80% of the total PP1 activity. We describe the first mutant alleles of PP1α96A and show that PP1α96A is not an essential gene, but seems to have a function in the regulation of nonmuscle myosin. We show that overexpression of the PP1α isozymes does not rescue semilethal PP1β9C mutants, whereas overexpression of either PP1α96A or PP1β9C does rescue a lethal PP1α87B mutant combination, showing that the lethality is due to a quantitative reduction in the level of PP1c. Overexpression of PP1β9C does not rescue a PP1α87B, PP1α96A double mutant, suggesting an essential PP1α-specific function in Drosophila. PMID:17513890

  5. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    PubMed

    Becherel, Olivier J; Yeo, Abrey J; Stellati, Alissa; Heng, Evelyn Y H; Luff, John; Suraweera, Amila M; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F

    2013-04-01

    Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome. PMID:23593030

  6. Essential genes in the infection model of Pseudomonas aeruginosa-PCR-based signature-tagged mutagenesis.

    PubMed

    Kukavica-Ibrulj, Irena; Levesque, Roger C

    2015-01-01

    PCR-based signature tagged mutagenesis is an "en masse" screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based STM applied to Pseudomonas aeruginosa permitted the identification of genes essential for in vivo maintenance by transposon insertion and negative selection in a mixed population of bacterial mutants. The innovative adaptations and refinement of the technology presented here with P. aeruginosa STM mutants selected in the rat model of chronic lung infection have given critical information about genes essential for causing a chronic infection and a wealth of information about biological processes in vivo. The additional use of competitive index analysis for measurement of the level of virulence in vivo, microarray-based screening of selected prioritized STM mutants coupled to metabolomics analysis can now be attempted systematically on a genomic scale. PCR-based STM and combined whole-genome methods can also be applied to any organism having selectable phenotypes for screening. PMID:25636615

  7. Senataxin Plays an Essential Role with DNA Damage Response Proteins in Meiotic Recombination and Gene Silencing

    PubMed Central

    Becherel, Olivier J.; Yeo, Abrey J.; Stellati, Alissa; Heng, Evelyn Y. H.; Luff, John; Suraweera, Amila M.; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F.

    2013-01-01

    Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx−/− revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome. PMID:23593030

  8. An Arabidopsis tissue-specific RNAi method for studying genes essential to mitosis.

    PubMed

    Burgos-Rivera, Brunilís; Dawe, R Kelly

    2012-01-01

    A large fraction of the genes in plants can be considered essential in the sense that when absent the plant fails to develop past the first few cell divisions. The fact that angiosperms pass through a haploid gametophyte stage can make it challenging to propagate such mutants even in the heterozygous condition. Here we describe a tissue-specific RNAi method that allows us to visualize cell division phenotypes in petals, which are large dispensable organs. Portions of the APETALA (AP3) and PISTILLATA (PI) promoters confer early petal-specific expression. We show that when either promoter is used to drive the expression of a beta-glucuronidase (GUS) RNAi transgene in plants uniformly expressing GUS, GUS expression is knocked down specifically in petals. We further tested the system by targeting the essential kinetochore protein CENPC and two different components of the Spindle Assembly Checkpoint (MAD2 and BUBR1). Plant lines expressing petal-specific RNAi hairpins targeting these genes exhibited an array of petal phenotypes. Cytological analyses of the affected flower buds confirmed that CENPC knockdown causes cell cycle arrest but provided no evidence that either MAD2 or BUBR1 are required for mitosis (although both genes are required for petal growth by this assay). A key benefit of the petal-specific RNAi method is that the phenotypes are not expressed in the lineages leading to germ cells, and the phenotypes are faithfully transmitted for at least four generations despite their pronounced effects on growth. PMID:23236491

  9. Maize Y9 Encodes a Product Essential for 15-cis-ζ-Carotene Isomerization1[OA

    PubMed Central

    Li, Faqiang; Murillo, Christina; Wurtzel, Eleanore T.

    2007-01-01

    Carotenoids are a diverse group of pigments found in plants, fungi, and bacteria. They serve essential functions in plants and provide health benefits for humans and animals. In plants, it was thought that conversion of the C40 carotenoid backbone, 15-cis-phytoene, to all-trans-lycopene, the geometrical isomer required by downstream enzymes, required two desaturases (phytoene desaturase and ζ-carotene desaturase [ZDS]) plus a carotene isomerase (CRTISO), in addition to light-mediated photoisomerization of the 15-cis-double bond; bacteria employ only a single enzyme, CRTI. Characterization of the maize (Zea mays) pale yellow9 (y9) locus has brought to light a new isomerase required in plant carotenoid biosynthesis. We report that maize Y9 encodes a factor required for isomerase activity upstream of CRTISO, which we term Z-ISO, an activity that catalyzes the cis- to trans-conversion of the 15-cis-bond in 9,15,9′-tri-cis-ζ-carotene, the product of phytoene desaturase, to form 9,9′-di-cis-ζ-carotene, the substrate of ZDS. We show that recessive y9 alleles condition accumulation of 9,15,9′-tri-cis-ζ-carotene in dark tissues, such as roots and etiolated leaves, in contrast to accumulation of 9,9′-di-cis-ζ-carotene in a ZDS mutant, viviparous9. We also identify a locus in Euglena gracilis, which is similarly required for Z-ISO activity. These data, taken together with the geometrical isomer substrate requirement of ZDS in evolutionarily distant plants, suggest that Z-ISO activity is not unique to maize, but will be found in all higher plants. Further analysis of this new gene-controlled step is critical to understanding regulation of this essential biosynthetic pathway. PMID:17434985

  10. PhenoM: a database of morphological phenotypes caused by mutation of essential genes in Saccharomyces cerevisiae.

    PubMed

    Jin, Ke; Li, Jingjing; Vizeacoumar, Frederick S; Li, Zhijian; Min, Renqiang; Zamparo, Lee; Vizeacoumar, Franco J; Datti, Alessandro; Andrews, Brenda; Boone, Charles; Zhang, Zhaolei

    2012-01-01

    About one-fifth of the genes in the budding yeast are essential for haploid viability and cannot be functionally assessed using standard genetic approaches such as gene deletion. To facilitate genetic analysis of essential genes, we and others have assembled collections of yeast strains expressing temperature-sensitive (ts) alleles of essential genes. To explore the phenotypes caused by essential gene mutation we used a panel of genetically engineered fluorescent markers to explore the morphology of cells in the ts strain collection using high-throughput microscopy. Here, we describe the design and implementation of an online database, PhenoM (Phenomics of yeast Mutants), for storing, retrieving, visualizing and data mining the quantitative single-cell measurements extracted from micrographs of the ts mutant cells. PhenoM allows users to rapidly search and retrieve raw images and their quantified morphological data for genes of interest. The database also provides several data-mining tools, including a PhenoBlast module for phenotypic comparison between mutant strains and a Gene Ontology module for functional enrichment analysis of gene sets showing similar morphological alterations. The current PhenoM version 1.0 contains 78,194 morphological images and 1,909,914 cells covering six subcellular compartments or structures for 775 ts alleles spanning 491 essential genes. PhenoM is freely available at http://phenom.ccbr.utoronto.ca/. PMID:22009677

  11. Transcriptional Analysis of Essential Genes of the Escherichia coli Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous Salmonella typhimurium Gene Cluster

    PubMed Central

    Zhang, Yan; Cronan, John E.

    1998-01-01

    The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome. A difficulty in analysis of the fab cluster by the polar allele duplication approach (Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:36143620, 1996) is that several of these genes are essential for the growth of E. coli. We overcame this complication by use of the fab gene cluster of Salmonella typhimurium, a close relative of E. coli, to provide functions necessary for growth. The S. typhimurium fab cluster was isolated by complementation of an E. coli fabD mutant and was found to encode proteins with >94% homology to those of E. coli. However, the S. typhimurium sequences cannot recombine with the E. coli sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the plsX transcripts initiate at promoters located far upstream and include the upstream rpmF ribosomal protein gene, a promoter located upstream of the plsX coding sequence (probably within the upstream gene, rpmF) is sufficient for normal growth. We have also found that the fabG gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (?-Cm cassette) between the fabD and fabG genes of the E. coli chromosome abolished fabG transcription and blocked cell growth, thus providing the first indication that fabG is an essential gene. Insertion of the ?-Cm cassette between fabH and fabD caused greatly decreased transcription of the fabD and fabG genes and slower cellular growth, indicating that fabD has only a weak promoter(s). PMID:9642179

  12. Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.

    PubMed Central

    Tsay, Y H; Robinson, G W

    1991-01-01

    Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells. Images PMID:1846667

  13. Cloning and characterization of Ehox, a novel homeobox gene essential for embryonic stem cell differentiation.

    PubMed

    Jackson, Melany; Baird, Janet W; Cambray, Noemi; Ansell, John D; Forrester, Lesley M; Graham, Gerard J

    2002-10-11

    We report here the identification and characterization of a novel paired-like homeobox-containing gene (Ehox). This gene, identified in embryonic stem (ES) cells, is differentially expressed during in vitro ES cell differentiation. We have assessed Ehox function using the ES cell in vitro differentiation system. This has involved molecular and biological analyses of the effects of sense or antisense Ehox expression (using episomal vectors) on ES cell differentiation. Analysis of antisense Ehox-expressing ES cells indicates that they are unable to express marker genes associated with hematopoietic, endothelial, or cardiac differentiation following removal of leukemia inhibitory factor. In contrast, overexpression of Ehox using the sense construct accelerated the appearance of these differentiation markers. ES cell self-renewal and differentiation assays reveal that inhibition of Ehox activity results in the maintenance of a stem cell phenotype in limiting concentrations of leukemia inhibitory factor and the almost complete impairment of the cardiomyocyte differentiation capacity of these cells. We therefore conclude that Ehox is a novel homeobox-containing gene that is essential for the earliest stages of murine ES cell differentiation. PMID:12087094

  14. The CXCR2 Gene Polymorphism Is Associated with Stroke in Patients with Essential Hypertension

    PubMed Central

    Timasheva, Yanina R.; Nasibullin, Timur R.; Mustafina, Olga E.

    2015-01-01

    Hypertension is the major risk factor for stroke, and genetic factors contribute to its development. Inflammation has been hypothesized to be the key link between blood pressure elevation and stroke. We performed an analysis of the association between inflammatory mediator gene polymorphisms and the incidence of stroke in patients with essential hypertension (EH). The study group consisted of 625 individuals (296 patients with noncomplicated EH, 71 hypertensive patients with ischemic stroke, and 258 control subjects). Both patients and controls were ethnic Tatars originating from the Republic of Bashkortostan (Russian Federation). The analysis has shown that the risk of ischemic stroke was associated with the CXCR2 rs1126579 polymorphism. Our results indicate that among patients with EH, the heterozygous genotype carriers had a higher risk of stroke (OR = 1.72, 95% CI 1.01-2.92), whereas the CXCR2*C/C genotype was protective against stroke (OR = 0.32, 95% CI 0.12-0.83). As shown by the gene-gene interaction analysis, the CXCR2 rs1126579 polymorphism was also present in all genotype/allele combinations associated with the risk of stroke. Genetic patterns associated with stroke also included polymorphisms in the CCL2, CCL18, CX3CR1, CCR5, and CXCL8 (IL8) genes, although no association between these loci and stroke was detected by individual analysis. PMID:26648969

  15. Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium

    PubMed Central

    Khatiwara, Anita; Jiang, Tieshan; Sung, Sam-Sun; Dawoud, Turki; Kim, Jeong Nam; Bhattacharya, Dhruva; Kim, Hee-Bal; Ricke, Steven C.

    2012-01-01

    As more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5 transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturating Salmonella enterica serotype Typhimurium mutant library recovered from selection under 3 different in vitro growth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes in S. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD, glnL, recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other approximately 70 bacterial species for which EZ:Tn5 mutagenesis has been established. PMID:22367088

  16. Sall4 is essential for mouse primordial germ cell specification by suppressing somatic cell program genes.

    PubMed

    Yamaguchi, Yasuka L; Tanaka, Satomi S; Kumagai, Maho; Fujimoto, Yuka; Terabayashi, Takeshi; Matsui, Yasuhisa; Nishinakamura, Ryuichi

    2015-01-01

    The Spalt-like 4 (Sall4) zinc finger protein is a critical transcription factor for pluripotency in embryonic stem cells (ESCs). It is also involved in the formation of a variety of organs, in mice, and humans. We report the essential roles of Sall4 in mouse primordial germ cell (PGC) specification. PGC specification is accompanied by the activation of the stem cell program and repression of the somatic cell program in progenitor cells. Conditional inactivation of Sall4 during PGC specification led to a reduction in the number of PGCs in embryonic gonads. Sall4(del/del) PGCs failed to translocate from the mesoderm to the endoderm and underwent apoptosis. In Sall4(del/del) PGC progenitors, somatic cell program genes (Hoxa1 and Hoxb1) were derepressed, while activation of the stem cell program was not impaired. We demonstrated that in differentiated ESCs, Sall4 bound to these somatic cell program gene loci, which are reportedly occupied by Prdm1 in embryonic carcinoma cells. Given that Sall4 and Prdm1 are known to associate with the histone deacetylase repressor complex, our findings suggest that Sall4 suppresses the somatic cell program possibly by recruiting the repressor complex in conjunction with Prdm1; therefore, it is essential for PGC specification. PMID:25263278

  17. Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae.

    PubMed Central

    Werner-Washburne, M; Stone, D E; Craig, E A

    1987-01-01

    Saccharomyces cerevisiae contains a large family of genes related to hsp70, the major heat shock-inducible gene of Drosophila melanogaster. One subfamily, identified by sequence homology, contains four genes, SSA1, SSA2, SSA3, and SSA4 (formerly YG100, YG102, YG106, and YG107, respectively). Previous studies showed that strains containing mutations in SSA1 and SSA2 are temperature sensitive for growth. SSA4, which is normally heat inducible and not expressed during vegetative growth, is expressed at high levels in ssa1 ssa2 strains at 23 degrees C. We constructed mutations in SSA3 and SSA4 and analyzed strains carrying mutations in the four genes. Strains carrying mutations in SSA3 SSA4 or SSA3 and SSA4 were indistinguishable from the wild type. However, ssa1 ssa2 ssa4 strains were inviable. SSA3, like SSA4, is a heat-inducible gene that is not normally expressed at 23 degrees C. Nevertheless, an intact copy of SSA3 regulated by the constitutive SSA2 promoter was capable of rescuing a ssa1 ssa2 ssa4 strain. This indicates that SSA3 encodes a functional protein and that the SSA1, SSA2, SSA3, and SSA4 gene products are functionally similar. Images PMID:3302682

  18. ZCURVE 3.0: identify prokaryotic genes with higher accuracy as well as automatically and accurately select essential genes.

    PubMed

    Hua, Zhi-Gang; Lin, Yan; Yuan, Ya-Zhou; Yang, De-Chang; Wei, Wen; Guo, Feng-Biao

    2015-07-01

    In 2003, we developed an ab initio program, ZCURVE 1.0, to find genes in bacterial and archaeal genomes. In this work, we present the updated version (i.e. ZCURVE 3.0). Using 422 prokaryotic genomes, the average accuracy was 93.7% with the updated version, compared with 88.7% with the original version. Such results also demonstrate that ZCURVE 3.0 is comparable with Glimmer 3.02 and may provide complementary predictions to it. In fact, the joint application of the two programs generated better results by correctly finding more annotated genes while also containing fewer false-positive predictions. As the exclusive function, ZCURVE 3.0 contains one post-processing program that can identify essential genes with high accuracy (generally >90%). We hope ZCURVE 3.0 will receive wide use with the web-based running mode. The updated ZCURVE can be freely accessed from http://cefg.uestc.edu.cn/zcurve/ or http://tubic.tju.edu.cn/zcurveb/ without any restrictions. PMID:25977299

  19. Genetic analysis of the FUS/TLS gene in essential tremor

    PubMed Central

    Kisselev, S.; Merner, N.; Dion, P.; Rouleau, G.; Clark, L.; Louis, E. D.

    2013-01-01

    Background and purpose Although essential tremor (ET) has a genetic basis, specific genes have not been identified. Recently, in a large ET family (FET1) from Quebec, a non-sense mutation (p.Q290X) in the amyotrophic lateral sclerosis (ALS) gene fused in sarcoma/translated in liposarcoma (FUS/TLS) was identified by exome sequencing. No confirmatory studies have been published. Methods Two-hundred and fifty-nine ET cases and 262 controls were enrolled in a study at Columbia University. We performed a comprehensive analysis of the FUS/TLS gene by sequencing all exons in a subsample of 116 ET cases with early-onset (≤40 years) ET. We evaluated an association between ET and SNPs in the FUS/TLS gene by genotyping four haplotype tagging SNPs in all 259 ET cases and 262 controls. Additionally, seven variants associated with ALS, two variants of unknown pathogenicity detected in ALS cases, eight mis-sense variants predicted to be damaging, and six rare variants were genotyped in these 259 ET cases and 262 controls. Results FUS/TLS mutations previously reported in ALS, the FET1 family, or novel mutations were not found in any of the 116 early-onset ET cases. In the case–control analyses, although the power of the performed associations was limited, no significant association between tagging SNPs in FUS/TLS and ET was observed, and none of the analyzed SNPs showed evidence of association with ET. Conclusion Our study suggests that pathogenic mutations in FUS/TLS are rare in a sample of early-onset ET cases in North America. We did not find evidence that the FUS/TLS gene is a risk factor for ET. PMID:23114103

  20. Complex Formation and Interactions between Transcription Factors Essential for Human Prolactin Receptor Gene Transcription ▿

    PubMed Central

    Kang, Jung-Hoon; Tsai-Morris, Chon-Hwa; Dufau, Maria L.

    2011-01-01

    The protein association of estrogen receptor α ERα with DNA-bound SP1 and C/EBPβ is essential for the 17β-estradiol (E2)-induced activation of human prolactin receptor (hPRLR) gene transcription. Protein-protein interaction and complex formation at the hPIII promoter of hPRLR was investigated. The basic region and leucine zipper (bZIP) of C/EBPβ, zinc finger (ZF) motifs of SP1, and the DNA binding domain of ERα were identified as regions responsible for the interactions between transfactors. The E2-induced interaction was confirmed by bioluminescence resonance energy transfer (BRET) assays of live cells. The combination of BRET/bimolecular luminescence complementation assay revealed that ERα exists as a constitutive homodimer, and E2 induced a change(s) in ERα homodimer conformation favorable for its association with C/EBPβ and SP1. Chromatin immunoprecipitation and small interfering RNA knockdown of members of the complex in breast cancer cells demonstrated the endogenous recruitment of components of the complex onto the hPIII promoter of the hPRLR gene. SP1 is the preferred transfactor for the recruitment of ERα to the complex that facilitates the C/EBPβ association. The E2/ERα-induced hPRLR transcription was demonstrated in ERα-negative breast cancer cells. This study indicates that the enhanced complex formation of ERα dimer with SP1 and C/EBPβ by E2 has an essential role in the transcriptional activation of the hPRLR gene. PMID:21670145

  1. The Colletotrichum lagenariu Ste12-like gene CST1 is essential for appressorium penetration.

    PubMed

    Tsuji, Gento; Fujii, Satoshi; Tsuge, Seiji; Shiraishi, Tomonori; Kubo, Yasuyuki

    2003-04-01

    Colletotrichum lagenarium is the causal agent of anthracnose of cucumber. This fungus produces a darkly melanized infection structure, appressoria, to penetrate the host leaves. The C. lagenarium CMK1 gene, a homologue of the Saccharomyces cerevisiae FUS3/KSS1 mitogen-activated protein (MAP) kinase genes, was shown to regulate conidial germination, appressorium formation, and invasive growth. In S. cerevisiae, Ste12p is known to be a transcriptional factor downstream of Fus3p/Kss1p MAP kinases. To evaluate the CMK1 MAP kinase pathway, we isolated the Ste12 homologue CST1 gene from C. lagenarium and characterized. The cst1delta strains were nonpathogenic on intact host leaves, but could form lesions when inoculated on wounded leaves. Conidia of the cst1delta strains could germinate and form melanized appressoria on both host leaf surface and artificial cellulose membrane, but could not produce infectious hyphae from appressoria, suggesting that CST1 is essential for appressorium penetration in C. lagenarium. In addition, matured appressoria of the cst1delta strains contained an extremely low level of lipid droplets compared with that of the wild-type strain. Lipid droplets were abundant in conidia of the cst1delta strains, but rapidly disappeared during appressorium formation. This misscheduled lipid degradation might be related to the failure of appressorium penetration in the cst1delta strain. PMID:12744460

  2. Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes.

    PubMed

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S; Boeke, Jef D

    2015-02-10

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10(-10)), consistent with their orthogonal nature and the individual escape frequencies of <10(-6). Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance. PMID:25624482

  3. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

    PubMed Central

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S.; Boeke, Jef D.

    2015-01-01

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer’s yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational “safeguard” control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10−10), consistent with their orthogonal nature and the individual escape frequencies of <10−6. Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance. PMID:25624482

  4. Allelic variants in the interleukin-6 gene and essential hypertension in Japanese women.

    PubMed

    Nakajima, T; Ota, N; Yoshida, H; Watanabe, S; Suzuki, T; Emi, M

    1999-11-01

    Genes that can be implicated in hypertension in experimental animals are plausible candidates in the pathogenesis of human hypertension. A recent genome-wide search for quantitative-trait loci (QTL) in hypertensive rats revealed a strong correlation between the interleukin-6 (IL-6) locus on rat chromosome 4 and systolic, diastolic, and mean arterial pressure in this mammalian species. To investigate a possible association between genetic variations of the IL-6 gene and hypertension in humans, we identified two novel single-nucleotide sequence variations, a C/G substitution at -634 in the promoter region and a G/A substitution at 4391 in a 3' non-coding portion of exon 5, and a previous reported sequence variant, an A/T variation in the composition of the AnTn tract around -447 in the promoter region (Fishman D et al. J Clin Invest 1998; 102: 1369-1376), within a test population of 96 Japanese subjects. Allelic associations involving these variations were analyzed in 150 hypertensive and 143 normotensive Japanese women. The distribution of alleles of the three polymorphisms, as well as a dinucleotide repeat present at the IL-6 locus, was similar in the two groups. Therefore, the IL-6 gene appears to play a minimal role in the genetic etiology of essential hypertension in Japanese women. PMID:11196657

  5. Expression of essential genes for biosynthesis of antimicrobial peptides of Bacillus is modulated by inactivated cells of target microorganisms.

    PubMed

    Leães, Fernanda Leal; Velho, Renata Voltolini; Caldas, Danielle Gregório Gomes; Ritter, Ana Carolina; Tsai, Siu Mui; Brandelli, Adriano

    2016-01-01

    Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms. PMID:26577655

  6. CYP2E1 gene promoter polymorphism -1293G>C increases the risk of essential hypertension in men with alcohol abuse.

    PubMed

    Polonikov, A V; Ivanov, V P; Solodilova, M A

    2013-10-01

    We studied the association between of cytochrome P450 2E1 (CYP2E1) gene polymorphism and risk of essential hypertension development depending on alcohol drinking habit in unrelated men in Russian population (patients with essential hypertension and healthy volunteers). All participants were genotyped for four CYP2E1 gene polymorphisms -1293G>C (rs3813867), -1053C>T (rs2031920), 7632T>A (rs6413432), and 9896C>G (rs2070676) by PCR and restriction analysis. We found statistically significant associations between -1293C allele (OR=5.04, 95% CI=1.23-20.70, p=0.03) and -1293GC genotype (OR=5.36, 95% CI=1.28-22.50, p=0.03) with increased risk of essential hypertension in men. Stratified analysis on alcohol drinking habit showed that the presence of -1293C allele (OR=6.82, 95% CI=1.12-41.70, p=0.04) and -1293GC genotype (OR=7.61, 95% CI=1.2-48.4, p=0.03) in men with alcohol abuse increases the risk of essential hypertension. The obtained data suggest that excessive alcohol consumption and increased induction of cytochrome in the carriers of -1293C allele of CYP2E1 gene lead to generation of highly reactive free radical oxidation products. These processes induced oxidative stress and endothelial induction, which served as the pathogenetic basis for essential hypertension. PMID:24288753

  7. surA, an Escherichia coli gene essential for survival in stationary phase.

    PubMed

    Tormo, A; Almirón, M; Kolter, R

    1990-08-01

    Mutations in genes not required for exponential growth but essential for survival in stationary phase were isolated in an effort to understand the ability of wild-type Escherichia coli cells to remain viable during prolonged periods of nutritional deprivation. The phenotype of these mutations is referred to as Sur- (survival) and the genes are designated sur. The detailed analysis of one of these mutations is presented here. The mutation (surA1) caused by insertion of a mini-Tn10 element defined a new gene located near 1 min on the E. coli chromosome. It was located directly upstream of pdxA and formed part of a complex operon. Evidence is presented supporting the interpretation that cells harboring the surA1 mutation die during stationary phase while similar insertion mutations in other genes of the operon do not lead to a Sur- phenotype. Strains harboring surA1 had a normal doubling time in both rich and minimal medium, but cultures lost viability after several days in stationary phase. Analysis of revertants and suppressors of surA1, which arose after prolonged incubation in stationary phase, indicates that DNA rearrangements (excisions and duplications) occurred in cultures of this strain even when the viable-cell counts were below 10(2) cells per ml. Cells containing suppressing mutations then grew in the same culture to 10(8) cells per ml, taking over the population. The implications of these observations to our understanding of stationary-phase mutagenesis are discussed. PMID:2165476

  8. The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis.

    PubMed

    McKim, Sarah M; Stenvik, Grethe-Elisabeth; Butenko, Melinka A; Kristiansen, Wenche; Cho, Sung Ki; Hepworth, Shelley R; Aalen, Reidunn B; Haughn, George W

    2008-04-01

    The Arabidopsis BLADE-ON-PETIOLE 1 (BOP1) and BOP2 genes encode redundant transcription factors that promote morphological asymmetry during leaf and floral development. Loss-of-function bop1 bop2 mutants display a range of developmental defects, including a loss of floral organ abscission. Abscission occurs along specialised cell files, called abscission zones (AZs) that develop at the junction between the leaving organ and main plant body. We have characterized the bop1 bop2 abscission phenotype to determine how BOP1 and BOP2 contribute to the known abscission developmental framework. Histological analysis and petal breakstrength measurements of bop1 bop2 flowers show no differentiation of floral AZs. Furthermore, vestigial cauline leaf AZs are also undifferentiated in bop1 bop2 mutants, suggesting that BOP proteins are essential to establish AZ cells in different tissues. In support of this hypothesis, BOP1/BOP2 activity is required for both premature floral organ abscission and the ectopic abscission of cauline leaves promoted by the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene under the control of the constitutive CaMV 35S promoter. Expression of several abscission-related marker genes, including IDA, is relatively unperturbed in bop1 bop2 mutants, indicating that these AZ genes respond to positional cues that are independent of BOP1/BOP2 activity. We also show that BOP1 and BOP2 promote growth of nectary glands, which normally develop at the receptacle adjacent to developing AZs. Taken together, these data suggest that BOP1/BOP2 activity is required for multiple cell differentiation events in the proximal regions of inflorescence lateral organs. PMID:18339677

  9. surA, an Escherichia coli gene essential for survival in stationary phase.

    PubMed Central

    Tormo, A; Almirón, M; Kolter, R

    1990-01-01

    Mutations in genes not required for exponential growth but essential for survival in stationary phase were isolated in an effort to understand the ability of wild-type Escherichia coli cells to remain viable during prolonged periods of nutritional deprivation. The phenotype of these mutations is referred to as Sur- (survival) and the genes are designated sur. The detailed analysis of one of these mutations is presented here. The mutation (surA1) caused by insertion of a mini-Tn10 element defined a new gene located near 1 min on the E. coli chromosome. It was located directly upstream of pdxA and formed part of a complex operon. Evidence is presented supporting the interpretation that cells harboring the surA1 mutation die during stationary phase while similar insertion mutations in other genes of the operon do not lead to a Sur- phenotype. Strains harboring surA1 had a normal doubling time in both rich and minimal medium, but cultures lost viability after several days in stationary phase. Analysis of revertants and suppressors of surA1, which arose after prolonged incubation in stationary phase, indicates that DNA rearrangements (excisions and duplications) occurred in cultures of this strain even when the viable-cell counts were below 10(2) cells per ml. Cells containing suppressing mutations then grew in the same culture to 10(8) cells per ml, taking over the population. The implications of these observations to our understanding of stationary-phase mutagenesis are discussed. Images PMID:2165476

  10. Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.

    PubMed

    Bakkali, F; Averbeck, S; Averbeck, D; Zhiri, A; Idaomar, M

    2005-08-01

    In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum>Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine. PMID:15975845

  11. Contact and Repellent Activities of the Essential Oil from Juniperus formosana against Two Stored Product Insects.

    PubMed

    Guo, Shanshan; Zhang, Wenjuan; Liang, Junyu; You, Chunxue; Geng, Zhufeng; Wang, Chengfang; Du, Shushan

    2016-01-01

    The chemical composition of the essential oil from Juniperus formosana leaves and its contact and repellent activities against Tribolium castaneum and Liposcelis bostrychophila adults were investigated. The essential oil of J. formosana leaves was obtained by hydrodistillation and analyzed by GC-MS. A total of 28 components were identified and the main compounds in the essential oil were α-pinene (21.66%), 4-terpineol (11.25%), limonene (11.00%) and β-phellandrene (6.63%). The constituents α-pinene, 4-terpineol and d-limonene were isolated from the essential oil. It was found that the essential oil exhibited contact activity against T. castaneum and L. bostrychophila adults (LD50 = 29.14 μg/adult and 81.50 µg/cm², respectively). The compound 4-terpineol exhibited the strongest contact activity (LD50 = 7.65 μg/adult). In addition, data showed that at 78.63 nL/cm², the essential oil and the three isolated compounds strongly repelled T. castaneum adults. The compounds α-pinene and d-limonene reached the same level (Class V) of repellency as DEET (p = 0.396 and 0.664) against L. bostrychophila at 63.17 nL/cm² after 2 h treatment. The results indicate that the essential oil and the isolated compounds have potential to be developed into natural insecticides and repellents to control insects in stored products. PMID:27092485

  12. Early estrogen-induced gene 1, a novel RANK signaling component, is essential for osteoclastogenesis

    PubMed Central

    Choi, Han Kyoung; Kang, Hye Ri; Jung, Eutteum; Kim, Tae Eon; Lin, Jing Jing; Lee, Soo Young

    2013-01-01

    The receptor activator of NF-κB (RANK) and immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors are essential factors involved in regulating osteoclast formation and bone remodeling. Here, we identify early estrogen-induced gene 1 (EEIG1) as a novel RANK ligand (RANKL)-inducible protein that physically interacts with RANK and further associates with Gab2, PLCγ2 and Tec/Btk kinases upon RANKL stimulation. EEIG1 positively regulates RANKL-induced osteoclast formation, likely due to its ability to facilitate RANKL-stimulated PLCγ2 phosphorylation and NFATc1 induction. In addition, an inhibitory peptide designed to block RANK-EEIG1 interaction inhibited RANKL-induced bone destruction by reducing osteoclast formation. Together, our results identify EEIG1 as a novel RANK signaling component controlling RANK-mediated osteoclast formation, and suggest that targeting EEIG1 might represent a new therapeutic strategy for the treatment of pathological bone resorption. PMID:23478294

  13. Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering.

    PubMed

    Juhas, Mario; Reuß, Daniel R; Zhu, Bingyao; Commichau, Fabian M

    2014-11-01

    Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell 'chassis'. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications. PMID:25092907

  14. Effect of commercially available plant-derived essential oil products on arthropod pests.

    PubMed

    Cloyd, Raymond A; Galle, Cindy L; Keith, Stephen R; Kalscheur, Nanette A; Kemp, Kenneth E

    2009-08-01

    Plant-derived essential oil products, in general, are considered minimum-risk pesticides and are exempt from Environmental Protection Agency registration under section 25(b) of the Federal Insecticide Fungicide and Rodenticide Act. However, many of the plant-derived essential products available to consumers (homeowners) have not been judiciously evaluated for both efficacy and plant safety. In fact, numerous plant-derived essential oil products labeled for control of arthropod pests have not been subject to rigorous evaluation, and there is minimal scientific information or supporting data associated with efficacy against arthropod pests. We conducted a series of greenhouse experiments to determine the efficacy and phytotoxicity of an array of plant-derived essential oil products available to consumers on arthropod pests including the citrus mealybug, Planococcus citri (Risso); western flower thrips, Frankliniella occidentalis (Pergande); twospotted spider mite, Tetranychus urticae Koch; sweetpotato whitefly B-biotype, Bemisia tabaci (Gennadius); and green peach aphid, Myzus persicae (Sulzer). Although the products Flower Pharm (cottonseed, cinnamon, and rosemary oil) and Indoor Pharm (soybean, rosemary, and lavender oil) provided > 90% mortality of citrus mealybug, they were also the most phytotoxic to the coleus, Solenostemon scutellarioides (L.) Codd, plants. Both GC-Mite (cottonseed, clove, and garlic oil) and Bugzyme (citric acid) were most effective against the twospotted spider mite (> or = 90% mortality). However, SMC (canola, coriander oil, and triethanolamine), neem (clarified hydrophobic extract of neem oil), and Bug Assassin (eugenol, sodium lauryl sulfate, peppermint, and citronella oil) provided > 80% mortality. Monterey Garden Insect Spray, which contained 0.5% spinosad, was most effective against western flower thrips with 100% mortality. All the other products evaluated failed to provide sufficient control of western flower thrips with < 30% mortality. In addition, the products Pest Out (cottonseed, clove, and garlic oil), Bang (Pipereaceae), and Fruit & Vegetable Insect Spray (rosemary, cinnamon, clove oil, and garlic extract) had the highest flower (transvaal daisy, Gerberajamesonii [H. Bolus ex Hook.f]) phytotoxicity ratings (> or = 4.5 of 5) among all the products. None of the plant-derived essential oil products provided sufficient control of sweetpotato whitefly B-biotype or green peach aphid 7, 14, and 21 d after application. Furthermore, the products Bug Assassin (eugenol, sodium lauryl sulfate, peppermint, and citronella oil) and Sharpshooter (sodium lauryl sulfate and clove oil) were phytotoxic to the poinsettia, Euphorbia pulcherrima Willd. ex Klotzsch, plants. This study is one of the first to quantitatively demonstrate that commercially available plant-derived essential oil products vary in their effectiveness against certain arthropod pests stated on the label and are phytotoxic. PMID:19736770

  15. Genome-Wide Identification of Streptococcus pneumoniae Genes Essential for Bacterial Replication during Experimental Meningitis▿ †

    PubMed Central

    Molzen, T. E.; Burghout, P.; Bootsma, H. J.; Brandt, C. T.; van der Gaast-de Jongh, Christa E.; Eleveld, M. J.; Verbeek, M. M.; Frimodt-Møller, N.; Østergaard, C.; Hermans, P. W. M.

    2011-01-01

    Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during infection. The results point to essential roles for capsular polysaccharides, nutrient uptake, and amino acid biosynthesis in bacterial replication during experimental meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive infection models of experimental rat meningitis. It appeared that adenylosuccinate synthetase, flavodoxin, and LivJ, the substrate binding protein of a branched-chain amino acid ABC transporter, are relevant as targets for future therapy and prevention of pneumococcal meningitis, since their mutants were attenuated in both models of infection as well as in competitive growth in human cerebrospinal fluid in vitro. PMID:21041497

  16. Discs large 5, an Essential Gene in Drosophila, Regulates Egg Chamber Organization

    PubMed Central

    Reilly, Eve; Changela, Neha; Naryshkina, Tatyana; Deshpande, Girish; Steward, Ruth

    2015-01-01

    Discs large 5 (Dlg5) is a member of the MAGUK family of proteins that typically serve as molecular scaffolds and mediate signaling complex formation and localization. In vertebrates, Dlg5 has been shown to be responsible for polarization of neural progenitors and to associate with Rab11-positive vesicles in epithelial cells. In Drosophila, however, the function of Dlg5 is not well-documented. We have identified dlg5 as an essential gene that shows embryonic lethality. dlg5 embryos display partial loss of primordial germ cells (PGCs) during gonad coalescence between stages 12 and 15 of embryogenesis. Loss of Dlg5 in germline and somatic stem cells in the ovary results in the depletion of both cell lineages. Reduced expression of Dlg5 in the follicle cells of the ovary leads to a number of distinct phenotypes, including defects in egg chamber budding, stalk cell overgrowth, and ectopic polar cell induction. Interestingly, loss of Dlg5 in follicle cells results in abnormal distribution of a critical component of cell adhesion, E-cadherin, shown to be essential for proper organization of egg chambers. PMID:25795662

  17. Isolation and Characterization of Pep5, a Gene Essential for Vacuolar Biogenesis in Saccharomyces Cerevisiae

    PubMed Central

    Woolford, C. A.; Dixon, C. K.; Manolson, M. F.; Wright, R.; Jones, E. W.

    1990-01-01

    pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37°. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursor forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein. PMID:2204580

  18. Processing of coriander fruits for the production of essential oil, triglyceride, and high protein seed meal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Coriander (Coriandrum sativum L.) is a summer annual traditionally grown for use as a fresh green herb or as a spice. The essential oil extracted from coriander fruit is also widely used as flavoring in a variety of food products. The fatty oil (triglyceride) fraction in the seed is rich in petrosel...

  19. Characterization of the cis and trans elements essential for rat insulin II gene expression.

    PubMed

    Crowe, D T; Hwung, Y P; Tsai, S Y; Tsai, M J

    1988-01-01

    We have constructed 14 different linker-scanner (LS) mutants throughout the enhancer and promoter of a rat insulin II-CAT fusion gene. These LS mutants were transiently transfected into an insulin-producing (HIT) cell line and three mutants (LS-261/252, LS-102/91, LS-54/45) displayed drastically reduced levels of CAT activity. Therefore, at least three regions are essential for the in vivo expression of an insulin-CAT fusion gene. To identify the trans-acting factors which interact with this cell-specific promoter, we performed band-shifting assays with either HIT or HeLa nuclear extracts and an end-labelled insulin fragment (-100 to +49). Three binding activities were common to both extracts, and another one was unique to HIT cells. DNase I protection studies localized one binding activity to -60 to -40 bp 5' to the cap site. We show that this factor is common to both cell lines and is identical to a previously characterized transcription factor (COUP) which binds to the chicken ovalbumin upstream promoter. PMID:3064102

  20. Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux

    PubMed Central

    Harris, Z. Leah; Durley, Alison P.; Man, Tsz Kwong; Gitlin, Jonathan D.

    1999-01-01

    Aceruloplasminemia is an autosomal recessive disorder of iron metabolism. Affected individuals evidence iron accumulation in tissue parenchyma in association with absent serum ceruloplasmin. Genetic studies of such patients reveal inherited mutations in the ceruloplasmin gene. To elucidate the role of ceruloplasmin in iron homeostasis, we created an animal model of aceruloplasminemia by disrupting the murine ceruloplasmin (Cp) gene. Although normal at birth, Cp−/− mice demonstrate progressive accumulation of iron such that by one year of age all animals have a prominent elevation in serum ferritin and a 3- to 6-fold increase in the iron content of the liver and spleen. Histological analysis of affected tissues in these mice shows abundant iron stores within reticuloendothelial cells and hepatocytes. Ferrokinetic studies in Cp+/+ and Cp−/− mice reveal equivalent rates of iron absorption and plasma iron turnover, suggesting that iron accumulation results from altered compartmentalization within the iron cycle. Consistent with this concept, Cp−/− mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Our findings reveal an essential physiologic role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores. PMID:10485908

  1. Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

    PubMed Central

    Boynak, Natalia Y.; Rojas, Federico; D’Alessio, Cecilia; Vilchez Larrea, Salomé C.; Rodriguez, Vanina; Ghiringhelli, Pablo D.; Téllez-Iñón, María T.

    2013-01-01

    Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M. PMID:24223931

  2. Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

    PubMed Central

    2013-01-01

    Background Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. Results Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. Conclusion We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways. PMID:24015778

  3. Insecticidal Activity of the Essential Oils from Different Plants Against Three Stored-Product Insects

    PubMed Central

    Ayvaz, Abdurrahman; Sagdic, Osman; Karaborklu, Salih; Ozturk, Ismet

    2010-01-01

    This study was conducted to determine the insecticidal activity of essential oils from oregano, Origanum onites L. (Lamiales: Lamiaceae), savory, Satureja thymbra L. (Lamiales: Lamiaceae), and myrtle, Myrtus communis L. (Rosales: Myrtaceae) against three stored-product insects. Essential oils from three species of plants were obtained by Clevenger-type water distillation. The major compounds in these essential oils were identified using gas chromatography-mass spectrometry and their insecticidal activity was tested against adults of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) and the bean weevil Acanthoscelides obtectus Say (Coleoptera: Bruchidae). While the major compound found in oregano and savory was carvacrol, the main constituent of the myrtle was linalool. Among the tested insects, A. obtectus was the most tolerant species against the essential oils. However, the insecticidal activity of the myrtle oil was more pronounced than other oils tested against A. obtectus adults. The essential oils of oregano and savory were highly effective against P. interpunctella and E. kuehniella, with 100% mortality obtained after 24 h at 9 and 25 µl/l air for P. interpunctella and E. kuehniella, respectively. LC50 and LC99 values of each essential oil were estimated for each insect species. PMID:20578885

  4. Insecticidal activity of the essential oils from different plants against three stored-product insects.

    PubMed

    Ayvaz, Abdurrahman; Sagdic, Osman; Karaborklu, Salih; Ozturk, Ismet

    2010-01-01

    This study was conducted to determine the insecticidal activity of essential oils from oregano, Origanum onites L. (Lamiales: Lamiaceae), savory, Satureja thymbra L. (Lamiales: Lamiaceae), and myrtle, Myrtus communis L. (Rosales: Myrtaceae) against three stored-product insects. Essential oils from three species of plants were obtained by Clevenger-type water distillation. The major compounds in these essential oils were identified using gas chromatography-mass spectrometry and their insecticidal activity was tested against adults of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) and the bean weevil Acanthoscelides obtectus Say (Coleoptera: Bruchidae). While the major compound found in oregano and savory was carvacrol, the main constituent of the myrtle was linalool. Among the tested insects, A. obtectus was the most tolerant species against the essential oils. However, the insecticidal activity of the myrtle oil was more pronounced than other oils tested against A. obtectus adults. The essential oils of oregano and savory were highly effective against P. interpunctella and E. kuehniella, with 100% mortality obtained after 24 h at 9 and 25 microl/l air for P. interpunctella and E. kuehniella, respectively. LC(50) and LC(99) values of each essential oil were estimated for each insect species. PMID:20578885

  5. Assessment of inhibitory potential of essential oils on natural mycoflora and Fusarium mycotoxins production in wheat

    PubMed Central

    2013-01-01

    Background In the last years essential oils from different plants were used in the prevention of fungi and mycotoxins accumulation in cereals. The most attractive aspect derived from using of essential oils as seed grains protectants is due to their non-toxicity. This study was focused on assessment the inhibitory effect of some essential oils: Melissa officinalis (O1), Salvia officinalis (O2), Coriandrum sativum (O3), Thymus vulgaris (O4) Mentha piperita (O5) and Cinnamomum zeylanicum (O6) against natural mycoflora and Fusarium mycotoxins production correlated with their antioxidants properties. Results All essential oils showed inhibitory effect on fungal contamination of wheat seeds. This ability was dose-dependent. The highest inhibitory effect on Fusarium and Aspergillus fungi was recorded after 5 days of treatment. Fungi such as yeast (Pichia, Saccharomyces and Hyphopichia) were predominantly on seeds mycoflora after 22 days. Each treatment had a selective inhibitory effect on frequency of fungus genera. After 5 days of treatment the most fungicidal effect was recorder for O4, followed by O1. In terms of essential oils effect on mycotoxins development, the best control on fumonisins (FUMO) production was recorded for O6. The antioxidant properties of essential oils decreased in order: O4 > O1 > O6 > O5 > O2 > O3. Also, our data suggested that there is a significant negative correlation between antioxidant properties and seed contamination index (SCI), but there was not recorded a good correlation between antioxidant properties and FUMO content. Conclusions Based on proven antifungal and antimycotoxin effects as well as their antioxidant properties, the essential oils could be recommended as natural preservatives for stored cereals. The highest inhibition of fungal growth was noted after 5 days of treatment and decreased after 22 days. PMID:23409841

  6. Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development

    PubMed Central

    Moule, Madeleine G.; Hemsley, Claudia M.; Seet, Qihui; Guerra-Assunção, José Afonso; Lim, Jiali; Sarkar-Tyson, Mitali; Clark, Taane G.; Tan, Patrick B. O.; Titball, Richard W.; Cuccui, Jon; Wren, Brendan W.

    2014-01-01

    ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. PMID:24520057

  7. Regulatory Oversight of Cell and Gene Therapy Products in Canada.

    PubMed

    Ridgway, Anthony; Agbanyo, Francisca; Wang, Jian; Rosu-Myles, Michael

    2015-01-01

    Health Canada regulates gene therapy products and many cell therapy products as biological drugs under the Canadian Food and Drugs Act and its attendant regulations. Cellular products that meet certain criteria, including minimal manipulation and homologous use, may be subjected to a standards-based approach under the Safety of Human Cells, Tissues and Organs for Transplantation Regulations. The manufacture and clinical testing of cell and gene therapy products (CGTPs) presents many challenges beyond those for protein biologics. Cells cannot be subjected to pathogen removal or inactivation procedures and must frequently be administered shortly after final formulation. Viral vector design and manufacturing control are critically important to overall product quality and linked to safety and efficacy in patients through concerns such as replication competence, vector integration, and vector shedding. In addition, for many CGTPs, the value of nonclinical studies is largely limited to providing proof of concept, and the first meaningful data relating to appropriate dosing, safety parameters, and validity of surrogate or true determinants of efficacy must come from carefully designed clinical trials in patients. Addressing these numerous challenges requires application of various risk mitigation strategies and meeting regulatory expectations specifically adapted to the product types. Regulatory cooperation and harmonisation at an international level are essential for progress in the development and commercialisation of these products. However, particularly in the area of cell therapy, new regulatory paradigms may be needed to harness the benefits of clinical progress in situations where the resources and motivation to pursue a typical drug product approval pathway may be lacking. PMID:26374212

  8. Regulation of photoreceptor gene expression by the retinal homeobox (Rx) gene product

    PubMed Central

    Pan, Yi; Martinez-De Luna, Reyna I.; Lou, Chih-Hong; Nekkalapudi, Srivamsi; Kelly, Lisa E.; Sater, Amy K.; El-Hodiri, Heithem M.

    2010-01-01

    The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type. PMID:20060393

  9. Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models.

    PubMed

    Chowdhury, Ratul; Chowdhury, Anupam; Maranas, Costas D

    2015-01-01

    Essentiality (ES) and Synthetic Lethality (SL) information identify combination of genes whose deletion inhibits cell growth. This information is important for both identifying drug targets for tumor and pathogenic bacteria suppression and for flagging and avoiding gene deletions that are non-viable in biotechnology. In this study, we performed a comprehensive ES and SL analysis of two important eukaryotic models (S. cerevisiae and CHO cells) using a bilevel optimization approach introduced earlier. Information gleaned from this study is used to propose specific model changes to remedy inconsistent with data model predictions. Even for the highly curated Yeast 7.11 model we identified 50 changes (metabolic and GPR) leading to the correct prediction of an additional 28% of essential genes and 36% of synthetic lethals along with a 53% reduction in the erroneous identification of essential genes. Due to the paucity of mutant growth phenotype data only 12 changes were made for the CHO 1.2 model leading to an additional correctly predicted 11 essential and eight non-essential genes. Overall, we find that CHO 1.2 was 76% less accurate than the Yeast 7.11 metabolic model in predicting essential genes. Based on this analysis, 14 (single and double deletion) maximally informative experiments are suggested to improve the CHO cell model by using information from a mouse metabolic model. This analysis demonstrates the importance of single and multiple knockout phenotypes in assessing and improving model reconstructions. The advent of techniques such as CRISPR opens the door for the global assessment of eukaryotic models. PMID:26426067

  10. Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models

    PubMed Central

    Chowdhury, Ratul; Chowdhury, Anupam; Maranas, Costas D.

    2015-01-01

    Essentiality (ES) and Synthetic Lethality (SL) information identify combination of genes whose deletion inhibits cell growth. This information is important for both identifying drug targets for tumor and pathogenic bacteria suppression and for flagging and avoiding gene deletions that are non-viable in biotechnology. In this study, we performed a comprehensive ES and SL analysis of two important eukaryotic models (S. cerevisiae and CHO cells) using a bilevel optimization approach introduced earlier. Information gleaned from this study is used to propose specific model changes to remedy inconsistent with data model predictions. Even for the highly curated Yeast 7.11 model we identified 50 changes (metabolic and GPR) leading to the correct prediction of an additional 28% of essential genes and 36% of synthetic lethals along with a 53% reduction in the erroneous identification of essential genes. Due to the paucity of mutant growth phenotype data only 12 changes were made for the CHO 1.2 model leading to an additional correctly predicted 11 essential and eight non-essential genes. Overall, we find that CHO 1.2 was 76% less accurate than the Yeast 7.11 metabolic model in predicting essential genes. Based on this analysis, 14 (single and double deletion) maximally informative experiments are suggested to improve the CHO cell model by using information from a mouse metabolic model. This analysis demonstrates the importance of single and multiple knockout phenotypes in assessing and improving model reconstructions. The advent of techniques such as CRISPR opens the door for the global assessment of eukaryotic models. PMID:26426067

  11. ABG1, a Novel and Essential Candida albicans Gene Encoding a Vacuolar Protein Involved in Cytokinesis and Hyphal Branching

    PubMed Central

    Veses, Verónica; Casanova, Manuel; Murgui, Amelia; Domínguez, Ángel; Gow, Neil A. R.; Martínez, José P.

    2005-01-01

    Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching. PMID:15947201

  12. Methylation silencing of ULK2, an autophagy gene, is essential for astrocyte transformation and tumor growth.

    PubMed

    Shukla, Sudhanshu; Patric, Irene Rosita Pia; Patil, Vikas; Shwetha, Shivayogi D; Hegde, Alangar S; Chandramouli, Bangalore A; Arivazhagan, Arimappamagan; Santosh, Vani; Somasundaram, Kumaravel

    2014-08-01

    Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5(+/+) but not in autophagy-deficient ATG5(-/-) cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development. PMID:24923441

  13. The boule gene is essential for spermatogenesis of haploid insect male.

    PubMed

    Sekiné, Kazuki; Furusawa, Tadashi; Hatakeyama, Masatsugu

    2015-03-01

    boule (bol), a member of the Deleted in Azoospermia (DAZ) gene family plays an important role in meiosis (reductional maturation divisions) in a spermatogenesis-specific manner in animals by regulating translation of the downstream cell division cycle 25 (cdc25) phosphatase mRNA. Orthologues of bol are conserved among animals and found in the genomes of hymenopteran insects, in which the general mode of reproduction is haplodiploidy: female is diploid and male is haploid. In this mode of reproduction, haploid males produce haploid sperm through non-reductional maturation divisions. The question thus arises of whether the bol gene actually functions during spermatogenesis in these haploid males. In this study, we identified two transcriptional isoforms of bol orthologue (Ar bol and Ar bol-2), and one cdc25 orthologue (Ar cdc25) in the hymenopteran sawfly, Athalia rosae. Ar bol was expressed exclusively in the testis when maturation divisions occurred, while Ar bol-2 was expressed ubiquitously. Knockdown of all bol transcripts (both Ar bol and Ar bol-2) resulted in a lack of mature sperm, whereas males with sole knockdown of Ar bol-2 were able to produce a small number of mature sperm. The cell cycle was arrested before maturation divisions in the testis in which all bol transcripts were knocked down, as revealed by flow cytometry. Although no mature sperm was produced, sperm elongation was partially observed when Ar cdc25 alone was knocked down. These results indicate that Ar bol is essential for the entry and progression of maturation divisions and sperm differentiation in haploid males. PMID:25592223

  14. The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity

    PubMed Central

    Turki-Judeh, Wiam; Courey, Albert J.

    2012-01-01

    Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C-termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression. PMID:22319573

  15. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme. PMID:25151124

  16. Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins.

    PubMed Central

    Maddock, J; Bhatt, A; Koch, M; Skidmore, J

    1997-01-01

    We have identified an essential Caulobacter crescentus gene (cgtA) that encodes a member of a recently identified subfamily of GTPases (the Obg family) conserved from Bacteria to Archaea to humans. This evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins possesses a fundamental, yet unknown, cellular role. In this report, we describe the isolation and sequence of the cgtA gene. The predicted CgtA protein displays striking similarity to the Obg family of small, monomeric GTP-binding proteins, both in the conserved guanine nucleotide-binding domains and throughout the N-terminal glycine-rich domain that is found in many members of the Obg family. Disruption of the cgtA gene was lethal, demonstrating that this gene is essential for cell growth. Immunoblot analysis revealed that CgtA protein levels remained constant throughout the C. crescentus cell cycle. PMID:9335292

  17. Genetic variants of the class A scavenger receptor gene are associated with essential hypertension in Chinese

    PubMed Central

    Zhang, Min; Han, Zhijun; Yan, Zihe; Cui, Qichen; Jiang, Yuhai; Gao, Mingzhu; Yu, Wei

    2015-01-01

    Background The class A scavenger receptor, which is encoded by the macrophage scavenger receptor 1 (MSR1) gene, is a pattern recognition receptor (PPR) primarily expressed in macrophages. It has been reported that genetic polymorphisms of MSR1 are significantly associated with many cardiovascular events. However, whether it links genetically to essential hypertension (EH) in Chinese is not defined. Methods We performed an independent case-control study in a Chinese population consisting of 617 EH cases and 620 controls by genotyping three single nucleotide polymorphisms (SNPs) of MSR1. Results We found that rs13306541 and rs3747531 were significantly associated with an increased risk of EH with per allele odds ratio (OR) of 1.63 [95% confidence interval (CI): 1.27-2.09; P<0.001] and 1.29 (95% CI: 1.09-1.52; P=0.003), respectively. Individuals with 2-4 risk alleles had a 2.03-fold (95% CI: 1.48-2.78) increased risk of EH compared with those having none of the risk alleles (P for trend <0.001). Conclusions Our results indicate that genetic variants of MSR1 may serve as predictive markers for the risk of EH in combination with traditional risk factors of EH in Chinese population. PMID:26716027

  18. Ewing sarcoma gene EWS is essential for meiosis and B lymphocyte development

    PubMed Central

    Li, Hongjie; Watford, Wendy; Li, Cuiling; Parmelee, Alissa; Bryant, Mark A.; Deng, Chuxia; O’Shea, John; Lee, Sean Bong

    2007-01-01

    Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing, but its physiological role in vivo remains undefined. Here, we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews–/– lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre–B lymphocyte) development. During meiosis, Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination, resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence, and the mutant animals showed hypersensitivity to ionizing radiation. Finally, we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre–B cell development and meiosis, with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore, we demonstrate a novel role of EWS in cellular senescence, possibly through its interaction and modulation of lamin A/C. PMID:17415412

  19. Ewing sarcoma gene EWS is essential for meiosis and B lymphocyte development.

    PubMed

    Li, Hongjie; Watford, Wendy; Li, Cuiling; Parmelee, Alissa; Bryant, Mark A; Deng, Chuxia; O'Shea, John; Lee, Sean Bong

    2007-05-01

    Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing, but its physiological role in vivo remains undefined. Here, we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews(-/-) lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre-B lymphocyte) development. During meiosis, Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination, resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence, and the mutant animals showed hypersensitivity to ionizing radiation. Finally, we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre-B cell development and meiosis, with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore, we demonstrate a novel role of EWS in cellular senescence, possibly through its interaction and modulation of lamin A/C. PMID:17415412

  20. Impact of obesity and nitric oxide synthase gene G894T polymorphism on essential hypertension.

    PubMed

    Wrzosek, M; Sokal, M; Sawicka, A; Wlodarczyk, M; Glowala, M; Wrzosek, M; Kosior, M; Talalaj, M; Biecek, P; Nowicka, G

    2015-10-01

    Hypertension is a multifactorial disease caused by environmental, metabolic and genetic factors, but little is currently known on the complex interplay between these factors and blood pressure. The aim of the present study was to assess the potential impact of obesity, and angiotensin-converting enzyme (ACE) I/D polymorphism and endothelial nitric oxide synthase gene (NOS3) 4a/4b, G894T and -T786C variants on the essential hypertension. The study group consisted of 1,027 Caucasian adults of Polish nationality (45.5 13.6 years old), of which 401 met the criteria for hypertension. Body weight, height and blood pressure were measured and data on self-reported smoking status were collected. Fasting blood glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides were determined by standard procedures. The ACE I/D polymorphism and three polymorphisms in NOS3 gene (4a/4b, G894T, -T786C) were detected by the PCR method. Multivariable logistic regression demonstrated that age above 45 years, diabetes, dyslipidemia, smoking and male sex are important risk factors for hypertension and no significant influence of variants in ACE and NOS3 genes on this risk was recognized. Obese subjects had a 3.27-times higher risk (OR = 3.27, 95% CI: 2.37 - 4.52) of hypertension than non-obese, and in obese the NOS3 894T allele was associated with 1.37 fold higher risk of hypertension (P = 0.031). The distribution of NOS3 G894T genotypes supported the co-dominant (OR = 1.35, P = 0.034, Pfit = 0.435) or recessive (OR = 2.00, P = 0.046, Pfit = 0.286), but not dominant model of inheritance (P = 0.100). The study indicates that in obese NOS3 G894T polymorphism may enhance hypertension risk. However, in the presence of such strong risk factors as age, diabetes and smoking, the impact of this genetic variant seems to be attenuated. Further studies are needed to reveal the usefulness of G894T polymorphism in hypertension risk assessment in obese. PMID:26579574

  1. Assessment of FBA Based Gene Essentiality Analysis in Cancer with a Fast Context-Specific Network Reconstruction Method

    PubMed Central

    Tobalina, Luis; Pey, Jon; Rezola, Alberto; Planes, Francisco J.

    2016-01-01

    Motivation Gene Essentiality Analysis based on Flux Balance Analysis (FBA-based GEA) is a promising tool for the identification of novel metabolic therapeutic targets in cancer. The reconstruction of cancer-specific metabolic networks, typically based on gene expression data, constitutes a sensible step in this approach. However, to our knowledge, no extensive assessment on the influence of the reconstruction process on the obtained results has been carried out to date. Results In this article, we aim to study context-specific networks and their FBA-based GEA results for the identification of cancer-specific metabolic essential genes. To that end, we used gene expression datasets from the Cancer Cell Line Encyclopedia (CCLE), evaluating the results obtained in 174 cancer cell lines. In order to more clearly observe the effect of cancer-specific expression data, we did the same analysis using randomly generated expression patterns. Our computational analysis showed some essential genes that are fairly common in the reconstructions derived from both gene expression and randomly generated data. However, though of limited size, we also found a subset of essential genes that are very rare in the randomly generated networks, while recurrent in the sample derived networks, and, thus, would presumably constitute relevant drug targets for further analysis. In addition, we compare the in-silico results to high-throughput gene silencing experiments from Project Achilles with conflicting results, which leads us to raise several questions, particularly the strong influence of the selected biomass reaction on the obtained results. Notwithstanding, using previous literature in cancer research, we evaluated the most relevant of our targets in three different cancer cell lines, two derived from Gliobastoma Multiforme and one from Non-Small Cell Lung Cancer, finding that some of the predictions are in the right track. PMID:27145226

  2. Analysis of renin-angiotensin aldosterone system gene polymorphisms in malaysian essential hypertensive and type 2 diabetic subjects

    PubMed Central

    Ramachandran, Vasudevan; Ismail, Patimah; Stanslas, Johnson; Shamsudin, Norashikin

    2009-01-01

    Background The renin-angiotensin aldosterone system (RAAS) plays an important role in regulating the blood pressure and the genetic polymorphisms of RAAS genes has been extensively studied in relation to the cardiovascular diseases in various populations with conflicting results. The aim of this study was to determine the association of five genetic polymorphisms (A6G and A20C of angiotensinogen (AGT), MboI of renin, Gly460Trp of aldosterone synthase and Lys173Arg of adducin) of RAAS genes in Malaysian essential hypertensive and type 2 diabetic subjects. Methods RAAS gene polymorphisms were determined using mutagenically separated PCR and PCR-RFLP method in a total of 270 subjects consisting of 70 hypertensive subjects without type 2 diabetes mellitus (T2DM), 60 T2DM, 65 hypertensive subjects with T2DM and 75 control subjects. Results There was significant difference found in age, body mass index, systolic/diastolic blood pressure, fasting plasma glucose and high density lipoprotein cholesterol levels between the hypertensive subjects with or without T2DM and control subjects. No statistically significant differences between groups were found in the allele frequency and genotype distribution for A20C variant of AGT gene, MboI of renin, Gly460Trp of aldosterone and Lys173Arg of adducin (p > 0.05). However, the results for A6G of AGT gene revealed significant differences in allele and genotype frequencies in essential hypertension with or without T2DM (p < 0.001). Conclusion Among the five polymorphisms of RAAS genes only A6G variant of AGT gene was significantly associated in Malaysian essential hypertensive and type 2 diabetic subjects. Therefore, A6G polymorphism of the AGT gene could be a potential genetic marker for increased susceptibility to essential hypertension with or without T2DMin Malaysian subjects. PMID:19243623

  3. Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum.

    PubMed

    Jeršek, Barbara; Poklar Ulrih, Nataša; Skrt, Mihaela; Gavarić, Neda; Božin, Biljana; Smole Možina, Sonja

    2014-06-01

    Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at ¼ and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at ¼ MIC (0.0313 mg mL-1). Essential oil of oregano (¼ MIC, 0.2930 μL mL-1) and carvacrol (½ MIC, 0.1953 μL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed. PMID:24945417

  4. The art gene product of human immunodeficiency virus is required for replication.

    PubMed Central

    Terwilliger, E; Burghoff, R; Sia, R; Sodroski, J; Haseltine, W; Rosen, C

    1988-01-01

    A mutation that alters the initiation codon of the art gene of the human immunodeficiency virus renders the virus replication defective. The replication defect of this mutation can be complemented in trans by the art gene product. These findings indicate that the art protein is essential for replication and, as such, represents a good target for development of new antiviral agents that could be useful in the control of human immunodeficiency virus infection. Images PMID:3257271

  5. Bifunctional Gene Cluster lnqBCDEF Mediates Bacteriocin Production and Immunity with Differential Genetic Requirements

    PubMed Central

    Iwatani, Shun; Horikiri, Yuko; Zendo, Takeshi; Nakayama, Jiro

    2013-01-01

    A comprehensive gene disruption of lacticin Q biosynthetic cluster lnqQBCDEF was carried out. The results demonstrated the necessity of the complete set of lnqQBCDEF for lacticin Q production, whereas immunity was flexible, with LnqEF (ABC transporter) being essential for and LnqBCD partially contributing to immunity. PMID:23335763

  6. FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division.

    PubMed

    Kadirjan-Kalbach, Deena K; Yoder, David W; Ruckle, Michael E; Larkin, Robert M; Osteryoung, Katherine W

    2012-12-01

    The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map-based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1-1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de-etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T-DNA insertion allele, ftsHi1-2, caused embryo-lethality, indicating that FtsHi1 is an essential gene product. A wild-type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1-1 and the embryo-lethal phenotype of ftsHi1-2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild-type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope-associated process that may couple plastid development with division. PMID:22900897

  7. Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

    PubMed Central

    Ebert, Matthias; Laaß, Sebastian; Burghartz, Melanie; Petersen, Jörn; Koßmehl, Sebastian; Wöhlbrand, Lars; Rabus, Ralf; Wittmann, Christoph; Jahn, Dieter

    2013-01-01

    Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na+/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies. PMID:23974024

  8. Repression of Lateral Organ Boundary Genes by PENNYWISE and POUND-FOOLISH Is Essential for Meristem Maintenance and Flowering in Arabidopsis1[OPEN

    PubMed Central

    Khan, Madiha; Ragni, Laura; Tabb, Paul; Salasini, Brenda C.; Chatfield, Steven; Datla, Raju; Lock, John; Kuai, Xiahezi; Després, Charles; Proveniers, Marcel; Yongguo, Cao; Xiang, Daoquan; Morin, Halima; Rullière, Jean-Pierre; Citerne, Sylvie; Hepworth, Shelley R.; Pautot, Véronique

    2015-01-01

    In the model plant Arabidopsis (Arabidopsis thaliana), endogenous and environmental signals acting on the shoot apical meristem cause acquisition of inflorescence meristem fate. This results in changed patterns of aerial development seen as the transition from making leaves to the production of flowers separated by elongated internodes. Two related BEL1-like homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), fulfill this transition. Loss of function of these genes impairs stem cell maintenance and blocks internode elongation and flowering. We show here that pny pnf apices misexpress lateral organ boundary genes BLADE-ON-PETIOLE1/2 (BOP1/2) and KNOTTED-LIKE FROM ARABIDOPSIS THALIANA6 (KNAT6) together with ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1). Inactivation of genes in this module fully rescues pny pnf defects. We further show that BOP1 directly activates ATH1, whereas activation of KNAT6 is indirect. The pny pnf restoration correlates with renewed accumulation of transcripts conferring floral meristem identity, including FD, SQUAMOSA PROMOTER-BINDING PROTEIN LIKE genes, LEAFY, and APETALA1. To gain insight into how this module blocks flowering, we analyzed the transcriptome of BOP1-overexpressing plants. Our data suggest a central role for the microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE-microRNA172 module in integrating stress signals conferred in part by promotion of jasmonic acid biosynthesis. These data reveal a potential mechanism by which repression of lateral organ boundary genes by PNY-PNF is essential for flowering. PMID:26417006

  9. Repression of Lateral Organ Boundary Genes by PENNYWISE and POUND-FOOLISH Is Essential for Meristem Maintenance and Flowering in Arabidopsis.

    PubMed

    Khan, Madiha; Ragni, Laura; Tabb, Paul; Salasini, Brenda C; Chatfield, Steven; Datla, Raju; Lock, John; Kuai, Xiahezi; Després, Charles; Proveniers, Marcel; Yongguo, Cao; Xiang, Daoquan; Morin, Halima; Rullière, Jean-Pierre; Citerne, Sylvie; Hepworth, Shelley R; Pautot, Véronique

    2015-11-01

    In the model plant Arabidopsis (Arabidopsis thaliana), endogenous and environmental signals acting on the shoot apical meristem cause acquisition of inflorescence meristem fate. This results in changed patterns of aerial development seen as the transition from making leaves to the production of flowers separated by elongated internodes. Two related BEL1-like homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), fulfill this transition. Loss of function of these genes impairs stem cell maintenance and blocks internode elongation and flowering. We show here that pny pnf apices misexpress lateral organ boundary genes BLADE-ON-PETIOLE1/2 (BOP1/2) and KNOTTED-LIKE FROM ARABIDOPSIS THALIANA6 (KNAT6) together with ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1). Inactivation of genes in this module fully rescues pny pnf defects. We further show that BOP1 directly activates ATH1, whereas activation of KNAT6 is indirect. The pny pnf restoration correlates with renewed accumulation of transcripts conferring floral meristem identity, including FD, SQUAMOSA PROMOTER-BINDING PROTEIN LIKE genes, LEAFY, and APETALA1. To gain insight into how this module blocks flowering, we analyzed the transcriptome of BOP1-overexpressing plants. Our data suggest a central role for the microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE-microRNA172 module in integrating stress signals conferred in part by promotion of jasmonic acid biosynthesis. These data reveal a potential mechanism by which repression of lateral organ boundary genes by PNY-PNF is essential for flowering. PMID:26417006

  10. The essential oil of bergamot stimulates reactive oxygen species production in human polymorphonuclear leukocytes.

    PubMed

    Cosentino, Marco; Luini, Alessandra; Bombelli, Raffaella; Corasaniti, Maria T; Bagetta, Giacinto; Marino, Franca

    2014-08-01

    Bergamot (Citrus aurantium L. subsp. bergamia) essential oil (BEO) is used in folk medicine as an antiseptic and anthelminthic and to facilitate wound healing. Evidence indicates that BEO has substantial antimicrobial activity; however its effects on immunity have never been examined. We studied the effects of BEO on reactive oxygen species (ROS) production in human polymorphonuclear leukocytes (PMN) and the role of Ca(2+) in the functional responses evoked by BEO in these cells. Results show that BEO increased intracellular ROS production in human PMN, an effect that required the contribution of extracellular (and, to a lesser extent, of intracellular) Ca(2+) . Bergamot essential oil also significantly increased ROS production induced by the chemotactic peptide N-formyl-Met-Leu-Phe and reduced the response to the protein kinase C activator phorbol myristate acetate. In conclusion, this is the first report showing the ability of BEO to increase ROS production in human PMN. This effect could both contribute to the activity of BEO in infections and in tissue healing as well as underlie an intrinsic proinflammatory potential. The relevance of these findings for the clinical uses of BEO needs careful consideration. PMID:24458921

  11. Finding a novel interacting protein of the hepatic carcinoma related gene MIP: NF-κB essential modulator (NEMO).

    PubMed

    Ye, Xiaoxia; Lu, Hong; Huo, Keke; Chen, Dong

    2011-01-01

    MafF interacting protein (MIP) is product of a candidate gene related to liver cancer development and progression. Here, we demonstrated that MIP could inhibit the proliferation of cancer cells through colony formation assays, and the inhibition ability of MIP to SMMC7721 cells was notably stronger than to HeLa cells. Using pGBKT7-MIP as bait, a human placenta cDNA library was screened using yeast two-hybrid system and a middle fragment of the NF-κB essential modulator (NEMO) was obtained as a novel important MIP interacting protein fragment, which contained 228 amino acid sequence from the 120 to 347 residue. Then the full coding sequence of NEMO was amplified from Clontech Placenta Marathon cDNA library and yeast mating assay verified the interaction of MIP and full length NEMO in yeast. GST pull-down assay and co-immunoprecipitation assay confirmed that MIP bound to NEMO specifically and directly. These results indicated that MIP interacted with NEMO and suggested that MIP could be involved in NF-κB signaling pathway, which is helpful to clarify the inhibition function of MIP to cancer cell proliferation. PMID:21109981

  12. Inhibitory Effect of Essential Oils on Aspergillus ochraceus Growth and Ochratoxin A Production

    PubMed Central

    Selvaraj, Jonathan Nimal; Wang, Yan; Zhao, Yueju; Zhou, Lu; Liu, Xiao; Liu, Yang

    2014-01-01

    Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains. PMID:25255251

  13. Inhibitory effect of essential oils on Aspergillus ochraceus growth and ochratoxin A production.

    PubMed

    Hua, Huijuan; Xing, Fuguo; Selvaraj, Jonathan Nimal; Wang, Yan; Zhao, Yueju; Zhou, Lu; Liu, Xiao; Liu, Yang

    2014-01-01

    Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150-250 µL/L with fumigation and 250-500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥ 100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥ 75 µg/mL and ≥ 150 µg/mL respectively, while natural cinnamaldehyde couldn't fully inhibit OTA production at ≤ 200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains. PMID:25255251

  14. Validation Framework for USGS Landsat-derived Essential Climate Variables: the Burned Area Product Example

    NASA Astrophysics Data System (ADS)

    Mladinich, C. S.; Brunner, N. M.; Beal, Y. G.

    2013-12-01

    The U.S. Geological Survey (USGS) is generating a suite of Essential Climate Variables (ECVs), as defined by the Global Climate Observing System program, from the Landsat data archive. The Landsat archive will provide high spatial resolution (30 m) and long-term (1972 to present) global land products, meeting the needs of climate and ecological studies at global, national, and regional scales. Validation protocols for these products are being established, paralleling the Committee on Earth Observing Satellites (CEOS) Calibration/Validation Working Groups' best practice guidelines, but also being modified to account for the unique characteristics of the Landsat data. The USGS validation plan is unique in that it incorporates protocols that span not only the breadth of ecoregions but the timespan of the ECV products and Landsat satellite sensors (MSS, TM, TM+, and OLI). To achieve these goals, the incorporation of existing data bases is essential. Protocols are being developed to perform a CEOS Working Group on Calibration/Validation Stage 2 validation with plans on performing a full Stage 4 validation ensuring the spatial and temporal consistency of the ECV products. A Stage 2 validation reports product accuracies over a large number of locations and time periods by comparison with in situ or other suitable reference data. The Stage 3 validation reports product uncertainties in a statistically robust way over multiple locations and time periods representing global conditions. Validation at this stage reports on the accuracies and confidence of products for the user communities as well as to the algorithm developers. The Stage 4 validation calls for continual assessments as new product versions of the algorithms are released. This presentation will report on the validation protocols used for the Burned Area ECV product. The burned area ECV product is unique from other ECV products such as land cover or LAI because of the transitory nature of fires. In the United States, the use of existing fire perimeter data bases from various state and federal agencies as reference data is economical and enables the validation of different time periods and locations. Additionally, the incorporation of existing satellite-derived reference data used to validate other coarser resolution global burned area data sets such as the MCD45 (Moderate Resolution Imaging Spectroradiometer (MODIS) sensor, 500 m spatial resolution), GlobCarbon (Along Track Scanning Radiometer (ATSR) sensor, 1 km spatial resolution), and L3JRC (SPOT-VEGETATION sensor, 1 km spatial resolution) is also being pursued. The validation the approach developed for the USGS ECV products and the challenges of using the vector polygons and raster layers from these reference datasets will be reported in the presentation.

  15. Novel Tissue Level Effects of the Staphylococcus aureus Enterotoxin Gene Cluster Are Essential for Infective Endocarditis

    PubMed Central

    Stach, Christopher S.; Vu, Bao G.; Merriman, Joseph A.; Herrera, Alfa; Cahill, Michael P.; Schlievert, Patrick M.; Salgado-Pabón, Wilmara

    2016-01-01

    Background Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens. Methods We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis. Results TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis. Conclusions Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis. PMID:27124393

  16. [Science of evaluating the characteristics, quality and safety of biotechnological products: RDNA-derived products, cell culture technology-derived products, gene therapy products, cellular therapy products, and transgenic animal-derived protein products and cellular products].

    PubMed

    Hayakawa, T

    1999-01-01

    Recent progress in biotechnology, including recombinant DNA (rDNA) technology and cell culture technology, has enabled us to produce new medically useful agents intended for human use. These agents include therapeutic peptides and proteins derived from rDNA-modified cell substrates, continuous cell lines, diploid cell lines, and hybridoma cell lines, gene therapy products, cellular therapy products, and therapeutic protein products and cellular products derived from transgenic animals. To enable these products to be of use in human therapy, it is essential that suitable measures be taken by manufacturers and regulatory authorities to assure their quality, efficacy, and safety. This article describes points based on the latest sound scientific principles to be considered when producing, testing, evaluating and controlling biotechnology products for human therapy, especially with respect to their characteristics, quality, and safety. PMID:10859934

  17. The Tumor Suppressor Gene, RASSF1A, Is Essential for Protection against Inflammation -Induced Injury

    PubMed Central

    Fiteih, Yahya; Law, Jennifer; Volodko, Natalia; Mohamed, Anwar; El-Kadi, Ayman O. S.; Liu, Lei; Odenbach, Jeff; Thiesen, Aducio; Onyskiw, Christina; Ghazaleh, Haya Abu; Park, Jikyoung; Lee, Sean Bong; Yu, Victor C.; Fernandez-Patron, Carlos; Alexander, R. Todd; Wine, Eytan; Baksh, Shairaz

    2013-01-01

    Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis. PMID:24146755

  18. GCN5 is essential for IRF-4 gene expression followed by transcriptional activation of Blimp-1 in immature B cells.

    PubMed

    Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo

    2014-03-01

    During B-cell differentiation, the gene expression of B-cell differentiation-related transcription factors must be strictly controlled by epigenetic mechanisms including histone acetylation and deacetylation, to complete the differentiation pathway. GCN5, one of the most important histone acetyltransferases, is involved in epigenetic events for transcriptional regulation through alterations in the chromatin structure. In this study, by analyzing the homozygous DT40 mutants GCN5(-/-), generated with gene targeting techniques, we found that GCN5 was necessary for transcriptional activation of IRF-4, an essential transcription factor for plasma cell differentiation. GCN5 deficiency caused drastic decreases in both the mRNA and the protein levels of Blimp-1 and IRF-4. The ectopic expression of Blimp-1 and IRF-4 suggests that IRF-4, but not Blimp-1, is the target gene of GCN5 in immature B cells. Moreover, a chromatin immunoprecipitation assay showed that GCN5 bound to the IRF-4 gene around its 5'-flanking region and acetylated H3K9 residues within chromatin surrounding the region in vivo, suggesting that gene expression of IRF-4 is certainly regulated by GCN5. These results reveal that GCN5 is essential for IRF-4 gene expression, followed by transcriptional activation of Blimp-1, and plays a key role in epigenetic regulation of B-cell differentiation. PMID:24072880

  19. Comparison of the Essential Cellular Functions of the Two murA Genes of Bacillus anthracis▿

    PubMed Central

    Kedar, G. C.; Brown-Driver, Vickie; Reyes, Daniel R.; Hilgers, Mark T.; Stidham, Mark A.; Shaw, Karen Joy; Finn, John; Haselbeck, Robert J.

    2008-01-01

    Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug. PMID:18378720

  20. Antagonistic activity of Ocimum sanctum L. essential oil on growth and zearalenone production by Fusarium graminearum in maize grains.

    PubMed

    Kalagatur, Naveen K; Mudili, Venkataramana; Siddaiah, Chandranayaka; Gupta, Vijai K; Natarajan, Gopalan; Sreepathi, Murali H; Vardhan, Batra H; Putcha, Venkata L R

    2015-01-01

    The present study was aimed to establish the antagonistic effects of Ocimum sanctum L. essential oil (OSEO) on growth and zearalenone (ZEA) production of Fusarium graminearum. GC-MS chemical profiling of OSEO revealed the existence of 43 compounds and the major compound was found to be eugenol (34.7%). DPPH free radical scavenging activity (IC50) of OSEO was determined to be 8.5 μg/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on F. graminearum were recorded as 1250 and 1800 μg/mL, respectively. Scanning electron microscope observations showed significant micro morphological damage in OSEO exposed mycelia and spores compared to untreated control culture. Quantitative UHPLC studies revealed that OSEO negatively effected the production of ZEA; the concentration of toxin production was observed to be insignificant at 1500 μg/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 μg/mL in OSEO untreated control culture. Reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13) revealed that increase in OSEO concentration (250-1500 μg/mL) significantly downregulated the expression of PKS4 and PKS13. These results were in agreement with the artificially contaminated maize grains as well. In conlusion, the antifungal and antimycotoxic effects of OSEO on F. graminearum in the present study reiterated that, the essential oil of O. sanctum could be a promising herbal fungicide in food processing industries as well as grain storage centers. PMID:26388846

  1. Antagonistic activity of Ocimum sanctum L. essential oil on growth and zearalenone production by Fusarium graminearum in maize grains

    PubMed Central

    Kalagatur, Naveen K.; Mudili, Venkataramana; Siddaiah, Chandranayaka; Gupta, Vijai K.; Natarajan, Gopalan; Sreepathi, Murali H.; Vardhan, Batra H.; Putcha, Venkata L. R.

    2015-01-01

    The present study was aimed to establish the antagonistic effects of Ocimum sanctum L. essential oil (OSEO) on growth and zearalenone (ZEA) production of Fusarium graminearum. GC–MS chemical profiling of OSEO revealed the existence of 43 compounds and the major compound was found to be eugenol (34.7%). DPPH free radical scavenging activity (IC50) of OSEO was determined to be 8.5 μg/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on F. graminearum were recorded as 1250 and 1800 μg/mL, respectively. Scanning electron microscope observations showed significant micro morphological damage in OSEO exposed mycelia and spores compared to untreated control culture. Quantitative UHPLC studies revealed that OSEO negatively effected the production of ZEA; the concentration of toxin production was observed to be insignificant at 1500 μg/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 μg/mL in OSEO untreated control culture. Reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13) revealed that increase in OSEO concentration (250–1500 μg/mL) significantly downregulated the expression of PKS4 and PKS13. These results were in agreement with the artificially contaminated maize grains as well. In conlusion, the antifungal and antimycotoxic effects of OSEO on F. graminearum in the present study reiterated that, the essential oil of O. sanctum could be a promising herbal fungicide in food processing industries as well as grain storage centers. PMID:26388846

  2. Lower ADD1 Gene Promoter DNA Methylation Increases the Risk of Essential Hypertension

    PubMed Central

    Yuan, Fang; Xu, Leiting; Xin, Yanfei; Fei, Li-Juan; Zhong, Qi-Long; Huang, Yi; Xu, Limin; Hao, Ling-Mei; Qiu, Xu-Jun; Le, Yanping; Ye, Meng; Duan, Shiwei

    2013-01-01

    The goal of our study is to investigate the contribution of promoter DNA methylation of α-adducin (ADD1) gene to the risk of essential hypertension (EH). Using the bisulphite pyrosequencing technology, DNA methylation levels of five CpG dinucleotides on ADD1 promoter were measured among 33 EH cases and 28 healthy controls. Significantly higher ADD1 DNA methylation levels were observed in the females than in the males (CpG1: P = 0.016; CpG2-5: P = 0.021). A breakdown analysis by gender showed that lower CpG1 methylation was associated with an increased risk of EH in females (adjusted P = 0.042). A much more significant association between lower CpG2-5 methylation levels and the increased risk of EH was found in males (adjusted P = 0.008). CpG1 methylation was inversely correlated with age in females (r = −0.407, P = 0.019) but not in males. ADD1 CpG1 and CpG2-5 methylation levels were significantly lower in post-menopausal (>50 years) women than pre-menopausal (≤50 years) women (CpG1: P = 0.006; CpG2-5: P = 0.034). A significant interaction between CpG1 methylation and age was found in females (CpG1*age: P = 0.029). CpG2-5 methylation was shown as a significant predictor of EH in males [area under curve (AUC) = 0.855, P = 0.001], in contrast that CpG1 methylation was a trend toward indicator in females (AUC = 0.699, P = 0.054). In addition, significant differences were observed between males and females for alanine aminotransferase (ALT, P = 0.001), aspartate aminotransferase (AST, P = 0.005) and uric acid (P<0.001). The concentration of AST was inversely correlated with ADD1 CpG2-5 methylation levels in female controls (r = −0.644, P = 0.024). These observations may bring new hints to elaborate the pathogenesis of EH. PMID:23691048

  3. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

    PubMed Central

    Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

    2014-01-01

    The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

  4. Combining Hierarchical and Associative Gene Ontology Relations with Textual Evidence in Estimating Gene and Gene Product Similarity

    SciTech Connect

    Sanfilippo, Antonio P.; Posse, Christian; Gopalan, Banu; Riensche, Roderick M.; Beagley, Nathaniel; Baddeley, Bob L.; Tratz, Stephen C.; Gregory, Michelle L.

    2007-03-01

    Gene and gene product similarity is a fundamental diagnostic measure in analyzing biological data and constructing predictive models for functional genomics. With the rising influence of the Gene Ontology, two complementary approaches have emerged where the similarity between two genes or gene products is obtained by comparing Gene Ontology (GO) annotations associated with the genes or gene products. One approach captures GO-based similarity in terms of hierarchical relations within each gene subontology. The other approach identifies GO-based similarity in terms of associative relations across the three gene subontologies. We propose a novel methodology where the two approaches can be merged with ensuing benefits in coverage and accuracy, and demonstrate that further improvements can be obtained by integrating textual evidence extracted from relevant biomedical literature.

  5. The Wilms’ Tumor Suppressor Gene (wt1) Product Regulates Dax-1 Gene Expression during Gonadal Differentiation

    PubMed Central

    Kim, Jungho; Prawitt, Dirk; Bardeesy, Nabeel; Torban, Elena; Vicaner, Caroline; Goodyer, Paul; Zabel, Bernard; Pelletier, Jerry

    1999-01-01

    Gonadal differentiation is dependent upon a molecular cascade responsible for ovarian or testicular development from the bipotential gonadal ridge. Genetic analysis has implicated a number of gene products essential for this process, which include Sry, WT1, SF-1, and DAX-1. We have sought to better define the role of WT1 in this process by identifying downstream targets of WT1 during normal gonadal development. We have noticed that in the developing murine gonadal ridge, wt1 expression precedes expression of Dax-1, a nuclear receptor gene. We document here that the spatial distribution profiles of both proteins in the developing gonad overlap. We also demonstrate that WT1 can activate the Dax-1 promoter. Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 regulates Dax-1 via GC-rich binding sites found upstream of the Dax-1 TATA box. We show that two WT1-interacting proteins, the product of a Denys-Drash syndrome allele of wt1 and prostate apoptosis response-4 protein, inhibit WT1-mediated transactivation of Dax-1. In addition, we demonstrate that WT1 can activate the endogenous Dax-1 promoter. Our results indicate that the WT1–DAX-1 pathway is an early event in the process of mammalian sex determination. PMID:10022915

  6. Insecticidal Constituents of Essential Oil Derived from Zanthoxylum armatum against Two Stored-Product Insects.

    PubMed

    Wang, Cheng-Fang; Zhang, Wen-Juan; You, Chun-Xue; Guo, Shan-Shan; Geng, Zhu-Feng; Fan, Li; Du, Shu-Shan; Deng, Zhi-Wei; Wang, Yong-Yan

    2015-01-01

    In the course of our search for natural bioactive chemicals and investigations on their insecticidal activities from some medicinal plants growing in China, the essential oil derived from the twigs of Zanthoxylum armatum (Rutaceae) was found to possess strong insecticidal activities against two stored-product insects, Lasioderma serricorne and Tribolium castaneum. A total of 32 constituents of the essential oil were identified by GC and GC-MS analysis, and it revealed (E)-anethole (20.5%), 1,8-cineole (14.0%), 2-tridecanone (12.5%), limonene (9.0%) and piperitone (8.0%) as major components, followed by β-phellandrene (6.3%), β-pinene (5.1%) and 4-terpineol (4.4%). From the essential oil, five compounds were isolated and identified as (E)-anethole, 1,8-cineole, 2-tridecanone, limonene and piperitone. The results of insecticidal bioassays showed that the essential oil of Z. armatum exhibited strong fumigant toxicity towards L. serricorne and T. castaneum with LC50 values of 13.83 and 4.28 mg/L air, respectively, and also possessed contact toxicity against two insect species with LD50 values of 18.74 and 32.16 μg/adult, respectively. Among the active compounds, piperitone performed the strongest fumigant toxicity against L. serricorne (LC50 = 1.21 mg/L air) and contact toxicity against T. castaneum (LD50 = 3.16 μg/adult). 1,8-Cineole, limonene and piperitone showed similar fumigant toxicity against T. castaneum with LC50 values of 5.47, 6.21 and 7.12 mg/L air, respectively. Meanwhile, L. serricorne was the most sensitive to 2-tridecanone (LD50 = 5.74 μg/adult) in the progress of contact toxicity assay. PMID:26179006

  7. A novel gene encoding a 54 kDa polypeptide is essential for butane utilization by Pseudomonas sp. IMT37.

    PubMed

    Padda, R S; Pandey, K K; Kaul, S; Nair, V D; Jain, R K; Basu, S K; Chakrabarti, T

    2001-09-01

    Twenty-three propane- and butane-utilizing bacteria were isolated from soil samples collected from oilfields. Three of them have been identified as Rhodococcus sp. IMT35, Pseudomonas sp. IMT37 and Pseudomonas sp. MT40. SDS-PAGE analysis of the membrane of Rhodococcus sp. IMT35 revealed the presence of at least four polypeptides induced by propane. Polyclonal antibody raised against a 58 kDa polypeptide from Rhodococcus sp. IMT35 specifically detected bacteria which were actively utilizing propane or butane. Immunoscreening of a genomic library in lambdagt11 with this antibody resulted in isolation of a clone containing a 4.9 kb EcoRI genomic DNA fragment. This 4.9 kb DNA fragment was found to hybridize specifically with organisms which could grow on propane or butane. This fragment could therefore be used as a probe for detection of such bacteria. A 2.3 kb fragment having an ORF encoding a polypeptide of 54 kDa was identified by screening a genomic library of Pseudomonas sp. IMT37 with this 4.9 kb EcoRI fragment. The sequence of the ORF (designated orf54) was found to be novel. Primer extension and S1 nuclease mapping showed that transcription of the ORF starts at base 283 and it had sequences upstream similar to that of a Pseudomonas promoter (-12, -24 type). Disruption of the ORF by a kanamycin ('kan') cassette prevented the organism from growing on any alkane but did not affect its ability to utilize the respective alkanols and acids, indicating that alcohol dehydrogenase and subsequent steps in the pathway remained unaltered. The mutants had no detectable level of butane monooxygenase activity. Therefore, the product of this gene plays a crucial role in the first step of the pathway and is an essential component of monooxygenase. The findings imply that this bacterium either employs a common genetic and metabolic route or at least shares the product of this gene for utilization of many alkanes. PMID:11535788

  8. By-product metals are technologically essential but have problematic supply.

    PubMed

    Nassar, N T; Graedel, T E; Harper, E M

    2015-04-01

    The growth in technological innovation that has occurred over the past decades has, in part, been possible because an increasing number of metals of the periodic table are used to perform specialized functions. However, there have been increasing concerns regarding the reliability of supply of some of these metals. A main contributor to these concerns is the fact that many of these metals are recovered only as by-products from a limited number of geopolitically concentrated ore deposits, rendering their supplies unable to respond to rapid changes in demand. Companionality is the degree to which a metal is obtained largely or entirely as a by-product of one or more host metals from geologic ores. The dependence of companion metal availability on the production of the host metals introduces a new facet of supply risk to modern technology. We evaluated companionality for 62 different metals and metalloids, and show that 61% (38 of 62) have companionality greater than 50%. Eighteen of the 38-including such technologically essential elements as germanium, terbium, and dysprosium-are further characterized as having geopolitically concentrated production and extremely low rates of end-of-life recycling. It is this subset of companion metals-vital in current technologies such as electronics, solar energy, medical imaging, energy-efficient lighting, and other state-of-the-art products-that may be at the greatest risk of supply constraints in the coming decades. PMID:26601159

  9. ROS Production Is Essential for the Apoptotic Function of E2F1 in Pheochromocytoma and Neuroblastoma Cell Lines

    PubMed Central

    Espada, Lilia; Meo-Evoli, Nathalie; Sancho, Patricia; Real, Sebastian; Fabregat, Isabel; Ambrosio, Santiago; Tauler, Albert

    2012-01-01

    In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator. PMID:23251571

  10. K-ras is an essential gene in the mouse with partial functional overlap with?N-ras

    PubMed Central

    Johnson, Leisa; Greenbaum, Doron; Cichowski, Karen; Mercer, Kim; Murphy, Elizabeth; Schmitt, Eric; Bronson, Roderick T.; Umanoff, Heywood; Edelmann, Windfried; Kucherlapati, Raju; Jacks, Tyler

    1997-01-01

    Mammalian ras genes are thought to be critical in the regulation of cellular proliferation and differentiation and are mutated in ?30% of all human tumors. However, N-ras and H-ras are nonessential for mouse development. To characterize the normal role of K-ras in growth and development, we have mutated it by gene targeting in the mouse. On an inbred genetic background, embryos homozygous for this mutation die between 12 and 14 days of gestation, with fetal liver defects and evidence of anemia. Thus, K-ras is the only member of the ras gene family essential for mouse embryogenesis. We have also investigated the effect of multiple mutations within the ras gene family. Most animals lacking N-ras function and heterozygous for the K-ras mutation exhibit abnormal hematopoietic development and die between days 10 and 12 of embryogenesis. Thus, partial functional overlap appears to occur within the ras gene family, but K-ras provides a unique and essential function. PMID:9334313

  11. Essential drugs production in Brazil, Russia, India, China and South Africa (BRICS): opportunities and challenges

    PubMed Central

    Ezziane, Zoheir

    2014-01-01

    The objective of this work is to elucidate various essential drugs in the Brazil, Russia, India, China and South Africa (BRICS) countries. It discusses the opportunities and challenges of the existing biotech infrastructure and the production of drugs and vaccines in member states of the BRICS. This research is based on a systematic literature review between the years 2000 and 2014 of documents retrieved from the databases Embase, PubMed/Medline, Global Health, and Google Scholar, and the websites of relevant international organizations, research institutions and philanthropic organizations. Findings vary from one member state to another. These include useful comparison between the BRICS countries in terms of pharmaceuticals expenditure versus total health expenditure, local manufacturing of drugs/vaccines using technology and know-how transferred from developed countries, and biotech entrepreneurial collaborations under the umbrella of the BRICS region. This study concludes by providing recommendations to support more of inter collaborations among the BRICS countries as well as between BRICS and many developing countries to shrink drug production costs. In addition, this collaboration would also culminate in reaching out to poor countries that are not able to provide their communities and patients with cost-effective essential medicines. PMID:25489593

  12. Essential drugs production in Brazil, Russia, India, China and South Africa (BRICS): opportunities and challenges.

    PubMed

    Ezziane, Zoheir

    2014-12-01

    The objective of this work is to elucidate various essential drugs in the Brazil, Russia, India, China and South Africa (BRICS) countries. It discusses the opportunities and challenges of the existing biotech infrastructure and the production of drugs and vaccines in member states of the BRICS. This research is based on a systematic literature review between the years 2000 and 2014 of documents retrieved from the databases Embase, PubMed/Medline, Global Health, and Google Scholar, and the websites of relevant international organizations, research institutions and philanthropic organizations. Findings vary from one member state to another. These include useful comparison between the BRICS countries in terms of pharmaceuticals expenditure versus total health expenditure, local manufacturing of drugs/vaccines using technology and know-how transferred from developed countries, and biotech entrepreneurial collaborations under the umbrella of the BRICS region. This study concludes by providing recommendations to support more of inter collaborations among the BRICS countries as well as between BRICS and many developing countries to shrink drug production costs. In addition, this collaboration would also culminate in reaching out to poor countries that are not able to provide their communities and patients with cost-effective essential medicines. PMID:25489593

  13. Gene Network Rewiring to Study Melanoma Stage Progression and Elements Essential for Driving Melanoma

    PubMed Central

    Kaushik, Abhinav; Bhatia, Yashuma; Ali, Shakir; Gupta, Dinesh

    2015-01-01

    Metastatic melanoma patients have a poor prognosis, mainly attributable to the underlying heterogeneity in melanoma driver genes and altered gene expression profiles. These characteristics of melanoma also make the development of drugs and identification of novel drug targets for metastatic melanoma a daunting task. Systems biology offers an alternative approach to re-explore the genes or gene sets that display dysregulated behaviour without being differentially expressed. In this study, we have performed systems biology studies to enhance our knowledge about the conserved property of disease genes or gene sets among mutually exclusive datasets representing melanoma progression. We meta-analysed 642 microarray samples to generate melanoma reconstructed networks representing four different stages of melanoma progression to extract genes with altered molecular circuitry wiring as compared to a normal cellular state. Intriguingly, a majority of the melanoma network-rewired genes are not differentially expressed and the disease genes involved in melanoma progression consistently modulate its activity by rewiring network connections. We found that the shortlisted disease genes in the study show strong and abnormal network connectivity, which enhances with the disease progression. Moreover, the deviated network properties of the disease gene sets allow ranking/prioritization of different enriched, dysregulated and conserved pathway terms in metastatic melanoma, in agreement with previous findings. Our analysis also reveals presence of distinct network hubs in different stages of metastasizing tumor for the same set of pathways in the statistically conserved gene sets. The study results are also presented as a freely available database at http://bioinfo.icgeb.res.in/m3db/. The web-based database resource consists of results from the analysis presented here, integrated with cytoscape web and user-friendly tools for visualization, retrieval and further analysis. PMID:26558755

  14. Integration of two essential virulence modulating signals at the Erwinia chrysanthemi pel gene promoters: a role for Fis in the growth-phase regulation.

    PubMed

    Lautier, Thomas; Blot, Nicolas; Muskhelishvili, Georgi; Nasser, William

    2007-12-01

    Production of the essential virulence factors, called pectate lyases (Pels), in the phytopathogenic bacterium Erwinia chrysanthemi is controlled by a complex regulation system and responds to various stimuli, such as the presence of pectin or plant extracts, growth phase, temperature and iron concentration. The presence of pectin and growth phase are the most important signals identified. Eight regulators modulating the expression of the pel genes (encoding Pels) have been characterized. These regulators are organized in a network allowing a sequential functioning of the regulators during infection. Although many studies have been carried out, the mechanisms of control of Pel production by growth phase have not yet been elucidated. Here we report that a fis mutant of E. chrysanthemi showed a strong increase in transcription of the pel genes during exponential growth whereas induction of expression in the parental strain occurred at the end of exponential growth. This reveals that Fis acts to prevent an efficient transcription of pel genes at the beginning of exponential growth and also provides evidence of the involvement of Fis in the growth-phase regulation of the pel genes. By using in vitro DNA-protein interactions and transcription experiments, we find that Fis directly represses the pel gene expression at the transcription initiation step. In addition, we show that Fis acts in concert with KdgR, the main repressor responding to the presence of pectin compounds, to shut down the pel gene transcription. Finally, we find that active Fis is required for the efficient translocation of the Pels in growth medium. Together, these data indicate that Fis tightly controls the availability of Pels during pathogenesis by acting on both their production and their translocation in the external medium. PMID:18028312

  15. Functional analysis of UMOD gene and its effect on inflammatory cytokines in serum of essential hypertension patients

    PubMed Central

    Jian, Liguo; Fa, Xian’en; Zhou, Zheng; Liu, Shichao

    2015-01-01

    Objective: The study aimed to investigate the function of uromodulin (UMOD) gene and its effect on inflammatory cytokines in serum of essential hypertension patients. Methods: The online database and software of computer were used for bioinformatics analysis on UMOD gene as well as the structure and function of its encoding proteins. Moreover, radioimmunoassay and enzyme linked immunosorbent assay was adopted to validate the content of urine UMOD protein of essential hypertension patients and their serum inflammatory cytokines. Results: As an alkaline and hydrophilic protein, UMOD has no transmembrane region, but it does have a signal peptide sequence. It is mainly located extracellularly, belonging to a secreted protein, whose secondary structure was based mainly on Random coil which account for 58.44%. According to function prediction, it is found that the UMOD protein has stress response which may be participate in the inflammatory reaction. It has been observed from the experiment which was designed on the basis of the correlation between inflammation reaction and essential hypertension that the content of urine UMOD protein of essential hypertension patients who is in stage I was (28.71±10.53) mg/24 h and when compared with the control group’s content (30.15±14.10 mg/24 h), the difference was not obviously; The content of urine UMOD protein of essential hypertension patients who’s in stage II and III was (18.24±6.12) mg/24 h and (9.43±3.16) mg/24 h, respectively, which were obviously lower than that of the control group (P<0.01). Additionally, the serum inflammatory cytokines, such as TNF-α, IL-6 and IL1-α content of essential hypertension patients were all markedly higher than that of control group (P<0.05). Conclusion: For essential hypertension patients, there’s a close relationship between the expression level of UMOD gene and inflammatory cytokines, which were manifested as the negative correlation between the level of the gene’s expression and inflammatory cytokines. That has certain reference value to realize the targeted treatment for essential hypertension through regulated blood pressure conversely in the view of expression level of inflammatory cytokines. PMID:26617860

  16. Genetic association study of glucocerebrosidase gene L444P mutation in essential tremor and multiple system atrophy in mainland China.

    PubMed

    Sun, Qi-ying; Guo, Ji-feng; Han, Wei-wei; Zuo, Xing; Wang, Lei; Yao, Ling-yan; Pan, Qian; Xia, Kun; Yan, Xin-xiang; Tang, Bei-sha

    2013-02-01

    The glucocerebrosidase (GBA) gene mutation is emerging as an important risk factor for Parkinson's disease. We previously reported that the GBA gene L444P mutation is an important risk factor for PD in the Chinese population. The prevalence of this mutation in other neurodegenerative diseases and movement disorders remains completely unexplored in mainland China. In the present study, we extended the screening of GBA gene L444P mutation to Chinese patients with essential tremor (ET) and multiple system atrophy (MSA). We searched for the GBA gene L444P mutation in 109 patients with ET, 54 patients with MSA, and 657 controls from mainland China. None of the 109 patients with ET or 54 patients with MSA carried the GBA gene L444P mutation. Among the 657 controls, we found one L444P heterozygote. The difference in mutation frequencies between patients with ET or MSA and the control group was not statistically significant (chi-squared test, p = 1, respectively). The results suggest that the GBA gene L444P mutation may be not responsible for ET in mainland China. Whether the GBA gene L444P mutation modifies the risk for MSA deserves further study in larger samples. PMID:23151436

  17. Gene-product relationships of fhlA and fdv genes of Escherichia coli.

    PubMed Central

    Sankar, P; Lee, J H; Shanmugam, K T

    1988-01-01

    Synthesis of formate dehydrogenase coupled to formate hydrogenlyase activity in Escherichia coli was found to require the product of the fhlA gene. Transcription of fdhF, the gene coding for the 80-kilodalton (kDa) selenopeptide of formate dehydrogenase, was not detected in an fhlA genetic background. Mutations in the fhlA gene also abolished production of the hydrogenase activity associated with formate hydrogenlyase activity. The fhlA gene resides next to the hydB gene at 59 min in the E. coli chromosome, and the two genes are transcribed in opposite directions. The fhlA gene codes for a 78-kDa protein. A neighboring gene, fdv, codes for an 82-kDa protein, and the physiological role of this gene product is unknown, although a role in H2 metabolism can be detected. Images PMID:3056900

  18. Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis.

    PubMed

    Shockey, Jay; Regmi, Anushobha; Cotton, Kimberly; Adhikari, Neil; Browse, John; Bates, Philip D

    2016-01-01

    The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. PMID:26586834

  19. Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis1[OPEN

    PubMed Central

    Browse, John

    2016-01-01

    The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. PMID:26586834

  20. Application of microencapsulated essential oils in cosmetic and personal healthcare products - a review.

    PubMed

    Carvalho, I T; Estevinho, B N; Santos, L

    2016-04-01

    Nowadays, the consumers around the world are increasingly focused on health and beauty. The renewed consumer interest in natural cosmetic products creates the demand for new products and reformulated others with botanical and functional ingredients. In cosmetic products, essential oils (EOs) play a major role as fragrance ingredients. They can optimize its proprieties and preservation, as well as the marketing image of the final product. Microencapsulation of EOs can protect and prevent the loss of volatile aromatic ingredients and improve the controlled release and stability of this core materials. The importance of EOs for cosmetic industry and its microencapsulation was reviewed in this study. Also a briefly introduction about the preparation of microparticles was presented. Some of the most important and usual microencapsulation techniques of EOs, as well as the conventional encapsulating agents, were discussed. Despite the fact that microencapsulation of EOs is a very promising and extremely attractive application area for cosmetic industry, further basic research needs to be carried out, for a better understanding of the biofunctional activities of microencapsulated EOs and its release modulation, as well as the effects of others cosmetic ingredients and the storage time in the microparticles properties. PMID:25923295

  1. By-product metals are technologically essential but have problematic supply

    PubMed Central

    Nassar, N. T.; Graedel, T. E.; Harper, E. M.

    2015-01-01

    The growth in technological innovation that has occurred over the past decades has, in part, been possible because an increasing number of metals of the periodic table are used to perform specialized functions. However, there have been increasing concerns regarding the reliability of supply of some of these metals. A main contributor to these concerns is the fact that many of these metals are recovered only as by-products from a limited number of geopolitically concentrated ore deposits, rendering their supplies unable to respond to rapid changes in demand. Companionality is the degree to which a metal is obtained largely or entirely as a by-product of one or more host metals from geologic ores. The dependence of companion metal availability on the production of the host metals introduces a new facet of supply risk to modern technology. We evaluated companionality for 62 different metals and metalloids, and show that 61% (38 of 62) have companionality greater than 50%. Eighteen of the 38—including such technologically essential elements as germanium, terbium, and dysprosium—are further characterized as having geopolitically concentrated production and extremely low rates of end-of-life recycling. It is this subset of companion metals—vital in current technologies such as electronics, solar energy, medical imaging, energy-efficient lighting, and other state-of-the-art products—that may be at the greatest risk of supply constraints in the coming decades. PMID:26601159

  2. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

    PubMed

    Ward, J E; Dale, E M; Christie, P J; Nester, E W; Binns, A N

    1990-09-01

    The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity. PMID:2203743

  3. Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat.

    PubMed

    Cheng, Wei; Song, Xiu-Shi; Li, He-Ping; Cao, Le-Hui; Sun, Ke; Qiu, Xiao-Li; Xu, Yu-Bin; Yang, Peng; Huang, Tao; Zhang, Jing-Bo; Qu, Bo; Liao, Yu-Cai

    2015-12-01

    Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co-expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T3 to T5 generations. Confocal microscopy revealed profoundly restricted mycelia in Fg-infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down-regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host-induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions. PMID:25735638

  4. An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

    PubMed

    Phadnis, Nitin; Baker, EmilyClare P; Cooper, Jacob C; Frizzell, Kimberly A; Hsieh, Emily; de la Cruz, Aida Flor A; Shendure, Jay; Kitzman, Jacob O; Malik, Harmit S

    2015-12-18

    Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems. PMID:26680200

  5. Identification and isolation of genes essential for H sub 2 oxidation in Rhodobacter capsulatus. [Hydrogenase

    SciTech Connect

    Xu, H.W.; Love, J.; Borghese, R.; Wall, J.D. )

    1989-02-01

    Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H{sub 2} and CO{sub 2} were isolated. Those lacking uptake hydrogenase activity as measured by H{sub 2}-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon Tn5 mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret to mean that one or both structural genes were among the genes isolated.

  6. Integrating Ontological Knowledge and Textual Evidence in Estimating Gene and Gene Product Similarity

    SciTech Connect

    Sanfilippo, Antonio P.; Posse, Christian; Gopalan, Banu; Tratz, Stephen C.; Gregory, Michelle L.

    2006-06-08

    With the rising influence of the Gene On-tology, new approaches have emerged where the similarity between genes or gene products is obtained by comparing Gene Ontology code annotations associ-ated with them. So far, these approaches have solely relied on the knowledge en-coded in the Gene Ontology and the gene annotations associated with the Gene On-tology database. The goal of this paper is to demonstrate that improvements to these approaches can be obtained by integrating textual evidence extracted from relevant biomedical literature.

  7. Nucleotide sequence of Bacillus subtilis dnaB: a gene essential for DNA replication initiation and membrane attachment.

    PubMed Central

    Hoshino, T; McKenzie, T; Schmidt, S; Tanaka, T; Sueoka, N

    1987-01-01

    The complete nucleotide sequence of the Bacillus subtilis dnaB gene and its flanking regions was determined. The dnaB gene is essential for both replication initiation and membrane attachment of the origin region of the chromosome and plasmid pUB110. It has been known that there are two different classes (dnaBI and dnaBII) in the dnaB mutants; dnaBI is essential for both chromosome and pUB110 replication, whereas dnaBII is necessary only for chromosome replication. The nucleotide sequence revealed that dnaBI and dnaBII are two functional domains in the single dnaB gene. The mutation sites of two mutants, belonging to dnaBI and dnaBII, respectively, were also determined as substitutions of amino acids. The putative DnaB protein deduced from nucleotide sequence consists of 472 amino acids (55 kDa) with no cysteine residue. A 55-kDa polypeptide produced in an in vitro transcription-translation system was labeled with [35S]methionine but not with [35S]cysteine. The DnaB protein has a highly hydrophobic sequence of 20 amino acids in its N-terminal region, a possible DNA binding site, and two possible ATP binding sites. The dnaBI domain is between the DNA binding site and one of the ATP binding sites; the dnaBII domain is close to the other ATP binding site. Comparison of the amino acid sequence between the "dnaB protein" and those of other dna genes of Escherichia coli showed no homology, suggesting that the dnaB gene of B. subtilis may be analogous to a hitherto undiscovered gene in E. coli. Images PMID:3027697

  8. NEON Data Products: Supporting the Validation of GCOS Essential Climate Variables

    NASA Astrophysics Data System (ADS)

    Petroy, S. B.; Fox, A. M.; Metzger, S.; Thorpe, A.; Meier, C. L.

    2014-12-01

    The National Ecological Observatory Network (NEON) is a continental-scale ecological observation platform designed to collect and disseminate data that contributes to understanding and forecasting the impacts of climate change, land use change, and invasive species on ecology. NEON will collect in-situ and airborne data over 60 sites across the US, including Alaska, Hawaii, and Puerto Rico. The NEON Biomass, Productivity, and Biogeochemistry protocols currently direct the collection of samples from distributed, gradient, and tower plots at each site, with sampling occurring either multiple times during the growing season, annually, or on three- or five-year centers (e.g. for coarse woody debris). These data are processed into a series of field-derived data products (e.g. Biogeochemistry, LAI, above ground Biomass, etc.), and when combined with the NEON airborne hyperspectral and LiDAR imagery, are used support validation efforts of algorithms for deriving vegetation characteristics from the airborne data. Sites are further characterized using airborne data combined with in-situ tower measurements, to create additional data products of interest to the GCOS community, such as Albedo and fPAR. Presented here are a summary of tower/field/airborne sampling and observation protocols and examples of provisional datasets collected at NEON sites that may be used to support the ongoing validation of GCOS Essential Climate Variables.

  9. Essential oils nanoformulations for stored-product pest control - characterization and biological properties.

    PubMed

    Werdin Gonzlez, Jorge Omar; Gutirrez, Mara Mercedes; Ferrero, Adriana Alicia; Fernndez Band, Beatriz

    2014-04-01

    The lethal and sublethal activity of poly(ethylene glycol) (PEG) nanoparticles containing essential oils (EO), also the physicochemical characterization, were determined against Tribolium castaneum and Rhizopertha dominica. The 10% ratio EO-PEG nanoparticles showed an average diameter<235 nm (PDI<0.280) and a loading efficacy>75%; after 6 month of storage their size did not change significantly and the amount of the EOs decreased 25%, approximately. Furthermore, during this period, no chemical derivates were observed. The EOs nanoparticles produced a notable increase of the residual contact toxicity apparently due to the slow and persistent release of the active terpenes. In addition, the nanoformulation enhanced the EO contact toxicity and altered the nutritional physiology of both stored product pest. The results indicated that these novel systems could be used in integrated pest management program for T. castaneum and R. dominica control. PMID:24359912

  10. Eukaryote to gut bacteria transfer of a glycoside hydrolase gene essential for starch breakdown in plants

    PubMed Central

    Arias, Maria Cecilia; Danchin, Étienne G.J.; Coutinho, Pedro; Henrissat, Bernard; Ball, Steven

    2012-01-01

    Lateral gene transfer (LGT) between bacteria constitutes a strong force in prokaryote evolution, transforming the hierarchical tree of life into a network of relationships between species. In contrast, only a few cases of LGT from eukaryotes to prokaryotes have been reported so far. The distal animal intestine is predominantly a bacterial ecosystem, supplying the host with energy from dietary polysaccharides through carbohydrate-active enzymes absent from its genome. It has been suggested that LGT is particularly important for the human microbiota evolution. Here we show evidence for the first eukaryotic gene identified in multiple gut bacterial genomes. We found in the genome sequence of several gut bacteria, a typically eukaryotic glycoside-hydrolase necessary for starch breakdown in plants. The distribution of this gene is patchy in gut bacteria with presence otherwise detected only in a few environmental bacteria. We speculate that the transfer of this gene to gut bacteria occurred by a sequence of two key LGT events; first, an original eukaryotic gene was transferred probably from Archaeplastida to environmental bacteria specialized in plant polysaccharides degradation and second, the gene was transferred from the environmental bacteria to gut microbes. PMID:22934241

  11. Correlating information contents of gene ontology terms to infer semantic similarity of gene products.

    PubMed

    Gan, Mingxin

    2014-01-01

    Successful applications of the gene ontology to the inference of functional relationships between gene products in recent years have raised the need for computational methods to automatically calculate semantic similarity between gene products based on semantic similarity of gene ontology terms. Nevertheless, existing methods, though having been widely used in a variety of applications, may significantly overestimate semantic similarity between genes that are actually not functionally related, thereby yielding misleading results in applications. To overcome this limitation, we propose to represent a gene product as a vector that is composed of information contents of gene ontology terms annotated for the gene product, and we suggest calculating similarity between two gene products as the relatedness of their corresponding vectors using three measures: Pearson's correlation coefficient, cosine similarity, and the Jaccard index. We focus on the biological process domain of the gene ontology and annotations of yeast proteins to study the effectiveness of the proposed measures. Results show that semantic similarity scores calculated using the proposed measures are more consistent with known biological knowledge than those derived using a list of existing methods, suggesting the effectiveness of our method in characterizing functional relationships between gene products. PMID:24963342

  12. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids. PMID:26945769

  13. Evolution of Mutational Robustness in the Yeast Genome: A Link to Essential Genes and Meiotic Recombination Hotspots

    PubMed Central

    Keller, Philipp J.; Knop, Michael

    2009-01-01

    Deleterious mutations inevitably emerge in any evolutionary process and are speculated to decisively influence the structure of the genome. Meiosis, which is thought to play a major role in handling mutations on the population level, recombines chromosomes via non-randomly distributed hot spots for meiotic recombination. In many genomes, various types of genetic elements are distributed in patterns that are currently not well understood. In particular, important (essential) genes are arranged in clusters, which often cannot be explained by a functional relationship of the involved genes. Here we show by computer simulation that essential gene (EG) clustering provides a fitness benefit in handling deleterious mutations in sexual populations with variable levels of inbreeding and outbreeding. We find that recessive lethal mutations enforce a selective pressure towards clustered genome architectures. Our simulations correctly predict (i) the evolution of non-random distributions of meiotic crossovers, (ii) the genome-wide anti-correlation of meiotic crossovers and EG clustering, (iii) the evolution of EG enrichment in pericentromeric regions and (iv) the associated absence of meiotic crossovers (cold centromeres). Our results furthermore predict optimal crossover rates for yeast chromosomes, which match the experimentally determined rates. Using a Saccharomyces cerevisiae conditional mutator strain, we show that haploid lethal phenotypes result predominantly from mutation of single loci and generally do not impair mating, which leads to an accumulation of mutational load following meiosis and mating. We hypothesize that purging of deleterious mutations in essential genes constitutes an important factor driving meiotic crossover. Therefore, the increased robustness of populations to deleterious mutations, which arises from clustered genome architectures, may provide a significant selective force shaping crossover distribution. Our analysis reveals a new aspect of the evolution of genome architectures that complements insights about molecular constraints, such as the interference of pericentromeric crossovers with chromosome segregation. PMID:19557188

  14. Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803

    PubMed Central

    Kato, Kazushige; Tanaka, Ryouichi; Sano, Shinsuke; Tanaka, Ayumi; Hosaka, Hideo

    2010-01-01

    Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. Protox is encoded by the hemY gene in eukaryotes and by the hemG gene in many ?-proteobacteria, including Escherichia coli. It has been suggested that other bacteria possess a yet unidentified type of Protox. To identify a unique bacterial gene encoding Protox, we first introduced the Arabidopsis hemY gene into the genome of the cyanobacterium, Synechocystis sp. PCC6803. We subsequently mutagenized the cells by transposon tagging and screened the tagged lines for mutants that were sensitive to acifluorfen, which is a specific inhibitor of the hemY-type Protox. Several cell lines containing the tagged slr1790 locus exhibited acifluorfen sensitivity. The slr1790 gene encodes a putative membrane-spanning protein that is distantly related to the M subunit of NADH dehydrogenase complex I. We attempted to disrupt this gene in the wild-type background of Synechocystis, but we were only able to obtain heteroplasmic disruptants. These cells accumulated a substantial amount of protoporphyrin IX, suggesting that the slr1790 gene is essential for growth and Protox activity of cells. We found that most cyanobacteria and many other bacteria possess slr1790 homologs. We overexpressed an slr1790 homolog of Rhodobacter sphaeroides in Escherichia coli and found that this recombinant protein possesses Protox activity in vitro. These results collectively demonstrate that slr1790 encodes a unique Protox enzyme and we propose naming the slr1790 gene hemJ. PMID:20823222

  15. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes

    PubMed Central

    Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-01-01

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone1,2, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down’s syndrome3-5. Which genes safeguard accurate progression through meiosis is largely unclear. Here, we developed high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNAi within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated dataset of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and now allows systematic studies of meiosis in mammals. PMID:26147080

  16. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    PubMed

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals. PMID:26147080

  17. In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice.

    PubMed

    Gandotra, Sheetal; Schnappinger, Dirk; Monteleone, Mercedes; Hillen, Wolfgang; Ehrt, Sabine

    2007-12-01

    The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-gamma-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics. PMID:18059281

  18. Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

    PubMed

    Tachikawa, H; Miura, T; Katakura, Y; Mizunaga, T

    1991-08-01

    A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth. PMID:1761527

  19. The production of pork with garlic flavour notes using garlic essential oil.

    PubMed

    Leong, Jasmine; Morel, Patrick C H; Purchas, Roger W; Wilkinson, Brian H P

    2010-04-01

    Garlic essential oil (GEO) added directly to pork mince or to the diet of pigs was evaluated for its effectiveness in masking undesirable mutton flavour. Pork was from 31 female pigs (Duroc x (Large White x Landrace)) grown on diets containing either animal-plus-plant products (AP diet) or plant products only (P diet) with four levels of GEO: 0, 0.55, 1.44 and 1.84 g/kg feed and 0, 0.55, 1.44 and 2.15 g/kg feed in the AP and P diet, respectively. Garlic flavour increased and mutton flavour decreased in pork from pigs that consumed more than about 150 g and 220 g of GEO in P and AP diets, respectively over the 57-day feeding period. The garlic flavour was stronger and the mutton flavour less intense for pork from pigs on the P diet. When GEO was added to pork mince at 125 ppm, it significantly reduced mutton flavour. PMID:20374845

  20. The hrp genes of Pseudomonas cichorii are essential for pathogenicity on eggplant but not on lettuce

    PubMed Central

    Hojo, Hiroshi; Koyanagi, Makoto; Tanaka, Masayuki; Kajihara, Shigeru; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2008-01-01

    Pseudomonas cichorii causes necrotic lesions in eggplant and rot in lettuce. Through transposon insertion into P. cichorii strain SPC9018 we produced two mutants, 4-57 and 2-99, that lost virulence on eggplant but not lettuce. Analyses showed that a transposon was inserted into the hrpG gene in 4-57 and the hrcT gene in 2-99. Nucleotide sequences of the hrp genes of SPC9018 are homologous to those of Pseudomonas viridiflava BS group strains. The pathogenicity of 4-57 on eggplant was restored by transformation with an hrpF operon, originating from either SPC9018 or the BS group member P. viridiflava strain 9504 (Pv9504). These data suggested the involvement of hrp genes in the pathogenicity of SPC9018 on eggplant, and functional conservation of hrpF operons between SPC9018 and Pv9504. Both the hrpS mutant and the hrpL mutant were unable to cause necrotic lesions on eggplant leaves but retained their pathogenicity against lettuce. These results suggest that the pathogenicity of P. cichorii is hrp-dependent in eggplant, but not in lettuce. PMID:18832299

  1. Haploinsufficiency screen highlights two distinct groups of ribosomal protein genes essential for embryonic stem cell fate

    PubMed Central

    Fortier, Simon; MacRae, Tara; Bilodeau, Mélanie; Sargeant, Tobias; Sauvageau, Guy

    2015-01-01

    In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding protein to the ribosome's mRNA exit site, is associated with the most profound phenotype. This EB phenotype was fully rescued by BAC or cDNA complementation but not by the reduction of p53 levels, although such reduction was effective with most other RP-deleted clones corresponding to non-mRNA exit-site proteins. RNA-sequencing studies further revealed that undifferentiated ESCs hemizygous for Rps5 showed reduced expression levels of several mesoderm-specific genes as compared with wild-type counterparts. Together, these results reveal that RP gene dosage limits the differentiation, not the self-renewal, of mouse ESCs. They also highlight two separate mechanisms underlying this process, one of which is p53 independent. PMID:25646475

  2. Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function.

    PubMed

    Yosef, Ido; Goren, Moran G; Edgar, Rotem; Qimron, Udi

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity. PMID:25981477

  3. The Popeye domain containing genes: essential elements in heart rate control.

    PubMed

    Schindler, Roland F; Poon, Kar Lai; Simrick, Subreena; Brand, Thomas

    2012-12-01

    The Popeye domain containing (Popdc) gene family displays preferential expression in skeletal muscle and heart. Only recently a significant gain in the understanding of the function of Popdc genes in the heart has been obtained. The Popdc genes encode membrane proteins harboring an evolutionary conserved Popeye domain, which functions as a binding domain for cyclic adenosine monophosphate (cAMP). Popdc proteins interact with the two-pore channel TREK-1 and enhance its current. This protein interaction is modulated by cAMP. Null mutations of members of the Popdc gene family in zebrafish and mouse are associated with severe cardiac arrhythmia phenotypes. While in zebrafish an atrioventricular block was prevalent, in mouse a stress-induced sinus bradycardia was observed, which was due to the presence of sinus pauses. Moreover, the phenotype develops in an age-dependent manner, being absent in the young animal and becoming increasingly severe, as the animals grow older. This phenotype is reminiscent of the sick sinus syndrome (SSS), which affects mostly the elderly and is characterized by the poor ability of the cardiac pacemaker to adapt the heart rate to the physiological demand. While being a prevalent disease, which is responsible for a large fraction of pacemaker implantations in Western countries, SSS is poorly understood at the molecular level. It is therefore expected that the study of the molecular basis of the stress-induced bradycardia in Popdc mice will shed new light on the etiology of pacemaker disease. PMID:24282731

  4. Identification of Actinobacillus suis Genes Essential for the Colonization of the Upper Respiratory Tract of Swine

    PubMed Central

    Ojha, Shivani; Sirois, Marc; MacInnes, Janet I.

    2005-01-01

    Actinobacillus suis has emerged as an important opportunistic pathogen of high-health-status swine. A colonization challenge method was developed, and using PCR-based signature-tagged transposon mutagenesis, 13 genes belonging to 9 different functional classes were identified that were necessary for A. suis colonization of the upper respiratory tract of swine. PMID:16177387

  5. Haploinsufficiency screen highlights two distinct groups of ribosomal protein genes essential for embryonic stem cell fate.

    PubMed

    Fortier, Simon; MacRae, Tara; Bilodeau, Mlanie; Sargeant, Tobias; Sauvageau, Guy

    2015-02-17

    In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding protein to the ribosome's mRNA exit site, is associated with the most profound phenotype. This EB phenotype was fully rescued by BAC or cDNA complementation but not by the reduction of p53 levels, although such reduction was effective with most other RP-deleted clones corresponding to non-mRNA exit-site proteins. RNA-sequencing studies further revealed that undifferentiated ESCs hemizygous for Rps5 showed reduced expression levels of several mesoderm-specific genes as compared with wild-type counterparts. Together, these results reveal that RP gene dosage limits the differentiation, not the self-renewal, of mouse ESCs. They also highlight two separate mechanisms underlying this process, one of which is p53 independent. PMID:25646475

  6. EXPORTIN1 Genes are Essential for Development and Function of the Gametophytes in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recru...

  7. Prediction of the Risk for Essential Hypertension among Carriers of C825T Genetic Polymorphism of G Protein β3 (GNB3) Gene

    PubMed Central

    El Din Hemimi, Neveen Salah; Mansour, Amal A.; Abdelsalam, Mona Mohamed

    2016-01-01

    BACKGROUND The guanine nucleotide-binding protein beta polypeptide 3 (GNB3) 825T allele encodes a product that enhances the activation of heterotrimeric G proteins, which is associated with the occurrence of the splice variant Gβ3 s that could play a role in vascular reactivity and hyperproliferation of smooth muscle cells, that makes such proteins attractive candidate gene products for susceptibility to essential hypertension (EH). OBJECTIVE To predict the risk for EH in individuals with C825T genetic polymorphism of G protein β3 gene. METHODS The study consisted of 222 normotensive individuals and 216 hypertensive patients. Individuals were genotyped for C825T genetic polymorphism of G protein β3 gene rs5443 by using restriction fragment length polymorphism. RESULTS Frequencies of C and T alleles were 58.1% and 41.9%, respectively, in the control group compared with 47.7% and 52.3%, respectively, in the hypertensive group. The carriers of rs5443 (T) allele exhibited a significant greater risk for EH compared with the carriers of rs5443 (C) allele (odds ratio = 1.5, 95% confidence interval = 1.2–2.0). CONCLUSION T allele is a risk factor for EH in the Egyptian population, which may be used as a prognostic and a therapeutic target of prophylaxis. PMID:27226707

  8. A plant-specific HUA2-LIKE (HULK) gene family in Arabidopsis thaliana is essential for development

    PubMed Central

    Jali, Sathya S; Rosloski, Sarah M; Janakirama, Preetam; Steffen, Joshua G; Zhurov, Vladimir; Berleth, Thomas; Clark, Richard M; Grbic, Vojislava

    2014-01-01

    In Arabidopsis thaliana, the HUA2 gene is required for proper expression of FLOWERING LOCUS C (FLC) and AGAMOUS, key regulators of flowering time and reproductive development, respectively. Although HUA2 is broadly expressed, plants lacking HUA2 function have only moderately reduced plant stature, leaf initiation rate and flowering time. To better understand HUA2 activity, and to test whether redundancy with similar genes underlies the absence of strong phenotypes in HUA2 mutant plants, we identified and subsequently characterized three additional HUA2-LIKE (HULK) genes in Arabidopsis. These genes form two clades (HUA2/HULK1 and HULK2/HULK3), with members broadly conserved in both vascular and non-vascular plants, but not present outside the plant kingdom. Plants with progressively reduced HULK activity had increasingly severe developmental defects, and plants homozygous for loss-of-function mutations in all four HULK genes were not recovered. Multiple mutants displayed reproductive, embryonic and post-embryonic abnormalities, and provide detailed insights into the overlapping and unique functions of individual HULK genes. With regard to flowering time, opposing influences were apparent: hua2 hulk1 plants were early-flowering, while hulk2 hulk3 mutants were late-flowering, and hua2 acted epistatically to cause early flowering in all combinations. Genome-wide expression profiling of mutant combinations using RNA-Seq revealed complex transcriptional changes in seedlings, with FLC, a known target of HUA2, among the most affected. Our studies, which include characterization of HULK expression patterns and subcellular localization, suggest that the HULK genes encode conserved nuclear factors with partially redundant but essential functions associated with diverse genetic pathways in plants. PMID:25070081

  9. Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.

    PubMed

    Ohno, Yusuke; Nakamichi, Shota; Ohkuni, Aya; Kamiyama, Nozomi; Naoe, Ayano; Tsujimura, Hisashi; Yokose, Urara; Sugiura, Kazumitsu; Ishikawa, Junko; Akiyama, Masashi; Kihara, Akio

    2015-06-23

    A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ?-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ?-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (?C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ?-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production. PMID:26056268

  10. Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation

    PubMed Central

    Ohno, Yusuke; Nakamichi, Shota; Ohkuni, Aya; Kamiyama, Nozomi; Naoe, Ayano; Tsujimura, Hisashi; Yokose, Urara; Sugiura, Kazumitsu; Ishikawa, Junko; Akiyama, Masashi; Kihara, Akio

    2015-01-01

    A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production. PMID:26056268

  11. Identification of genes essential for prey-independent growth of Bdellovibrio bacteriovorus HD100.

    PubMed

    Roschanski, Nicole; Klages, Sven; Reinhardt, Richard; Linscheid, Michael; Strauch, Eckhard

    2011-04-01

    Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Whole-genome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth. PMID:21278289

  12. An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity

    SciTech Connect

    Kenna, M.; Douglas, M.G. ); Stevens, A. ); McCammon, M. )

    1993-01-01

    This is a study of a temperature-sensitive (ts) mutant from Saccharomyces cerevisiae which was obtained in a screen for mutants reduced in the synthesis of binding of a hybrid protein which competes for the transport of protein precursors into mitochondria. Examination of this mutant lead to the characterization of a gene with significant primary sequence homology to a previously identified gene, XRN1 or KEM1. Often called XRN1/KEM1, it encodes a protein of 175kDa which appears to have a multitude of properties, including involvement in recombination, RNA processing and turnover, involvement in recombination, RNA processing and turnover, microtubule function, karyogamy and DNA replication. The related gene describes further characterization of the HKE1/RAT1 gene and an hkal mutant and shows that p116 is a protein having 5[prime]-->3[prime] exoribonuclease activity, a major activity of the product of the related XRN1/KEM1 gene.

  13. Inducible product gene expression technology tailored to bioprocess engineering.

    PubMed

    Weber, Wilfried; Fussenegger, Martin

    2007-10-01

    Bioprocess engineering has developed as a discipline to design optimal culture conditions and bioreactor operation protocols for production cell lines engineered for constitutive expression of desired protein pharmaceuticals. With the advent of heterologous gene regulation systems it has become possible to fine-tune expression of difficult-to-produce protein pharmaceuticals to optimal levels and to conditionally engineer cell metabolism for the best production performance. However, most of the small-molecules used to trigger expression of product or metabolic engineering product genes are incompatible with downstream processing regulations or process economics. Recent progress in product gene control design has resulted in the development of bioprocess-compatible regulation systems, which are responsive to physical parameters such as temperature or physiologic trigger molecules that are either an inherent part of host cell metabolism or intrinsic components of licensed protein-free cell culture media, such as redox status, vitamin H and gaseous acetaldehyde. While all of these systems have been shown to fine-tune product gene expression independent of the host cell metabolism some of them can be plugged into metabolic networks to capture critical physiologic parameters and convert them into an optimal production response. Assembly of individual product gene control modalities into synthetic networks has recently enabled construction of autonomously regulated time-delay or cell density-sensitive gene circuits, which trigger population-wide induction of product gene expression at a predefined time or culture density. We provide a comprehensive overview on the latest developments in the design of bioprocess-compatible product gene control systems. PMID:17933507

  14. Determination of the sequence of the yeast YCL313 gene localized on chromosome III. Homology with the protein disulfide isomerase (PDI gene product) of other organisms.

    PubMed

    Scherens, B; Dubois, E; Messenguy, F

    1991-02-01

    We have determined the nucleotide sequence of the YCL313 gene as part of the YIp5 A1G clone localized on the left arm of chromosome III. This YCL313 gene encodes a protein of 522 amino acids (MW 58.3 kDa) which has large homologies with the human, mouse, chicken, bovine and rat PDI gene products. In these organisms the PDI gene encodes the protein disulfide isomerase (EC 5.3.4.1) also called S-S rearrangase, an enzyme that catalyses the rearrangements of S-S bonds in proteins. This enzyme is probably involved in protein folding within the lumen of the endoplasmic reticulum. These sequence homologies suggest that YCL313 is the yeast equivalent of the PDI gene. Gene disruption of YCL313 leads to a lethal phenotype indicating that this gene is essential for cell survival. PMID:2063627

  15. A Brain-Specific Homeobox Gene, Bsx, Is Essential for Proper Postnatal Growth and Nursing▿

    PubMed Central

    McArthur, Tara; Ohtoshi, Akihira

    2007-01-01

    To investigate in vivo roles of a murine hypothalamic homeobox gene, Bsx, we generated and analyzed two mutant alleles, BsxΔHD and BsxlacZ. BsxΔHD lacks the homeodomain, and BsxlacZ is an insertion of a lacZ reporter gene. Bsx-lacZ expression was detected in the hypothalamus and pineal gland and reiterates Bsx expression. Bsx homozygous mutant mice were born at the expected Mendelian ratio, but their growth was impaired. Offspring from Bsx homozygous mutant females exhibited a low survival rate due to a nursing defect. Mammary glands of the mutant females developed normally during pregnancy; however, they involuted quickly after parturition. These results demonstrate that Bsx is required for postnatal growth and maintenance of lactating mammary glands. Thus, mouse Bsx is likely involved in systemic control of suppression of apoptosis of postpartum mammary epithelial cells. PMID:17485440

  16. Small RNAs: essential regulators of gene expression and defenses against environmental stresses in plants.

    PubMed

    Wang, Hsiao-Lin V; Chekanova, Julia A

    2016-05-01

    Eukaryotic genomes produce thousands of diverse small RNAs (smRNAs), which play vital roles in regulating gene expression in all conditions, including in survival of biotic and abiotic environmental stresses. SmRNA pathways intersect with most of the pathways regulating different steps in the life of a messenger RNA (mRNA), starting from transcription and ending at mRNA decay. SmRNAs function in both nuclear and cytoplasmic compartments; the regulation of mRNA stability and translation in the cytoplasm and the epigenetic regulation of gene expression in the nucleus are the main and best-known modes of smRNA action. However, recent evidence from animal systems indicates that smRNAs and RNA interference (RNAi) also participate in the regulation of alternative pre-mRNA splicing, one of the most crucial steps in the fast, efficient global reprogramming of gene expression required for survival under stress. Emerging evidence from bioinformatics studies indicates that a specific class of plant smRNAs, induced by various abiotic stresses, the sutr-siRNAs, has the potential to target regulatory regions within introns and thus may act in the regulation of splicing in response to stresses. This review summarizes the major types of plant smRNAs in the context of their mechanisms of action and also provides examples of their involvement in regulation of gene expression in response to environmental cues and developmental stresses. In addition, we describe current advances in our understanding of how smRNAs function in the regulation of pre-mRNA splicing. WIREs RNA 2016, 7:356-381. doi: 10.1002/wrna.1340 For further resources related to this article, please visit the WIREs website. PMID:26924473

  17. RONIN Is an Essential Transcriptional Regulator of Genes Required for Mitochondrial Function in the Developing Retina.

    PubMed

    Poch, Ross A; Zhang, Min; Rueda, Elda M; Tong, Xuefei; McElwee, Melissa L; Wong, Leeyean; Hsu, Chih-Wei; Dejosez, Marion; Burns, Alan R; Fox, Donald A; Martin, James F; Zwaka, Thomas P; Dickinson, Mary E

    2016-02-23

    A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression. PMID:26876175

  18. aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed Central

    Rather, P N; Solinsky, K A; Paradise, M R; Parojcic, M M

    1997-01-01

    The 2'-N-acetyltransferase [AAC(2')-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2')-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2')-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2')-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein. The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE. PMID:9079912

  19. The Association of Mitofusion-2 Gene Polymorphisms with Susceptibility of Essential Hypertension in Northern Han Chinese Population

    PubMed Central

    Li, Mei; Zhang, Bei; Li, Chuang; Liu, Jielin; Liu, Ya; Sun, Dongdong; Ma, Hanying; Wen, Shaojun

    2016-01-01

    Background: Mitofusion-2 (Mfn2) played an important role in regulating vascular smooth muscle cells proliferation, insulin resistance and endoplasmic reticulum stress, which were found to be involved in the development of hypertension. So we inferred that the Mfn2 gene may participate in the pathogenesis of hypertension. The aim of this study was to determine whether common single nucleotide polymorphisms (SNPs) in Mfn2 gene were associated with essential hypertension (EH) in northern Han Chinese. Methods: We genotyped 6 tagging SNPs of Mfn2 gene (rs2336384, rs2295281, rs17037564, rs2236057, rs2236058 and rs3766741) with the TaqMan assay in 626 hypertensive patients and 618 controls. Results: Logistic regression analysis indicated that CC+CA genotype of rs2336384 and AA+AG genotype of rs2236057 were significantly associated with increased risk of EH (OR=1.617, P=0.005; OR=1.418, P=0.031, respectively). GG genotype of rs2236058 and GG+CG genotype of rs3766741 were found to be significantly associated with decreased risk of EH (OR=0.662, P=0.023; OR=0.639, P=0.024).When stratified by gender, for rs2336384, rs2236057 and rs2236058, significant association was observed in males, but not in females. Haplotype analysis indicated that the CCAACC haplotype was positively correlated with EH and there was a negative correlation between ACAGGG haplotype and EH. Conclusions: This study demonstrated that Mfn2 gene polymorphisms were associated with essential hypertension in northern Han Chinese population, especially in male subjects. PMID:26816493

  20. Functions of the gene products of Escherichia coli.

    PubMed Central

    Riley, M

    1993-01-01

    A list of currently identified gene products of Escherichia coli is given, together with a bibliography that provides pointers to the literature on each gene product. A scheme to categorize cellular functions is used to classify the gene products of E. coli so far identified. A count shows that the numbers of genes concerned with small-molecule metabolism are on the same order as the numbers concerned with macromolecule biosynthesis and degradation. One large category is the category of tRNAs and their synthetases. Another is the category of transport elements. The categories of cell structure and cellular processes other than metabolism are smaller. Other subjects discussed are the occurrence in the E. coli genome of redundant pairs and groups of genes of identical or closely similar function, as well as variation in the degree of density of genetic information in different parts of the genome. PMID:7508076

  1. A Conditional Mouse Model for Measuring the Frequency of Homologous Recombination Events In Vivo in the Absence of Essential Genes?

    PubMed Central

    Brown, Adam D.; Claybon, Alison B.; Bishop, Alexander J. R.

    2011-01-01

    The ability to detect and repair DNA damage is crucial to the prevention of various diseases. Loss of function of genes involved in these processes is known to result in significant developmental defects and/or predisposition to cancer. One such DNA repair mechanism, homologous recombination, has the capacity to repair a wide variety of lesions. Knockout mouse models of genes thought to be involved in DNA repair processes are frequently lethal, making in vivo studies very difficult, if not impossible. Therefore, we set out to develop an in vivo conditional mouse model system to facilitate investigations into the involvement of essential genes in homologous recombination. To test our model, we measured the frequency of spontaneous homologous recombination using the pink-eyed unstable mouse model, in which we conditionally excised either Blm or full-length Brca1 (breast cancer 1, early onset). These two genes are hypothesized to have opposing roles in homologous recombination. In summary, our in vivo data supports in vitro studies suggesting that BLM suppresses homologous recombination, while full-length BRCA1 promotes this process. PMID:21709021

  2. A conditional mouse model for measuring the frequency of homologous recombination events in vivo in the absence of essential genes.

    PubMed

    Brown, Adam D; Claybon, Alison B; Bishop, Alexander J R

    2011-09-01

    The ability to detect and repair DNA damage is crucial to the prevention of various diseases. Loss of function of genes involved in these processes is known to result in significant developmental defects and/or predisposition to cancer. One such DNA repair mechanism, homologous recombination, has the capacity to repair a wide variety of lesions. Knockout mouse models of genes thought to be involved in DNA repair processes are frequently lethal, making in vivo studies very difficult, if not impossible. Therefore, we set out to develop an in vivo conditional mouse model system to facilitate investigations into the involvement of essential genes in homologous recombination. To test our model, we measured the frequency of spontaneous homologous recombination using the pink-eyed unstable mouse model, in which we conditionally excised either Blm or full-length Brca1 (breast cancer 1, early onset). These two genes are hypothesized to have opposing roles in homologous recombination. In summary, our in vivo data supports in vitro studies suggesting that BLM suppresses homologous recombination, while full-length BRCA1 promotes this process. PMID:21709021

  3. The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Wang, Hong; Wang, Congcong; Li, Ya; Yue, Xiaofeng; Ma, Zhonghua; Talbot, Nicholas J.; Wang, Zhengyi

    2013-01-01

    Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae. PMID:24116181

  4. A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400.

    PubMed

    Gaballa, A; Koedam, N; Cornelis, P

    1996-08-01

    Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens. PMID:8878040

  5. Cross-Ontological Analytics: Combining Associative and Hierarchical Relations in the Gene Ontologies to Assess Gene Product Similarity

    SciTech Connect

    Posse, Christian; Sanfilippo, Antonio P.; Gopalan, Banu; Riensche, Roderick M.; Beagley, Nathaniel; Baddeley, Bob L.

    2006-05-28

    Gene and gene product similarity is a fundamental diagnostic measure in analyzing biological data and constructing predictive models for functional genomics. With the rising influence of the gene ontologies, two complementary approaches have emerged where the similarity between two genes/gene products is obtained by comparing gene ontology (GO) annotations associated with the gene/gene products. One approach captures GO-based similarity in terms of hierarchical relations within each gene ontology. The other approach identifies GO-based similarity in terms of associative relations across the three gene ontologies. We propose a novel methodology where the two approaches can be merged with ensuing benefits in coverage and accuracy.

  6. Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus.

    PubMed

    Espina, Laura; Pagán, Rafael; López, Daniel; García-Gonzalo, Diego

    2015-01-01

    Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings. PMID:26102069

  7. Formulating essential oil microemulsions as washing solutions for organic fresh produce production.

    PubMed

    Zhang, Linhan; Critzer, Faith; Davidson, P Michael; Zhong, Qixin

    2014-12-15

    Applications of plant-derived organic essential oils (EOs) as antimicrobials for post-harvest produce operations are limited by their low water solubility. To dissolve EOs in water, microemulsions were studied using two surfactants permitted for organic production, sucrose octanoate ester (SOE) and soy lecithin that were mixed at various mass ratios before dilution with water to 40% w/w. EOs were then mixed with the surfactant solution by hand shaking. Based on visual transparency, intermediate lecithin:SOE mass ratios favoured the formation of microemulsions, e.g., up to 4.0% clove bud oil at ratios of 2:8 and 3:7, and 4.0% cinnamon bark oil and 3.0% thyme oil at ratios of 2:8 and 1:9, respectively. Microemulsions with intermediate lecithin:SOE mass ratios had a relatively low viscosity and better ability to wet fresh produce surfaces. The microemulsions established in this work may be used as washing solutions to enhance the microbial safety of organic fresh produce. PMID:25038656

  8. Mode of action for natural products isolated from essential oils of two trees is different from available mosquito adulticides.

    PubMed

    McAllister, Janet C; Adams, Mary F

    2010-11-01

    Insecticidal properties of natural products may present alternatives to the use of synthetic molecule pesticides that are of diminishing effectiveness due to resistance. Three compounds, thymoquinone, nootkatone, and carvacrol, components of Alaska yellow cedar, Chamaecyyparis nootkatensis (D. Don) Spach, and incense cedar, Calocedrus decurrens (Torr.), essential oils, have been shown to have biological activity against a variety of mosquito and tick species. Although these components act as both repellents and insecticides, how they function in either capacity is unknown. Their use as mosquito control insecticides would be greatly increased if their mode of action is not the same as that of currently used commercial products. This study compared the lethal dosages for nootkatone, carvacrol, and thymoquinone by using colony strains of Anopheles gambiae Giles with known mutations at three different target sites. The altered target sites evaluated were the sodium channel para-locus mutation (L1014 F KDR) that confers permethrin resistance, the ACE-1 gene that confers organophosphate and carbamate resistance, and a gamma-aminobutyric acid receptor mutation of the Rdl locus conferring dieldrin resistance. Significant increases in lethal dose were not observed in any of the mosquito strains for any of the compounds tested compared with the doses required of chemicals with known modes of action at the mutated sites. Although the mode of action was not determined, this screening study indicates that none of these compounds interact at the target sites represented in the test mosquito strains. These compounds represent a different mode of action than existing chemicals currently used in mosquito control. PMID:21175062

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

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

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

  10. Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination

    PubMed Central

    Kondratov, Roman V.; Jamasbi, Roudabeh J.

    2015-01-01

    Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis. PMID:26439128

  11. Effect of different liming levels on the biomass production and essential oil extraction yield of Cunila galioides Benth.

    PubMed

    Mossi, A J; Pauletti, G F; Rota, L; Echeverrigaray, S; Barros, I B I; Oliveira, J V; Paroul, N; Cansian, R L

    2012-11-01

    Poejo is an aromatic and medicinal plant native to highland areas of south Brazil, in acid soils with high Al3+ concentration. The main objective of the present work was to evaluate the effect of liming on the extraction yield of essential oil of three chemotypes of poejo (Cunila galioides Benth). For this purpose, the experiments were performed in a greenhouse, using 8-litre pots. The treatments were four dosages of limestone (0, 3.15, 12.5, and 25 g.L(-1)) and a completely random experimental design was used, with four replications and three chemotypes, set up in a 3 × 4 factorial arrangement. The parameters evaluated were dry weight of aerial parts, essential oil content and chemical composition of essential oil. Results showed that liming affects the biomass production, essential oil yield and chemical composition, with cross interaction verified between chemotype and limestone dosage. For the higher dosage lower biomass production, lower yield of essential oil as well as the lowest content of citral (citral chemotype) and limonene (menthene chemotype) was observed. In the ocimene chemotype, no liming influence was observed on the essential oil yield and on the content of major compounds. The dosage of 3.15 g.L(-1) can be considered the best limestone dosage for the production of poejo for the experimental conditions evaluated. PMID:23295505

  12. Rlm1 mediates positive autoregulatory transcriptional feedback that is essential for Slt2-dependent gene expression.

    PubMed

    García, Raúl; Sanz, Ana Belén; Rodríguez-Peña, José Manuel; Nombela, César; Arroyo, Javier

    2016-04-15

    Activation of the yeast cell wall integrity (CWI) pathway induces an adaptive transcriptional programme that is largely dependent on the transcription factor Rlm1 and the mitogen-activated protein kinase (MAPK) Slt2. Upon cell wall stress, the transcription factor Rlm1 is recruited to the promoters ofRLM1andSLT2, and exerts positive-feedback mechanisms on the expression of both genes. Activation of the MAPK Slt2 by cell wall stress is not impaired in strains with individual blockade of any of the two feedback pathways. Abrogation of the autoregulatory feedback mechanism onRLM1severely affects the transcriptional response elicited by activation of the CWI pathway. In contrast, a positive trans-acting feedback mechanism exerted by Rlm1 onSLT2also regulates CWI output responses but to a lesser extent. Therefore, a complete CWI transcriptional response requires not only phosphorylation of Rlm1 by Slt2 but also concurrentSLT2- andRLM1-mediated positive-feedback mechanisms; sustained patterns of gene expression are mainly achieved by positive autoregulatory circuits based on the transcriptional activation of Rlm1. PMID:26933180

  13. The Tolkin Gene Is a Tolloid/Bmp-1 Homologue That Is Essential for Drosophila Development

    PubMed Central

    Finelli, A. L.; Xie, T.; Bossie, C. A.; Blackman, R. K.; Padgett, R. W.

    1995-01-01

    The Drosophila decapentaplegic (dpp) gene, a member of the tranforming growth factor ? superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interaction between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp. PMID:8536976

  14. The ORF012 Gene of Marek's Disease Virus Type 1 Produces a Spliced Transcript and Encodes a Novel Nuclear Phosphoprotein Essential for Virus Growth

    PubMed Central

    Schippers, Timo; Jarosinski, Keith

    2014-01-01

    ABSTRACT Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV carries a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potentials and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique long region. Using reverse transcriptase PCR, we showed that ORF011 and ORF012 are not individual genes but form a single gene through mRNA splicing of a small intron, resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defect in vitro and could not be passaged in cultured cells, suggesting an essential role for the ORF012 product in virus replication. Further studies revealed that protein 012 (p012) localized to the nucleus in transfected and infected cells, and we identified by site-directed mutagenesis and green fluorescent protein (GFP) reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23-amino-acid sequence at the protein's C terminus. Nuclear export was blocked using leptomycin B, suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution. IMPORTANCE Marek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle, which eventually could help us to interfere with MDV and herpesviral pathogenesis. We have identified a previously unidentified phosphoprotein encoded by MDV ORF012. We were able to show experimentally that predicted splicing of the gene based on bioinformatics data does indeed occur during replication. The newly identified p012 is essential for MDV replication and localizes to the nucleus due to the presence of a transferable nuclear localization signal at its C terminus. Our results also imply that p012 could constitute a nucleocytoplasmic shuttle protein, a feature that could prove interesting and important. PMID:25392220

  15. The Drosophila Clathrin Heavy Chain Gene: Clathrin Function Is Essential in a Multicellular Organism

    PubMed Central

    Bazinet, C.; Katzen, A. L.; Morgan, M.; Mahowald, A. P.; Lemmon, S. K.

    1993-01-01

    The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc(1), Chc(2) or Chc(3) alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc(4), exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc(4) germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc(4) males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis. PMID:8375651

  16. Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, plants (14) and essential oils (EO; 88) from plants that are naturalized to, or can be successfully grown in North America were evaluated in a batch culture in vitro screening experiments with ruminal fluid as potential anti-methanogenic additives for ruminant diets. Essential oils we...

  17. A FUSCA gene of Arabidopsis encodes a novel protein essential for plant development.

    PubMed Central

    Castle, L A; Meinke, D W

    1994-01-01

    Arabidopsis fusca mutants display striking purple coloration due to anthocyanin accumulation in their cotyledons. We describe six recessive fusca mutants isolated from Agrobacterium-transformed Arabidopsis families. These mutants first become defective during embryogenesis and exhibit limited seedling development. Double mutant constructs revealed that developmental defects were not simply a consequence of anthocyanin accumulation. fusca seedlings showed altered responses to several environmental and endogenous factors. Allelism tests established that three fusca loci are represented by mutants previously described as defective in light-regulated responses. To study the molecular basis of the fusca phenotype, we cloned the FUS6 gene. FUS6 encodes a novel protein that is hydrophilic, alpha-helical, and contains potential protein kinase C phosphorylation sites. The FUSCA proteins appear to act in a network of signal transduction pathways critical for plant development. PMID:8130643

  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. Unveiling a new essential cis element for the transactivation of the CYP3A4 gene by xenobiotics.

    TOXLINE Toxicology Bibliographic Information

    Toriyabe T; Nagata K; Takada T; Aratsu Y; Matsubara T; Yoshinari K; Yamazoe Y

    2009-03-01

    Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor alpha (RXRalpha) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at -7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them alpha-eta half-sites. Introduction of a mutation into either an alpha or beta half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXRalpha heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with alpha and beta half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.

  20. Direct activator/co-activator interaction is essential for bacteriophage T4 middle gene expression.

    PubMed

    Yuan, Andy H; Hochschild, Ann

    2009-11-01

    The bacteriophage T4 AsiA protein is a bifunctional regulator that inhibits transcription from the major class of bacterial promoters and also serves as an essential co-activator of transcription from T4 middle promoters. AsiA binds the primary s factor in Escherichia coli, sigma(70), and modifies the promoter recognition properties of the sigma(70)-containing RNA polymerase(RNAP) holoenzyme. In its role as co-activator, AsiA directs RNAP to T4 middle promoters in the presence of the T4-encoded activator MotA. According to the current model for T4 middle promoter activation, AsiA plays an indirect role in stabilizing the activation complex by facilitating interaction between DNA-bound MotA and sigma(70). Here we show that AsiA also plays a direct role in T4 middle promoter activation by contacting the MotA activation domain. Furthermore,we show that interaction between AsiA and the beta-flap domain of RNAP is important for co-activation. Based on our findings, we propose a revised model for T4 middle promoter activation, with AsiA organizing the activation complex via three distinct protein-protein interactions. PMID:19843221

  1. The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants.

    PubMed Central

    Vogel, A M; Das, A

    1992-01-01

    Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence. In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used. An A. tumefaciens strain, A348 delta D, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed. Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers. As expected, deletion of the virD operon led to an avirulent phenotype. The virulence of this strain could be restored by providing virD1, virD2, and virD4 in trans. No requirement for virD3 in tumor formation was observed in these assays. Images PMID:1629176

  2. The Zebrafish fleer Gene Encodes an Essential Regulator of Cilia Tubulin Polyglutamylation

    PubMed Central

    Pathak, Narendra; Obara, Tomoko; Mangos, Steve; Liu, Yan

    2007-01-01

    Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule B-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Ttll6, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis. PMID:17761526

  3. Kaposi's Sarcoma-Associated Herpesvirus ORF6 Gene Is Essential in Viral Lytic Replication

    PubMed Central

    Peng, Can; Chen, Jungang; Tang, Wei; Liu, Chunlan; Chen, Xulin

    2014-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV. PMID:24911362

  4. PFP1, a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune

    PubMed Central

    Siersleben, Sylvia; Penselin, Daniel; Wenzel, Claudia; Albert, Sylvie

    2014-01-01

    Scald caused by Rhynchosporium commune is an important foliar disease of barley. Insertion mutagenesis of R. commune generated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene named PFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex in R. commune with an essential role in the epigenetic control of fungal pathogenicity. Targeted PFP1 disruption also yielded nonpathogenic mutants which showed wild-type-like growth ex planta, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-level PFP1 expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control of PFP1 expression. PMID:24906413

  5. Estrogen-related receptor {alpha} is essential for the expression of antioxidant protection genes and mitochondrial function

    SciTech Connect

    Rangwala, Shamina M. . E-mail: shamina.rangwala@novartis.com; Li, Xiaoyan; Lindsley, Loren; Wang, Xiaomei; Shaughnessy, Stacey; Daniels, Thomas G.; Szustakowski, Joseph; Nirmala, N.R.; Wu, Zhidan; Stevenson, Susan C.

    2007-05-25

    Estrogen-related receptor {alpha} (ERR{alpha}) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERR{alpha} null mice. Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) stimulated mitochondrial gene expression program in control cells, but not in the ERR{alpha} null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1{alpha} levels was dependent on ERR{alpha}. Furthermore, we found that the PGC-1{alpha}-mediated induction of estrogen-related receptor {gamma} and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERR{alpha}. Basal levels of NRF-2 were decreased in the absence of ERR{alpha}. The absence of ERR{alpha} resulted in a decrease in citrate synthase enzyme activity in response to PGC-1{alpha} overexpression. Our results indicate an essential role for ERR{alpha} as a key regulator of oxidative metabolism.

  6. IGF-I Signaling Is Essential for FSH Stimulation of AKT and Steroidogenic Genes in Granulosa Cells

    PubMed Central

    Zhou, Ping; Baumgarten, Sarah C.; Wu, Yanguang; Bennett, Jill; Winston, Nicola; Hirshfeld-Cytron, Jennifer

    2013-01-01

    FSH and IGF-I synergistically stimulate gonadal steroid production; conversely, silencing the FSH or the IGF-I genes leads to infertility and hypogonadism. To determine the molecular link between these hormones, we examined the signaling cross talk downstream of their receptors. In human and rodent granulosa cells (GCs), IGF-I potentiated the stimulatory effects of FSH and cAMP on the expression of steroidogenic genes. In contrast, inhibition of IGF-I receptor (IGF-IR) activity or expression using pharmacological, genetic, or biochemical approaches prevented the FSH- and cAMP-induced expression of steroidogenic genes and estradiol production. In vivo experiments demonstrated that IGF-IR inactivation reduces the stimulation of steroidogenic genes and follicle growth by gonadotropins. FSH or IGF-I alone stimulated protein kinase B (PKB), which is also known as AKT and in combination synergistically increased AKT phosphorylation. Remarkably, blocking IGF-IR expression or activity decreased AKT basal activity and abolished AKT activation by FSH. In GCs lacking IGF-IR activity, FSH stimulation of Cyp19 expression was rescued by overexpression of constitutively active AKT. Our findings demonstrate, for the first time, that in human, mouse, and rat GCs, the well-known stimulatory effect of FSH on Cyp19 and AKT depends on IGF-I and on the expression and activation of the IGF-IR. PMID:23340251

  7. Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis

    PubMed Central

    Karki, Anju; Humphrey, Sean E.; Steele, Rebecca E.; Hess, David A.; Taparowsky, Elizabeth J.; Konieczny, Stephen F.

    2015-01-01

    Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis—events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer. PMID:26717480

  8. MicroRNA Regulation of Human Genes Essential for Influenza A (H7N9) Replication

    PubMed Central

    Wolf, Stefan; Wu, Weilin; Jones, Cheryl; Perwitasari, Olivia; Mahalingam, Suresh; Tripp, Ralph A.

    2016-01-01

    Influenza A viruses are important pathogens of humans and animals. While seasonal influenza viruses infect humans every year, occasionally animal-origin viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. In March 2013, the public health authorities of China reported three cases of laboratory confirmed human infection with avian influenza A (H7N9) virus, and subsequently there have been many cases reported across South East Asia and recently in North America. Most patients experience severe respiratory illness, and morbidity with mortality rates near 40%. No vaccine is currently available and the use of antivirals is complicated due the frequent emergence of drug resistant strains. Thus, there is an imminent need to identify new drug targets for therapeutic intervention. In the current study, a high-throughput screening (HTS) assay was performed using microRNA (miRNA) inhibitors to identify new host miRNA targets that reduce influenza H7N9 replication in human respiratory (A549) cells. Validation studies lead to a top hit, hsa-miR-664a-3p, that had potent antiviral effects in reducing H7N9 replication (TCID50 titers) by two logs. In silico pathway analysis revealed that this microRNA targeted the LIF and NEK7 genes with effects on pro-inflammatory factors. In follow up studies using siRNAs, anti-viral properties were shown for LIF. Furthermore, inhibition of hsa-miR-664a-3p also reduced virus replication of pandemic influenza A strains H1N1 and H3N2. PMID:27166678

  9. IL-17A is Essential for Cell Activation and Inflammatory Gene Circuits in Psoriasis

    PubMed Central

    Krueger, James G.; Fretzin, Scott; Surez-Farias, Mayte; Haslett, Patrick A.; Phipps, Krista M.; Cameron, Gregory S.; McColm, Juliet; Katcherian, Artemis; Cueto, Inna; White, Traci; Banerjee, Subhashis; Hoffman, Robert W.

    2012-01-01

    Background In psoriasis, inflammation and epidermal hyperplasia are thought to be controlled by T cell-derived cytokines. Evidence suggests that the Th17 cell cytokine interleukin-17 (IL-17) may play a role in disease pathogenesis. Objective To understand the impact that neutralization of IL-17 has on the clinical features of psoriasis and to understand the role that IL-17 has in inflammatory pathways underlying psoriasis in human subjects. Methods We examined skin lesions obtained from 40 subjects participating in a phase 1, randomized, double-blind, placebo-controlled trial of an anti-IL-17 monoclonal antibody, ixekizumab (previously LY2439821), in which subjects received subcutaneous 5mg, 15mg, 50mg or 150mg ixekizumab or placebo at weeks 0, 2, and 4. Results There were significant, dose-dependent reductions from baseline in keratinocyte proliferation, hyperplasia, epidermal thickness, infiltration into the dermis and epidermis by T cells and dendritic cells and keratinocyte expression of innate defense peptides at 2 weeks. By week 6, the skin appeared normal. Quantitative reverse transcriptase polymerase chain reaction and microarrays revealed an ablation of the disease-defining mRNA expression profile by 2 weeks after the first dose of study drug. The effect of IL-17 blockade on expression of genes synergistically regulated by IL-17 and Tumor necrosis factor (TNF) was of higher magnitude at 2 weeks than in prior studies with TNF antagonism. Conclusion Our data suggest that IL-17 is a key driver cytokine in psoriasis that activates pathogenic inflammation. Neutralizing IL-17 with ixekizumab may be a successful therapeutic strategy. PMID:22677045

  10. Association of intronic single-nucleotide polymorphisms in the EMILIN1 gene with essential hypertension in a Chinese population.

    PubMed

    Oh, V M S; Chua, B-M; Heng, C-K; Yeo, S-B; Yim, O-S; Yap, E P H

    2012-09-01

    Studies in mice suggest that the elastin microfibril interfacer-1 gene (EMILIN1), the gene encoding elastin microfibril interfacer-1 protein, contributes to the pathogenesis of essential hypertension (EH) in humans. EMILIN1 in part maintains elastic fibres in vessel walls, and hence peripheral arterial compliance. In a case-control study, we assessed 942 non-obese non-diabetic Chinese, comprising 467 patients with EH and 475 normotensive control subjects (166 without, and 309 with, family history of hypertension in first-degree relatives (FHH)). Hypertension in first-degree relatives occurred in 88%, 65% and 0% of cases, all controls and controls without FHH, respectively. We scanned for single-nucleotide polymorphisms (SNPs) and genotyped them in the EMILIN1 gene using high-resolution melt-curve analysis. No exonic variants were detected. We assessed the association of SNPs and their haplotypes with EH. Three SNPs in introns 1 and 5 (rs2289360, rs2011616 and rs7424556) were in strong pair-wise linkage disequilibrium (r(2)>0.89). All three SNPs were significantly associated with hypertension. Genotypic frequencies at the three SNPs differed significantly between cases and only those controls without FHH. Healthy controls with FHH should be excluded to increase the odds of detecting association. All the G alleles of rs2289360 (odds ratio = 1.69, P = 0.010), rs2011616 (odds ratio = 1.52, P = 0.038) and rs7424556 (odds ratio = 1.59, P = 0.023) were high-risk alleles in the recessive genetic model. We observed significant overall haplotypic association with EH (empirical P = 0.0072); GGG is a risk haplotype (P = 0.043). The overall results support EMILIN1 as a candidate gene for human EH. PMID:21753788

  11. Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

    PubMed

    Lin, Yi-Chu; Wang, Po-Yu; Tsai, Shih-Chih; Lin, Chien-Liang; Tai, Hsuen-Yung; Lo, Chi-Fang; Wu, Shiow-Ing; Chiang, Yu-Mei; Liu, Li-Ling

    2015-01-01

    Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients. PMID:26374219

  12. The sf32 Unique Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Is a Non-Essential Gene That Could Be Involved in Nucleocapsid Organization in Occlusion-Derived Virions

    PubMed Central

    Beperet, Inés; Barrera, Gloria; Simón, Oihane; Williams, Trevor; López-Ferber, Miguel; Gasmi, Laila; Herrero, Salvador; Caballero, Primitivo

    2013-01-01

    A recombinant virus lacking the sf32 gene (Sf32null), unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac). Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs) of Sf32null contained 62% more genomic DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration), speed-of-kill or budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity) to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion, resulting in increased numbers of nucleocapsids within ODVs. PMID:24204916

  13. Novel Essential Role of Ethanol Oxidation Genes at Low Temperature Revealed by Transcriptome Analysis in the Antarctic Bacterium Pseudomonas extremaustralis

    PubMed Central

    Tribelli, Paula M.; Solar Venero, Esmeralda C.; Ricardi, Martiniano M.; Gómez-Lozano, Maria; Raiger Iustman, Laura J.; Molin, Søren; López, Nancy I.

    2015-01-01

    Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures. PMID:26671564

  14. Novel Essential Role of Ethanol Oxidation Genes at Low Temperature Revealed by Transcriptome Analysis in the Antarctic Bacterium Pseudomonas extremaustralis.

    PubMed

    Tribelli, Paula M; Solar Venero, Esmeralda C; Ricardi, Martiniano M; Gómez-Lozano, Maria; Raiger Iustman, Laura J; Molin, Søren; López, Nancy I

    2015-01-01

    Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures. PMID:26671564

  15. Identification of PRKDC (Protein Kinase, DNA-Activated, Catalytic Polypeptide) as an essential gene for colorectal cancer (CRCs) cells.

    PubMed

    Sun, Shangfeng; Cheng, Shuguang; Zhu, Yunxiao; Zhang, Peng; Liu, Ning; Xu, Tong; Sun, Chao; Lv, Yanfeng

    2016-06-10

    Oncogene and non-oncogene addictions describe the phenomenon that tumor cells become reliant on certain genes for maintenance of malignancy. Reversal of these mutations profoundly affects tumor growth and survival, providing a fundamental rationale for development of targeted cancer therapy. However, inadequate knowledge on cancer signaling networks and lack of potential drug targets limited its clinical application. A screen was conducted using a custom small interfering RNA (siRNA) library in colorectal cancer (CRC). Transient knockdown followed by cell proliferation assays were performed to validate the essentiality of PRKDC (Protein Kinase, DNA-Activated, Catalytic Polypeptide) in CRC. Western blot analysis was performed to examine the mechanism by which PRKDC confers selective survival advantage in CRC cells. Inducible knockdown and overexpression cell lines were introduced into nude mice to assess PRKDC dependency of CRC cells in vivo. PRKDC expression level in patient samples and overall survival of patients with low or high PRKDC expression were analyzed. Transient knockdown of PRKDC reduced cell proliferation/survival in HCT116 and DLD1, but not FHC cells. PRKDC down-regulation induced apoptosis partially through inhibiting AKT activation, and sensitized HCT116 cells to chemotherapeutic agents interfering with DNA replication. Inducible knockdown of PRKDC inhibited tumor growth in vivo. PRKDC was up-regulated in cancerous tissues compared with normal tissues. Patients with high PRKDC expression showed poorer overall survival. PRKDC is an essential gene required for CRC cell proliferation/survival, which may represent as a potential prognostic biomarker and an ideal therapeutic target for CRC. PMID:26992638

  16. Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation.

    PubMed Central

    Gu, Z; Harrod, R; Rogers, E J; Lovett, P S

    1994-01-01

    Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA. Images PMID:7928994

  17. The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation

    PubMed Central

    Rohrer, S.; Ehlert, K.; Tschierske, M.; Labischinski, H.; Berger-Bächi, B.

    1999-01-01

    The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in Staphylococcus aureus peptidoglycan was found to be encoded by the essential gene fmhB. We have analyzed murein composition and structure synthesized when fmhB expression is reduced. The endogenous fmhB promoter was substituted with the xylose regulon from Staphylococcus xylosus, which allowed glucose-controlled repression of fmhB transcription. Repression of fmhB reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropeptide was confirmed by MS to be N-acetylglucosamine-(β-1,4)-N-acetylmuramic acid(-l-Ala-d-iGln-l-Lys-d-Ala-d-Ala), proving that FmhB is involved in the attachment of the first glycine to the pentaglycine interpeptide. This interpeptide plays an important role in crosslinking and stability of the S. aureus cell wall, acts as an anchor for cell wall-associated proteins, determinants of pathogenicity, and is essential for the expression of methicillin resistance. Any shortening of the pentaglycine side chain reduces or even abolishes methicillin resistance, as occurred with fmhB repression. Because of its key role FmhB is a potential target for novel antibacterial agents that could control the threat of emerging multiresistant S. aureus. PMID:10430946

  18. XSIP1 is essential for early neural gene expression and neural differentiation by suppression of BMP signaling.

    PubMed

    Nitta, Kazuhiro R; Tanegashima, Kousuke; Takahashi, Shuji; Asashima, Makoto

    2004-11-01

    Neural differentiation is induced by inhibition of BMP signaling. Secreted inhibitors of BMP such as Chordin from the Spemann organizer contribute to the initial step of neural induction. Xenopus Smad-interacting protein-1 gene (XSIP1) is expressed in neuroectoderm from the early gastrula stage through to the neurula stage. XSIP1 is able to inhibit BMP signaling and overexpression of XSIP1 induces neural differentiation. To clarify the function of XSIP1 in neural differentiation, we performed a loss-of-function study of XSIP1. Knockdown of XSIP1 inhibited SoxD expression and neural differentiation. These results indicate that XSIP1 is essential for neural induction. Furthermore, loss-of-function experiments showed that SoxD is essential for XSIP1 transcription and for neural differentiation. However, inhibition of XSIP1 translation prevented neural differentiation induced by SoxD; thus, SoxD was not sufficient to mediate neural differentiation. Expression of XSIP1 was also required for inhibition of BMP signaling. Together, these results suggest that XSIP1 and SoxD interdependently function to maintain neural differentiation. PMID:15464588

  19. Fumigant toxicity and oviposition deterrency of the essential oil from cardamom, Elettaria cardamomum, against three stored–product insects.

    PubMed

    Abbasipour, Habib; Mahmoudvand, Mohammad; Rastegar, Fahimeh; Hosseinpour, Mohammad Hossein

    2011-01-01

    Use of insecticides can have disruptive effects on the environment. Replacing the chemical compounds in these insecticides with plant materials, however, can be a safe method with low environmental risk. In the current study, chemical composition and insecticidal activities of the essential oil from cardamom, Elettaria cardamomum L. (Maton) (Zingiberales: Zingiberaceae) on the adults of three stored product pests was investigated. Results indicated that essential oil of E. cardamomum toxic to the bruchid beetle, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae), the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), and the flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Adults of E. kuehniella were more sensitive than the Coleoptera. Also, the highest mortality of these insects was seen after 12 hours. Results of the LT₅₀ tests showed that the lethal time of mortality occurred between 10-20 hours in various test concentrations. Essential oil of E. cardamomum had a good efficacy on oviposition deterrence of C. maculatus females, too. The chemical constituents of the essential oils were analyzed by gas chromatography-mass spectrometry. The major constituents of cardamom were identified as 1,8-cineol, α-terpinyl acetate, terpinene and fenchyl alcohol. These results suggest that essential oil of E. cardamomum is a good choice for control of stored product pests. PMID:22242564

  20. Fumigant Toxicity and Oviposition Deterrency of the Essential Oil from Cardamom, Elettaria cardamomum, Against Three Stored—product Insects

    PubMed Central

    Abbasipour, Habib; Mahmoudvand, Mohammad; Rastegar, Fahimeh; Hosseinpour, Mohammad Hossein

    2011-01-01

    Use of insecticides can have disruptive effects on the environment. Replacing the chemical compounds in these insecticides with plant materials, however, can be a safe method with low environmental risk. In the current study, chemical composition and insecticidal activities of the essential oil from cardamom, Elettaria cardamomum L. (Maton) (Zingiberales: Zingiberaceae) on the adults of three stored product pests was investigated. Results indicated that essential oil of E. cardamomum toxic to the bruchid beetle, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae), the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), and the flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Adults of E. kuehniella were more sensitive than the Coleoptera. Also, the highest mortality of these insects was seen after 12 hours. Results of the LT50 tests showed that the lethal time of mortality occurred between 10–20 hours in various test concentrations. Essential oil of E. cardamomum had a good efficacy on oviposition deterrence of C. maculatus females, too. The chemical constituents of the essential oils were analyzed by gas chromatography—mass spectrometry. The major constituents of cardamom were identified as 1,8-cineol, α-terpinyl acetate, terpinene and fenchyl alcohol. These results suggest that essential oil of E. cardamomum is a good choice for control of stored product pests. PMID:22242564

  1. Effect of essential oils on Aspergillus spore germination, growth and mycotoxin production: a potential source of botanical food preservative

    PubMed Central

    Gemeda, Negero; Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw

    2014-01-01

    Objective To investigate effect of essential oils on Aspergillus spore germination, growth and mycotoxin production. Method In vitro antifungal and antiaflatoxigenic activity of essential oils was carried out using poisoned food techniques, spore germination assay, agar dilution assay, and aflatoxin arresting assay on toxigenic strains of Aspergillus species. Results Cymbopogon martinii, Foeniculum vulgare and Trachyspermum ammi (T. ammi) essential oils were tested against toxicogenic isolates of Aspergillus species. T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 µl/mL by essential oils of T. ammi. The oil also showed, complete inhibition of spore germination at a concentration of 2 µl/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting aflatoxin production from Aspergillus niger and Aspergillus flavus at 0.5 and 0.75 µl/mL, respectively. Cymbopogon martinii, Foeniculum vulgare and T. ammi oils as antifungal were found superior over synthetic preservative. Moreover, a concentration of 5 336.297 µl/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity and strengthening its traditional reputations. Conclusions In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by storage fungi. PMID:25183114

  2. Association of TNF-α G308A gene polymorphism in essential hypertensive patients without type 2 diabetes mellitus.

    PubMed

    Ghodsian, N; Akhlaghi, M; Ramachandran, V; Heidari, F; Haghvirdizadeh, P; Eshkoor, S A; Etemad, A; Jamaluddin, J A; Ismail, P

    2015-01-01

    This study aims to investigate the effects of tumor necrosis factor alpha (TNF-α) G308A gene polymorphism on essential hypertension (EHT) with or without type 2 diabetes mellitus (T2DM). The project was conducted on buccal epithelial and blood cells for case and control patients, respectively. Epithelial cells were obtained from the inner part of the cheeks. Techniques including DNA extraction, polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLP) were utilized to assess biomarkers of DNA damage. Our results demonstrated significant differences between wild and mutated genotypes among EHT patients without T2DM. We also found a significant association between wild and mutated allele frequencies in EHT patients (P < 0.05). Clinical characteristics between the groups (EHT with or without T2DM and controls) showed statistically significant association (P < 0.05). Overall, we show that G308A polymorphism of the TNF-αgene may be a significant genetic risk factor for EHT without T2DM patients in Malaysia. PMID:26782547

  3. Regulatory Oversight of Cell- and Tissue-Based Therapeutic Products and Gene Therapy Products in Singapore.

    PubMed

    Goh, Choon Wee; Kellathur, Srinivasan N; Ong, Lee Lee; Wu, Xiaofeng

    2015-01-01

    The regulatory environment for cell- and tissue-based therapeutic products and gene therapy products is rapidly evolving and drug regulatory agencies are working towards establishing a risk-based system in the regulatory framework. Similarly in Singapore, a risk-based tiered approach has been applied whereby clinical trials and product licence of high-risk cell- and tissue-based therapeutic products (substantially manipulated products, products intended for nonhomologous use or combined products) and gene therapy products are regulated as medicinal products under the Medicines Act. There is no legal definition for cell- and tissue-based therapeutic and gene therapy products. The current working definition for a cell- and tissue-based therapeutic product is an article containing or consisting of an autologous or allogeneic human cell or tissue that are used for or administered to, or intended to be used for or administered to, human beings for the diagnosis, treatment, or prevention of human diseases or conditions. Gene therapy products are included under the current biological medicinal product definition. PMID:26374220

  4. Plastid tRNA genes trnC-GCA and trnN-GUU are essential for plant cell development.

    PubMed

    Legen, Julia; Wanner, Gerhard; Herrmann, Reinhold G; Small, Ian; Schmitz-Linneweber, Christian

    2007-09-01

    Higher plant chloroplast genomes code for a conserved set of 30 tRNAs. This set is believed to be sufficient to support translation, although import of cytosolic tRNA has been proposed to provide additional tRNA species to the chloroplast. Previous knock-outs of tRNA genes, or the pronounced reduction of the level of selected tRNAs, has not led to severe phenotypes. We deleted the two tRNA genes trnN-GUU and trnC-GCA independently from the plastid chromosome of tobacco. No homoplastomic tissue of either DeltatrnN or DeltatrnC plants could be isolated. Both mutants exhibit occasional loss of leaf sectors, and mutant plastid chromosomes are rapidly lost upon relief of selective pressure. This suggests that the knock-out of both trn genes is lethal, and that both tRNA species are required for cell survival. Surprisingly, the impact on chloroplast and cell development differs pronouncedly between the two mutants. Heteroplastomic DeltatrnC and DeltatrnN tissue exhibit different aberrations of the internal membrane systems and, more importantly, heteroplastomic DeltatrnN plants are variegated. Accumulation of tRNA-N and plastid-encoded proteins is reduced in white sectors of DeltatrnN plants, and differentiation of palisade cells is abolished. Our data demonstrate that plastid tRNAs are essential, i.e. not complemented by cytosolic tRNA, and have a differential impact on chloroplast and plant cell development. PMID:17573798

  5. Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

    PubMed Central

    2013-01-01

    Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA::GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA::GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion. PMID:23800192

  6. Design and construction of a first-generation high-throughput integrated molecular biology platform for production of optimized synthetic genes and improved industrial strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular biological techniques for plasmid-based assembly and cloning of synthetic assembled gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. These techniques involve the production of full-length cDNA libraries as a source of plasmid-bas...

  7. Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch Migrase

    PubMed Central

    Dresser, Ashley R.; Hardy, Pierre-Olivier; Chaconas, George

    2009-01-01

    Persistent infection by pathogenic organisms requires effective strategies for the defense of these organisms against the host immune response. A common strategy employed by many pathogens to escape immune recognition and clearance is to continually vary surface epitopes through recombinational shuffling of genetic information. Borrelia burgdorferi, a causative agent of Lyme borreliosis, encodes a surface-bound lipoprotein, VlsE. This protein is encoded by the vlsE locus carried at the right end of the linear plasmid lp28-1. Adjacent to the expression locus are 15 silent cassettes carrying information that is moved into the vlsE locus through segmental gene conversion events. The protein players and molecular mechanism of recombinational switching at vlsE have not been characterized. In this study, we analyzed the effect of the independent disruption of 17 genes that encode factors involved in DNA recombination, repair or replication on recombinational switching at the vlsE locus during murine infection. In Neisseria gonorrhoeae, 10 such genes have been implicated in recombinational switching at the pilE locus. Eight of these genes, including recA, are either absent from B. burgdorferi, or do not show an obvious requirement for switching at vlsE. The only genes that are required in both organisms are ruvA and ruvB, which encode subunits of a Holliday junction branch migrase. Disruption of these genes results in a dramatic decrease in vlsE recombination with a phenotype similar to that observed for lp28-1 or vls-minus spirochetes: productive infection at week 1 with clearance by day 21. In SCID mice, the persistence defect observed with ruvA and ruvB mutants was fully rescued as previously observed for vlsE-deficient B. burgdorferi. We report the requirement of the RuvAB branch migrase in recombinational switching at vlsE, the first essential factor to be identified in this process. These findings are supported by the independent work of Lin et al. in the accompanying article, who also found a requirement for the RuvAB branch migrase. Our results also indicate that the mechanism of switching at vlsE in B. burgdorferi is distinct from switching at pilE in N. gonorrhoeae, which is the only other organism analyzed genetically in detail. Finally, our findings suggest a unique mechanism for switching at vlsE and a role for currently unidentified B. burgdorferi proteins in this process. PMID:19997508

  8. CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

    PubMed Central

    Khairnar, Vishal; Duhan, Vikas; Maney, Sathish Kumar; Honke, Nadine; Shaabani, Namir; Pandyra, Aleksandra A.; Seifert, Marc; Pozdeev, Vitaly; Xu, Haifeng C.; Sharma, Piyush; Baldin, Fabian; Marquardsen, Florian; Merches, Katja; Lang, Elisabeth; Kirschning, Carsten; Westendorf, Astrid M.; Häussinger, Dieter; Lang, Florian; Dittmer, Ulf; Küppers, Ralf; Recher, Mike; Hardt, Cornelia; Scheffrahn, Inka; Beauchemin, Nicole; Göthert, Joachim R.; Singer, Bernhard B.; Lang, Philipp A.; Lang, Karl S.

    2015-01-01

    B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses. PMID:25692415

  9. Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

    A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

  10. Effects of Zataria multiflora Boiss. essential oil on growth and gene expression of enterotoxins A, C and E in Staphylococcus aureus ATCC 29213.

    PubMed

    Azizkhani, Maryam; Misaghi, Ali; Basti, Afshin Akhondzadeh; Gandomi, Hassan; Hosseini, Hedayat

    2013-05-15

    Staphylococcal food poisoning results from the consumption of food in which enterotoxigenic staphylococci have grown and produced toxins. The present study was conducted with three principal aims: i) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Zataria multiflora Boiss. essential oil (EO) against Staphylococcus aureus ATCC 29213, ii) to evaluate the effect of subinhibitory concentrations (subMIC) of EO on the growth of bacteria over 72 h (at 25 and 35 °C), and iii) to investigate the expression of genes involved in the production of staphylococcal enterotoxins SEA, SEC and SEE over 72 h at 35 °C. The MIC and MBC of Z. multiflora Boiss. EO were 0.03 and 0.04%, respectively. Colony counting at 24, 48 and 72 h of 3 day cultures grown in the presence of 75%MIC of the EO showed that the growth rate was reduced 2.16, 2.78 and 2.91 log 10 cfu/ml at 25 °C, and 1.34, 2.35 and 2.57 log 10 cfu/ml at 35 °C, respectively, compared to control cultures. SubMIC levels of EO also significantly decreased the expression of staphylococcal enterotoxin (SE)-related genes and therefore the production of SEs in a dose dependent manner. For example, when cultured with 75% MIC, the transcriptional levels of sea, sec, see and agrA were decreased 11.7, 9.3, 10.45 and 10.3 fold after 18 h and 13.9, 11.21, 12.44 and 12.52 fold after 72 h in comparison to control, respectively. PMID:23558199

  11. Effect of Cymbopogon martinii, Foeniculum vulgare, and Trachyspermum ammi Essential Oils on the Growth and Mycotoxins Production by Aspergillus Species

    PubMed Central

    Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw

    2014-01-01

    This study was performed to investigate effect of essential oils on Aspergillus spore germination, growth, and mycotoxin production. In vitro antifungal and antiaflatoxigenic activities of Cymbopogon martinii, Foeniculum vulgare, and Trachyspermum ammi essential oils were carried out on toxigenic strains of Aspergillus species. Plant materials were hydrodistilled for 4-5 h in Clevenger apparatus. 0.25 μL/mL, 0.5 μL/mL, 1 μL/mL, 2 μL/mL, and 4 μL/mL concentrations of each essential oil were prepared in 0.1% Tween 80 (V/V). T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 μL/mL by essential oils of T. ammi. The oil also showed complete inhibition of spore germination at a concentration of 2 μL/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting toxin production from A. niger and A. flavus at 0.5 and 0.75 μL/mL, respectively. C. martinii, F. vulgare, and T. ammi oils as antifungals were found superior over synthetic preservative. Moreover, a concentration of 5336.297 μL/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity. In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by Aspergillus species. PMID:26904653

  12. Incorporation of essential oils and nanoparticles in pullulan films to control foodborne pathogens on meat and poultry products.

    PubMed

    Morsy, Mohamed K; Khalaf, Hassan H; Sharoba, Ashraf M; El-Tanahi, Hassan H; Cutter, Catherine N

    2014-04-01

    The incorporation of essential oils and nanotechnology into edible films has the potential to improve the microbiological safety of foods. The aim of this study was to evaluate the effectiveness of pullulan films containing essential oils and nanoparticles against 4 foodborne pathogens. Initial experiments using plate overlay assays demonstrated that 2% oregano essential oil was active against Staphylococcus aureus and Salmonella Typhimurium, whereas Listeria monocytogenes and Escherichia coli O157:H7 were not inhibited. Two percent rosemary essential oil was active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 1%. Zinc oxide nanoparticles at 110 nm were active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 100 or 130 nm. Conversely, 100 nm silver (Ag) nanoparticles were more active against S. aureus than L. monocytogenes. Using the results from these experiments, the compounds exhibiting the greatest activity were incorporated into pullulan films and found to inhibit all or some of the 4 pathogens in plate overlay assays. In challenge studies, pullulan films containing the compounds effectively inhibited the pathogens associated with vacuum packaged meat and poultry products stored at 4 °C for up to 3 wk, as compared to control films. Additionally, the structure and cross-section of the films were evaluated using electron microscopy. The results from this study demonstrate that edible films made from pullulan and incorporated with essential oils or nanoparticles may improve the safety of refrigerated, fresh or further processed meat and poultry products. PMID:24621108

  13. Effect of Cymbopogon martinii, Foeniculum vulgare, and Trachyspermum ammi Essential Oils on the Growth and Mycotoxins Production by Aspergillus Species.

    PubMed

    Gemeda, Negero; Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw

    2014-01-01

    This study was performed to investigate effect of essential oils on Aspergillus spore germination, growth, and mycotoxin production. In vitro antifungal and antiaflatoxigenic activities of Cymbopogon martinii, Foeniculum vulgare, and Trachyspermum ammi essential oils were carried out on toxigenic strains of Aspergillus species. Plant materials were hydrodistilled for 4-5 h in Clevenger apparatus. 0.25 μL/mL, 0.5 μL/mL, 1 μL/mL, 2 μL/mL, and 4 μL/mL concentrations of each essential oil were prepared in 0.1% Tween 80 (V/V). T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 μL/mL by essential oils of T. ammi. The oil also showed complete inhibition of spore germination at a concentration of 2 μL/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting toxin production from A. niger and A. flavus at 0.5 and 0.75 μL/mL, respectively. C. martinii, F. vulgare, and T. ammi oils as antifungals were found superior over synthetic preservative. Moreover, a concentration of 5336.297 μL/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity. In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by Aspergillus species. PMID:26904653

  14. Role of osteoprotegerin and its gene polymorphisms in the occurrence of left ventricular hypertrophy in essential hypertensive patients.

    PubMed

    Shen, Anna; Hou, Xuwei; Yang, Deguang; Liu, Tingrong; Zheng, Dezhong; Deng, Liehua; Zhou, Tao

    2014-12-01

    The aim of the study was to investigate the role of osteoprotegerin (OPG) in left ventricular hypertrophy (LVH) development in patients with essential hypertension (EH). A total of 1092 patients diagnosed with EH were recruited. The LVHs were determined and OPG gene polymorphisms were genotyped. Patients with LVH had a significantly higher mean serum OPG level than those without LVH. The 1181CC genotype carriers had significantly lower risk for LVH compared with GC and GG genotype carriers. The serum OPG level and OPG 1181 G>C polymorphism were found to be independent risk factors for the occurrence of LVH in hypertensive patients. In vitro study shows that OPG overexpression upregulates cell surface size, protein synthesis per cell, and hypertrophy- and fibrosis-related proteins in both cardiomyocytes and cardiac fibroblasts, whereas OPG inhibition can abolish the above-mentioned changes. Consistent with the in vitro data, our in vivo study revealed that the OPG administration induced the LVH in hypertensive rats. This study is the first to report the close association between OPG and LVH development in EH patients and the regulatory effect of OPG on cardiomyocytes and cardiac fibroblasts. PMID:25546658

  15. Role of Osteoprotegerin and Its Gene Polymorphisms in the Occurrence of Left Ventricular Hypertrophy in Essential Hypertensive Patients

    PubMed Central

    Shen, Anna; Hou, Xuwei; Yang, Deguang; Liu, Tingrong; Zheng, Dezhong; Deng, Liehua; Zhou, Tao

    2014-01-01

    Abstract The aim of the study was to investigate the role of osteoprotegerin (OPG) in left ventricular hypertrophy (LVH) development in patients with essential hypertension (EH). A total of 1092 patients diagnosed with EH were recruited. The LVHs were determined and OPG gene polymorphisms were genotyped. Patients with LVH had a significantly higher mean serum OPG level than those without LVH. The 1181CC genotype carriers had significantly lower risk for LVH compared with GC and GG genotype carriers. The serum OPG level and OPG 1181 G>C polymorphism were found to be independent risk factors for the occurrence of LVH in hypertensive patients. In vitro study shows that OPG overexpression upregulates cell surface size, protein synthesis per cell, and hypertrophy- and fibrosis-related proteins in both cardiomyocytes and cardiac fibroblasts, whereas OPG inhibition can abolish the above-mentioned changes. Consistent with the in vitro data, our in vivo study revealed that the OPG administration induced the LVH in hypertensive rats. This study is the first to report the close association between OPG and LVH development in EH patients and the regulatory effect of OPG on cardiomyocytes and cardiac fibroblasts. PMID:25546658

  16. The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

    PubMed Central

    Atanasova, Lea; Fekete, Erzsébet; Paholcsek, Melinda; Sándor, Erzsébet; Aquino, Benigno; Druzhinina, Irina S.; Karaffa, Levente; Kubicek, Christian P.

    2014-01-01

    Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex. PMID:25386652

  17. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    PubMed Central

    Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of > 95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. PMID:25173815

  18. Isolation and identification of precocenes and piperitone from essential oils as specific inhibitors of trichothecene production by Fusarium graminearum.

    PubMed

    Yaguchi, Atsushi; Yoshinari, Tomoya; Tsuyuki, Rie; Takahashi, Haruo; Nakajima, Takashi; Sugita-Konishi, Yoshiko; Nagasawa, Hiromichi; Sakuda, Shohei

    2009-02-11

    Inhibitors of deoxynivalenol production by Fusarium graminearum are useful for protecting crops from deoxynivalenol contamination. We isolated precocenes and piperitone from the essential oils of Matricaria recutita and Eucalyptus dives, respectively, as specific inhibitors of the production of 3-acetyldeoxynivalenol, a biosynthetic precursor of deoxynivalenol. Precocenes I and II and piperitone inhibited 3-acetyldeoxynivalenol production by F. graminearum in a liquid culture with IC(50) values of 16.6, 1.2, and 306 microM, respectively, without inhibiting fungal growth. Precocene II also inhibited deoxynivalenol production by the fungus in a solid culture on rice with an IC(50) value of 2.0 ppm. Precocene II and piperitone decreased the mRNA levels of Tri4, Tri5, Tri6, and Tri10 encoding proteins required for deoxynivalenol biosynthesis. PMID:19191669

  19. Production of the Ramoplanin Activity Analogue by Double Gene Inactivation

    PubMed Central

    Han, Jungang; Chen, Junsheng; Shao, Lei; Zhang, Junliang; Dong, Xiaojing; Liu, Pengyu; Chen, Daijie

    2016-01-01

    Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin. PMID:27149627

  20. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene.

    PubMed Central

    Totten, P A; Lara, J C; Lory, S

    1990-01-01

    The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism. Images FIG. 1 FIG. 2 PMID:2152909

  1. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

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

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  2. Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

    PubMed Central

    Vallone, D; Battista, S; Pierantoni, G M; Fedele, M; Casalino, L; Santoro, M; Viglietto, G; Fusco, A; Verde, P

    1997-01-01

    The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation. PMID:9311991

  3. Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products.

    PubMed Central

    Ronson, C W; Astwood, P M; Nixon, B T; Ausubel, F M

    1987-01-01

    We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum. The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm. The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM. The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E. coli. PMID:3671068

  4. Natural Products Version 2.0: Connecting Genes to Molecules

    PubMed Central

    Walsh, Christopher T.; Fischbach, Michael A.

    2009-01-01

    Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this perspective, we explore how connecting Nature’s small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and nonribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future? PMID:20121095

  5. Mutations in the Essential Spindle Checkpoint Gene bub1 Cause Chromosome Missegregation and Fail to Block Apoptosis in Drosophila

    PubMed Central

    Basu, Joydeep; Bousbaa, Hassan; Logarinho, Elsa; Li, ZeXiao; Williams, Byron C.; Lopes, Carla; Sunkel, Claudio E.; Goldberg, Michael L.

    1999-01-01

    We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element–induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies. PMID:10402457

  6. Chemoprevention by essential oil of turmeric leaves (Curcuma longa L.) on the growth of Aspergillus flavus and aflatoxin production.

    PubMed

    Sindhu, S; Chempakam, B; Leela, N K; Suseela Bhai, R

    2011-05-01

    Turmeric is well known for a wide range of medicinal properties. Essential oil of turmeric leaves (Curcuma longa L.) were evaluated at varying concentrations of 0.01, 0.05, 0.1, 0.5, 0.75, 1.0 and 1.5% (v/v) in Yeast Extract Sucrose (YES) broth inoculated with spore suspension of Aspergillus flavus of 10(6)conidia/ml. These were evaluated for their potential in the control of aflatoxigenic fungus A. flavus and aflatoxin production. Turmeric leaf oil exhibited 95.3% and 100% inhibition of toxin production respectively at 1.0% and 1.5%. The extent of inhibition of fungal growth and aflatoxin production was dependent on the concentration of essential oil used. The oil exhibited significant inhibition of fungal growth as well as aflatoxins B(1) and G(1) production. The LD(50) and LD(90) were also determined. GC-MS analysis of the oil showed ?-phellandrene, p-cymene and terpinolene as the major components in turmeric leaf oil. The possibility of using these phytochemical components as bio-preservatives for storage of spices is discussed. PMID:21354246

  7. Association study between some renin-angiotensin system gene variants and essential hypertension in a sample of Algerian population: case control study.

    PubMed

    Amrani, Asma; Baba Hamed, Mohamed Bey; Mesli Talebbendiab, Farida

    2015-01-01

    Essential hypertension is an important risk factor for the development of cardiovascular disease. We aim in this study to analyse the relationship between AGT M235T gene variant and ACE I/D gene variant with essential hypertension in a sample of the Algerian population of the Oran city. A case-control study has been performed in 145 subjects including; 75 hypertensives and 70 controls from Algerian population of Oran city. Polymerase chain reaction (PCR) combined with restrictive fragment length polymorphism (RFLP) was used to detect the M235T variant of angiotensinogen (AGT) gene and a nested PCR to determine ACE I/D gene variant. The genotypic and allelic distribution of the M235Tvariant of the AGT gene did not differ in hypertensives and normotensives group (X(2) =7.81, P<0.05; X(2) =4.67, respectively) thus there was no association between the T allele and hypertension (OR=1.64; 95%CI [1.01-2.69]). The genotypic and allelic frequencies of the ACE I/D variant did differ significantly between hypertensives and controls (X(2)=13.98, P<0.05; X(2) =12.66, P<0.05, respectively) where a significant association between the D allele of the ACE I/D gene and essential hypertension has been observed (OR=0.46; 95%CI [0.27-0.75]). We reported a high prevalence of the D and T allele in hypertensives female. This study shows that the M235T variant of the AGT gene is not associated with essential hypertension while a significant association has been reported with the D allele in this sample of Algerian population of the Oran city. PMID:26489814

  8. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    PubMed

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M; Verstrepen, Kevin J

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts. PMID:25764309

  9. Glycerol Production by Fermenting Yeast Cells Is Essential for Optimal Bread Dough Fermentation

    PubMed Central

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M.; Verstrepen, Kevin J.

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts. PMID:25764309

  10. Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus Sequence Type 398 survival.

    PubMed

    Christiansen, Mette T; Kaas, Rolf S; Chaudhuri, Roy R; Holmes, Mark A; Hasman, Henrik; Aarestrup, Frank M

    2014-01-01

    Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 isolate to evaluate genes important for bacterial survival in laboratory and host-specific environments. The transposon mutant library consisted of approximately 1 million mutants with around 140,000 unique insertion sites and an average number of unique inserts per gene of 44.8. We identified LA-MRSA ST398 essential genes comparable to other high-throughput S. aureus essential gene studies. As ST398 is the most common MRSA isolated from pigs, the transposon mutant library was screened in whole porcine blood. Twenty-four genes were specifically identified as important for bacterial survival in porcine blood. Mutations in 23 of these genes resulted in attenuated bacterial fitness. Seven of the 23 genes were of unknown function, whereas 16 genes were annotated with functions predominantly related to carbon metabolism, pH shock and a variety of regulations and only indirectly to virulence factors. Mutations in one gene of unknown function resulted in a hypercompetitive mutant. Further evaluation of these genes is required to determine their specific relevance in blood survival. PMID:24563689

  11. Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

    SciTech Connect

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alex S.

    2015-03-27

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as ‘topologically important.’ Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as ‘functionally important’ genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.

  12. Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

    DOE PAGESBeta

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alex S.

    2015-03-27

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as ‘topologically important.’ Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termedmore » as ‘functionally important’ genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.« less

  13. The PBN1 gene of Saccharomyces cerevisiae: an essential gene that is required for the post-translational processing of the protease B precursor.

    PubMed Central

    Naik, R R; Jones, E W

    1998-01-01

    The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O-linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large amino-terminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p. PMID:9649520

  14. The Flavin-Containing Monooxygenase 3 Gene and Essential Hypertension: The Joint Effect of Polymorphism E158K and Cigarette Smoking on Disease Susceptibility.

    PubMed

    Bushueva, Olga; Solodilova, Maria; Churnosov, Mikhail; Ivanov, Vladimir; Polonikov, Alexey

    2014-01-01

    Gene encoding flavin-containing monooxygenase 3 (FMO3), a microsomal antioxidant defense enzyme, has been suggested to contribute to essential hypertension (EH). The present study was designed to investigate whether common functional polymorphism E158K (rs2266782) of the FMO3 gene is associated with EH susceptibility in a Russian population. A total of 2 995 unrelated subjects from Kursk (1 362 EH patients and 843 healthy controls) and Belgorod (357 EH patients and 422 population controls) regions of Central Russia were recruited for this study. DNA samples from all study participants were genotyped for the FMO3 gene polymorphism through PCR followed by RFLP analysis. We found that the polymorphism E158K is associated with increased risk of essential hypertension in both discovery population from Kursk region (OR 1.36 95% CI 1.09-1.69, P = 0.01) and replication population from Belgorod region (OR 1.54 95% CI 1.07-1.89, P = 0.02) after adjustment for gender and age using logistic regression analysis. Further analysis showed that the increased hypertension risk in carriers of genotype 158KK gene occurred in cigarette smokers, whereas nonsmoker carriers of this genotype did not show the disease risk. This is the first study reporting the association of the FMO3 gene polymorphism and the risk of essential hypertension. PMID:25243081

  15. The Flavin-Containing Monooxygenase 3 Gene and Essential Hypertension: The Joint Effect of Polymorphism E158K and Cigarette Smoking on Disease Susceptibility

    PubMed Central

    Bushueva, Olga; Solodilova, Maria; Churnosov, Mikhail; Ivanov, Vladimir; Polonikov, Alexey

    2014-01-01

    Gene encoding flavin-containing monooxygenase 3 (FMO3), a microsomal antioxidant defense enzyme, has been suggested to contribute to essential hypertension (EH). The present study was designed to investigate whether common functional polymorphism E158K (rs2266782) of the FMO3 gene is associated with EH susceptibility in a Russian population. A total of 2 995 unrelated subjects from Kursk (1 362 EH patients and 843 healthy controls) and Belgorod (357 EH patients and 422 population controls) regions of Central Russia were recruited for this study. DNA samples from all study participants were genotyped for the FMO3 gene polymorphism through PCR followed by RFLP analysis. We found that the polymorphism E158K is associated with increased risk of essential hypertension in both discovery population from Kursk region (OR 1.36 95% CI 1.09–1.69, P = 0.01) and replication population from Belgorod region (OR 1.54 95% CI 1.07–1.89, P = 0.02) after adjustment for gender and age using logistic regression analysis. Further analysis showed that the increased hypertension risk in carriers of genotype 158KK gene occurred in cigarette smokers, whereas nonsmoker carriers of this genotype did not show the disease risk. This is the first study reporting the association of the FMO3 gene polymorphism and the risk of essential hypertension. PMID:25243081

  16. Redefining body composition: nutrients hormones, and genes in meat production.

    PubMed

    Wray-Cahen, C D; Kerr, D E; Evock-Clover, C M; Steele, N C

    1998-01-01

    Growth rate and body composition of livestock can be optimized to meet consumer needs for a leaner product and to improve the efficiency of meat-animal production. Optimization strategies have traditionally focused on genetic selection and cost-effective ration formulation to achieve the genetic potential. Advances in understanding the mechanisms of growth and its control have led to additional opportunities for its manipulation. These include nutritional manipulation,the use of growth promotants, and, more recently, the ability to change the genetic potential through genetic engineering. Selection of appropriate candidate genes for manipulation depends on understanding the mechanisms underlying differentiation and growth of embryonic muscle cells. Recent advances in genetic engineering techniques, including gene therapy and germline transgenesis, will likely hasten the genetic progress toward a leaner carcass in domestic livestock. Such strategies may prove to be more beneficial then the controlled enhancement of somatotropin expression. PMID:9706219

  17. Plasmid genes required for microcin B17 production.

    PubMed Central

    San Millán, J L; Kolter, R; Moreno, F

    1985-01-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production. PMID:2993228

  18. An integrated approach utilising chemometrics and GC/MS for classification of chamomile flowers, essential oils and commercial products.

    PubMed

    Wang, Mei; Avula, Bharathi; Wang, Yan-Hong; Zhao, Jianping; Avonto, Cristina; Parcher, Jon F; Raman, Vijayasankar; Zweigenbaum, Jerry A; Wylie, Philip L; Khan, Ikhlas A

    2014-01-01

    As part of an ongoing research program on authentication, safety and biological evaluation of phytochemicals and dietary supplements, an in-depth chemical investigation of different types of chamomile was performed. A collection of chamomile samples including authenticated plants, commercial products and essential oils was analysed by GC/MS. Twenty-seven authenticated plant samples representing three types of chamomile, viz. German chamomile, Roman chamomile and Juhua were analysed. This set of data was employed to construct a sample class prediction (SCP) model based on stepwise reduction of data dimensionality followed by principle component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The model was cross-validated with samples including authenticated plants and commercial products. The model demonstrated 100.0% accuracy for both recognition and prediction abilities. In addition, 35 commercial products and 11 essential oils purported to contain chamomile were subsequently predicted by the validated PLS-DA model. Furthermore, tentative identification of the marker compounds correlated with different types of chamomile was explored. PMID:24444953

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

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

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

  20. Stock-outs of essential health products in Mozambique-longitudinal analyses from 2011 to 2013

    PubMed Central

    Wagenaar, Bradley H.; Gimbel, Sarah; Hoek, Roxanne; Pfeiffer, James; Michel, Cathy; Manuel, João Luis; Cuembelo, Fatima; Quembo, Titos; Afonso, Pires; Gloyd, Stephen; Sherr, Kenneth

    2015-01-01

    objectives To assess the relationship between health system factors and facility-level EHP stock-outs in Mozambique. methods Service provisions were assessed in 26 health facilities and 13 district warehouses in Sofala Province, Mozambique, from July to August in 2011–2013. Generalised estimating equations were used to model factors associated with facility-level availability of essential drugs, supplies and equipment. results Stock-out rates for drugs ranged from 1.3% for oral rehydration solution to 20.5% for Depo-Provera and condoms, with a mean stock-out rate of 9.1%; mean stock-out rates were 15.4% for supplies and 4.1% for equipment. Stock-outs at the district level accounted for 27.1% (29/107) of facility-level drug stock-outs and 44.0% (37/84) of supply stock-outs. Each 10-km increase in the distance from district distribution warehouses was associated with a 31% (CI: 22–42%), 28% (CI: 17–40%) or 27% (CI: 7–50%) increase in rates of drug, supply or equipment stock-outs, respectively. The number of heath facility staff was consistently negatively associated with the occurrence of stock-outs. conclusions Facility-level stock-outs of EHPs in Mozambique are common and appear to disproportionately affect those living far from district capitals and near facilities with few health staff. The majority of facility-level EHP stock-outs in Mozambique occur when stock exists at the district distribution centre. Innovative methods are urgently needed to improve EHP supply chains, requesting and ordering of drugs, facility and district communication, and forecasting of future EHP needs in Mozambique. Increased investments in public-sector human resources for health could potentially decrease the occurrence of EHP stock-outs. PMID:24724617

  1. Potential of Cultivated Ganoderma lucidum Mushrooms for the Production of Supplements Enriched with Essential Elements.

    PubMed

    Rzymski, Piotr; Mleczek, Mirosław; Niedzielski, Przemysław; Siwulski, Marek; Gąsecka, Monika

    2016-03-01

    Ganoderma lucidum is an important medicinal mushroom species and there is continuous interest in its bioactive properties. This study evaluated whether it may additionally serve as a nutritional supplement for the trace elements: selenium (Se), copper (Cu), and zinc (Zn). Mushrooms were cultivated on substrates enriched with 0.1 to 0.8 mM of inorganic Se alone or in combination with Zn and/or Cu. Supplementation increased accumulation of the elements in fruiting bodies regardless of the applied cultivation model. G. lucidum demonstrated the ability to accumulate significant amounts of organic Se, maximally amounting to (i) over 44 mg/kg when the substrate was supplemented only with Se, (ii) over 20 mg/kg in the Se+Cu model, (iii) over 25 mg/kg in the Se+Zn model, and (iv) 15 mg/kg in the Se+Cu+Zn model. The accumulation of Cu and Zn steadily increased with their initial substrate concentrations. Maximum concentrations found after supplementation with 0.8 mM amounted to over 55 mg/kg (Se+Zn) and 52 mg/kg (Se+Cu+Zn) of Zn, and 29 mg/kg (Se+Cu) and over 31 mg/kg (Se+Cu+Zn) of Cu. The greater the supplemented concentration and number of supplemented elements, the lower the biomass of G. lucidum fruiting bodies. Nevertheless, it still remained high when the substrate was supplemented up to 0.4 mM with each element. These results highlight that G. lucidum can easily incorporate elements from the substrate and that, when biofortified, its dried fruiting bodies may serve as a nutritional source of these essential elements. PMID:26799621

  2. Peroxisome Proliferator-activated Receptor-γ Coactivator-1α Controls Transcription of the Sirt3 Gene, an Essential Component of the Thermogenic Brown Adipocyte Phenotype*

    PubMed Central

    Giralt, Albert; Hondares, Elayne; Villena, Josep A.; Ribas, Francesc; Díaz-Delfín, Julieta; Giralt, Marta; Iglesias, Roser; Villarroya, Francesc

    2011-01-01

    Sirt3 (silent mating type information regulation 2, homolog 3), a member of the sirtuin family of protein deacetylases with multiple actions on metabolism and gene expression is expressed in association with brown adipocyte differentiation. Using Sirt3-null brown adipocytes, we determined that Sirt3 is required for an appropriate responsiveness of cells to noradrenergic, cAMP-mediated activation of the expression of brown adipose tissue thermogenic genes. The transcriptional coactivator Pgc-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) induced Sirt3 gene expression in white adipocytes and embryonic fibroblasts as part of its overall induction of a brown adipose tissue-specific pattern of gene expression. In cells lacking Sirt3, Pgc-1α failed to fully induce the expression of brown fat-specific thermogenic genes. Pgc-1α activates Sirt3 gene transcription through coactivation of the orphan nuclear receptor Err (estrogen-related receptor)-α, which bound the proximal Sirt3 gene promoter region. Errα knockdown assays indicated that Errα is required for full induction of Sirt3 gene expression in response to Pgc-1α. The present results indicate that Pgc-1α controls Sirt3 gene expression and this action is an essential component of the overall mechanisms by which Pgc-1α induces the full acquisition of a brown adipocyte differentiated phenotype. PMID:21454513

  3. The Steroid Content of Adrenal Adenomas and Measurements of Aldosterone Production in Patients with Essential Hypertension and Primary Aldosteronism*

    PubMed Central

    Kaplan, Norman M.

    1967-01-01

    The content of aldosterone, corticosterone, and cortisol has been assayed in normal adrenal tissue obtained at autopsy and in adrenal adenomas from both normotensive and hypertensive patients obtained at autopsy and from patients with primary aldosteronism obtained at surgery. The content of aldosterone and corticosterone in the adenomas of patients with essential hypertension was similar to that of normal adrenal tissue and much less than that of adenomas from patients with primary aldosteronism. Since these results do not appear to reflect various extraneous factors such as assay technique, degradation of steroids, and patient selection, they suggest that most adrenal adenomas found in patients with essential hypertension are not associated with increased aldosterone production. The presence of such adenomas should not, in itself, be considered evidence for primary aldosteronism. The excretion or secretion of aldosterone in 43 hypertensive patients was found to be similar to that of 39 normotensive patients. These results suggest that primary aldosteronism is rarely the cause of essential hypertension. PMID:6025479

  4. Expression of a Testis-Specific Form of Gal3st1 (CST), a Gene Essential for Spermatogenesis, Is Regulated by the CTCF Paralogous Gene BORIS▿ †

    PubMed Central

    Suzuki, Teruhiko; Kosaka-Suzuki, Natsuki; Pack, Svetlana; Shin, Dong-Mi; Yoon, Jeongheon; Abdullaev, Ziedulla; Pugacheva, Elena; Morse, Herbert C.; Loukinov, Dmitri; Lobanenkov, Victor

    2010-01-01

    Previously, it was shown that the CTCF paralogous gene, BORIS (brother of the regulator of imprinted sites) is expressed in male germ cells, but its function in spermatogenesis has not been defined. To develop an understanding of the functional activities of BORIS, we generated BORIS knockout (KO) mice. Mice homozygous for the null allele had a defect in spermatogenesis that resulted in small testes associated with increased cell death. The defect was evident as early as postnatal day 21 and was manifested by delayed production of haploid cells. By gene expression profiling, we found that transcript levels for Gal3st1 (also known as cerebroside sulfotransferase [CST]), known to play a crucial role in meiosis, were dramatically reduced in BORIS KO testes. We found that CST is expressed in testis as a novel testis-specific isoform, CST form FTS, that has a short exon 1f. We showed that BORIS bound to and activated the promoter of CST form FTS. Mutation of the BORIS binding site in the promoter reduced the ability of BORIS to activate the promoter. These findings define transcriptional regulation of CST expression as a critical role for BORIS in spermatogenesis. PMID:20231363

  5. Angiotensinogen (AGT) M235T, AGT T174M and Angiotensin-1-Converting Enzyme (ACE) I/D Gene Polymorphisms in Essential Hypertension: Effects on Ramipril Efficacy

    PubMed Central

    Kolovou, Vana; Lagou, Evangelia; Mihas, Constantinos; Vasiliki, Giannakopoulou; Katsiki, Niki; Kollia, Aikaterini; Triposkiadis, Filippos; Degiannis, Dimitris; Mavrogeni, Sophie; Kolovou, Genovefa

    2015-01-01

    Background: Hypertension, one of the most important risk factors for premature cardiovascular disease, is a major worldwide public health problem. Angiotensin-1-converting enzyme (ACE) and angiotensinogen (AGT) gene polymorphisms are thought to be associated with primary hypertension. In the present study, we examined the frequency of these gene polymorphisms in an adult population with and without essential hypertension. Furthermore, we evaluated the effect of ACE and AGT gene polymorphisms on ramipril treatment efficacy in the hypertensive patients. Methods: A total of 166 adults (83 hypertensives and 83 normotensives) were involved in the study and genotyped for AGTM235T (rs699), AGTT174M (rs4762) and ACEI/D (rs1799752) gene polymorphisms. Results: The genotype and allele distribution of the AGTM235T variant significantly differed between hypertensives and normotensives [odds ratio (OR) = 1.57% (T vs M allele), 95% confidence intervals (CIs): 1.01 - 2.44; p=0.045 for hypertensives]. However, none of the 3 studied Simple Nucleotide Polymorphisms were associated with the blood pressure-lowering response to ramipril. Conclusion: These results suggest that AGTM235T gene polymorphism is associated with essential hypertension. However, none of the AGTM235T, AGTT174M and ACEI/D gene polymorphisms influenced ramipril effectiveness. PMID:27006715

  6. Peroxisome biogenesis in the yeast Hansenula polymorpha is controlled by a complex set of interacting gene products.

    PubMed Central

    Titorenko, V I; Waterham, H R; Cregg, J M; Harder, W; Veenhuis, M

    1993-01-01

    We have studied the genetic interactions between mutant alleles in 12 genes, designated PER1-PER12, which are essential for peroxisome biogenesis in the yeast Hansenula polymorpha. Recessive mutations in any of these genes determined three different morphological phenotypes: (i) complete absence of peroxisomes (Per-); (ii) presence of small peroxisomes in conjunction with a major fraction of peroxisomal matrix proteins in the cytosol (Pim-); and (iii) presence of peroxisomes with aberrant crystalline matrix substructure (Pss-). Extensive complementation analysis showed many cases of noncomplementation--that is, diploids that contained both wild-type and mutant alleles of two different PER genes were unable to grow on methanol and showed peroxisomal defects. The observed cases of unlinked noncomplementation appeared to be gene and allele specific and were predominantly observed at lower temperatures (cold sensitive). The genetic results obtained were used to formulate a model of PER gene product interactions. In this model, five PER gene products are key or core components of the complex. Other PER gene products appear to play a more peripheral role. Images Fig. 1 Fig. 2 PMID:8356040

  7. Modular optimization of multi-gene pathways for fumarate production.

    PubMed

    Chen, Xiulai; Zhu, Pan; Liu, Liming

    2016-01-01

    Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate. PMID:26241189

  8. Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.

    PubMed

    Burghout, Peter; Bootsma, Hester J; Kloosterman, Tomas G; Bijlsma, Jetta J E; de Jongh, Christa E; Kuipers, Oscar P; Hermans, Peter W M

    2007-09-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae. PMID:17631629

  9. Search for Genes Essential for Pneumococcal Transformation: the RadA DNA Repair Protein Plays a Role in Genomic Recombination of Donor DNA▿

    PubMed Central

    Burghout, Peter; Bootsma, Hester J.; Kloosterman, Tomas G.; Bijlsma, Jetta J. E.; de Jongh, Christa E.; Kuipers, Oscar P.; Hermans, Peter W. M.

    2007-01-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae. PMID:17631629

  10. CO2 – Intrinsic Product, Essential Substrate, and Regulatory Trigger of Microbial and Mammalian Production Processes

    PubMed Central

    Blombach, Bastian; Takors, Ralf

    2015-01-01

    Carbon dioxide formation mirrors the final carbon oxidation steps of aerobic metabolism in microbial and mammalian cells. As a consequence, CO2/HCO3− dissociation equilibria arise in fermenters by the growing culture. Anaplerotic reactions make use of the abundant CO2/HCO3− levels for refueling citric acid cycle demands and for enabling oxaloacetate-derived products. At the same time, CO2 is released manifold in metabolic reactions via decarboxylation activity. The levels of extracellular CO2/HCO3− depend on cellular activities and physical constraints such as hydrostatic pressures, aeration, and the efficiency of mixing in large-scale bioreactors. Besides, local CO2/HCO3− levels might also act as metabolic inhibitors or transcriptional effectors triggering regulatory events inside the cells. This review gives an overview about fundamental physicochemical properties of CO2/HCO3− in microbial and mammalian cultures effecting cellular physiology, production processes, metabolic activity, and transcriptional regulation. PMID:26284242

  11. Sesquiterpenes from the essential oil of Curcuma wenyujin and their inhibitory effects on nitric oxide production.

    PubMed

    Xia, Guiyang; Zhou, Li; Ma, Jianghao; Wang, Ying; Ding, Liqin; Zhao, Feng; Chen, Lixia; Qiu, Feng

    2015-06-01

    Three new sesquiterpenes including a new elemane-type sesquiterpene, 5βH-elem-1,3,7,8-tetraen-8,12-olide (1), and two new carabrane-type sesquiterpenes, 7α,11-epoxy-6α-methoxy-carabrane-4,8-dione (2) and 8,11-epidioxy-8-hydroxy-4-oxo-6-carabren (3), together with eight known sesquiterpenes (4-11) were isolated from Curcuma wenyujin Y. H. Chen et C. Ling. Their structures were elucidated based on extensive spectroscopic analyses. A possible biogenetic scheme for the related compounds was postulated. All of the isolated compounds were tested for inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages. Meanwhile, preliminary structure-activity relationships for these compounds are discussed. PMID:25819782

  12. Determination of elemental toxicity migration limits, bioaccessibility and risk assessment of essential childcare products.

    PubMed

    Aboel Dahab, Ali; Elhag, Dhia Eldin A; Ahmed, Ammar Bourai; Al-Obaid, Humaida A

    2016-02-01

    Children especially infants are particularly sensitive to contaminant exposure, they are exposed to toxic substances including heavy metals via multiple pathways, i.e. food, air, water, soil and childcare products. To date, determination of metal bioaccessibility in teethers and feeding teats is missing in the literature; therefore, it is vitally important to assess their metal bioaccessibility and characterise the risk for children. The aim of this study is to determine the migration levels of toxic elements in teethers and feeding teats of different brands as a measure of metal bioaccessibility and characterise the risk for children exposed to these products. The migration limits of several heavy metals (Al, As, Ba, Cd, Co, Cu, Cr, Mn, Ni, Pb, Se, Sr, Zn) in different brands of teethers and feeding teats were determined simultaneously using inductively coupled plasma optical emission spectroscopy (ICP-OES) adopting a protocol in the European standards for safety of toys. With the exception of Pb, the migration limits of all elements in all brands of teethers and feeding teats were below the specified limits. However, in the case of Pb, the migration was above the specified limits in all samples except one brand of feeding teats. Risk assessment expressed as hazard index (HI) was calculated for detected elements and all samples. Although HI was below 1.0 for all samples except one sample, the high Pb concentration would pose a considerable risk to children. Therefore, we recommend a more thorough research and risk characterisation taking into consideration the factors that affect HI values. Graphical Abstract Determination of metal bioaccessibility and risk characterisation of teethers and feeding teats ensure children safety against metal toxicity. PMID:26490916

  13. Cooperation between NRF-2 and YY-1 transcription factors is essential for triggering the expression of the PREPL-C2ORF34 bidirectional gene pair

    PubMed Central

    Huang, Chien-Chang; Chang, Wun-Shaing Wayne

    2009-01-01

    Background Many mammalian genes are organized as bidirectional (head-to-head) gene pairs with the two genes separated only by less than 1 kb. The transcriptional regulation of these bidirectional gene pairs remains largely unclear, but a few studies have suggested that the two closely adjacent genes in divergent orientation can be co-regulated by a single transcription factor binding to a specific regulatory fragment. Here we report an evolutionarily conserved bidirectional gene pair, known as the PREPL-C2ORF34 gene pair, whose transcription relies on the synergic cooperation of two transcription factors binding to an intergenic bidirectional minimal promoter. Results While PREPL is present primarily in brain and heart, C2ORF34 is ubiquitously and abundantly expressed in almost all tissues. Genomic analyses revealed that these two non-homologous genes are adjacent in a head-to-head configuration on human chromosome 2p21 and separated by only 405 bp. Within this short intergenic region, a 243-bp GC-rich segment was demonstrated to function as a bidirectional minimal promoter to initiate the transcription of both flanking genes. Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner. The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair. Conclusion This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region. PMID:19575798

  14. Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice.

    PubMed

    Kato, Yuki; Konishi, Mineko; Shigyo, Mikao; Yoneyama, Tadakatsu; Yanagisawa, Shuichi

    2010-07-01

    Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species. PMID:20570652

  15. Identification of the neurofibromatosis type 1 gene product

    SciTech Connect

    Gutmann, D.H.; Wood, D.L.; Collins, F.S. )

    1991-11-01

    The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of {approx}250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to the synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.

  16. The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

    PubMed

    Gazanion, E; Garcia, D; Silvestre, R; Gérard, C; Guichou, J F; Labesse, G; Seveno, M; Cordeiro-Da-Silva, A; Ouaissi, A; Sereno, D; Vergnes, B

    2011-10-01

    NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target. PMID:21819459

  17. dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

    PubMed Central

    Henrich, B; Becker, S; Schroeder, U; Plapp, R

    1993-01-01

    Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties. Images PMID:8226676

  18. Ferulic acid 5-hydroxylase 1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis.

    PubMed

    Maruta, Takanori; Noshi, Masahiro; Nakamura, Maki; Matsuda, Shun; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2014-04-01

    Anthocyanins are important for preventing photoinhibition and photodamage. By comprehensive reverse genetic analysis of chloroplast-produced H2O2-responsive genes, we isolated here an anthocyanin-deficient mutant under photooxidative stress, which lacked ferulate 5-hydroxylase 1 (FAH1) involved in the phenylpropanoid pathway. Interestingly, the expression of anthocyanin biosynthesis-associated genes was also inhibited in this mutant. These findings suggest that FAH1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis. Furthermore, we found that estrogen-inducible silencing of thylakoid membrane-bound ascorbate peroxidase, which is a major H2O2-scavenging enzyme in chloroplasts, enhances the expression of FAH1 and anthocyanin biosynthesis-associated genes and accumulation of anthocyanin without any application of stress. Thus, it is likely that chloroplastic H2O2 activates FAH1 expression to induce anthocyanin accumulation for protecting cells from photooxidative stress. PMID:24576765

  19. A gene and its product required for transposition of resistance transposon Tn2603.

    PubMed Central

    Tanaka, M; Harafuji, H; Yamamoto, T

    1982-01-01

    Tn2603 is a multiple-resistance transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury and having a molecular size of 20 kilobase pairs, with 200-base-pair inverted repeats at both ends. The essential sites and functions of Tn2603 which are required for its transposition were determined through construction and characterization of various deletion mutants affecting the efficiency of transposition. Deletions were introduced in plasmid pMK1::Tn2603 by partial digestion with restriction endonuclease EcoRI in vitro. Analysis of deletion mutants showed that the inverted repeat segments at both ends of the trans-acting diffusible product(s) encoded in the right-hand side of the central portion were required for the transposition of Tn2603. An essential gene product was revealed as a protein having a molecular weight of 110,000 by analysis of polypeptides synthesized in Escherichia coli minicells. This protein was assumed to be the so-called transposase. Images PMID:6284710

  20. Characterization and localization of the Huntington disease gene product.

    PubMed

    Hoogeveen, A T; Willemsen, R; Meyer, N; de Rooij, K E; Roos, R A; van Ommen, G J; Galjaard, H

    1993-12-01

    The recent identification of the Huntington's disease (HD) gene, enabled us to synthesize oligopeptides corresponding with the carboxy-terminal end of the predicted HD-gene (IT15) product. Immunobiochemcial studies with polyclonal antibodies directed against this synthetic peptide (position 3114-3141) on lymphoblastoid cells from normal individuals and patients with Huntington disease, revealed the presence of a protein (huntingtin) with a molecular mass of approximately 330 kDa. Immunocytochemical studies showed a cytoplasmic localization of huntingtin in various cell types including neurons. In most of the neuronal cells the protein was also present in the nucleus. No difference in molecular mass or intracellular localization was found between normal and mutant cells. PMID:8111375

  1. Involvement of distinct PKC gene products in T cell functions

    PubMed Central

    Pfeifhofer-Obermair, Christa; Thuille, Nikolaus; Baier, Gottfried

    2012-01-01

    It is well established that members of the protein kinase C (PKC) family seem to have important roles in T cells. Focusing on the physiological and non-redundant PKC functions established in primary mouse T cells via germline gene-targeting approaches, our current knowledge defines two particularly critical PKC gene products, PKCθ and PKCα, as the “flavor of PKC” in T cells that appear to have a positive role in signaling pathways that are necessary for full antigen receptor-mediated T cell activation ex vivo and T cell-mediated immunity in vivo. Consistently, in spite of the current dogma that PKCθ inhibition might be sufficient to achieve complete immunosuppressive effects, more recent results have indicated that the pharmacological inhibition of PKCθ, and additionally, at least PKCα, appears to be needed to provide a successful approach for the prevention of allograft rejection and treatment of autoimmune diseases. PMID:22888329

  2. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    PubMed Central

    Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

  3. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    SciTech Connect

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  4. Viral gene delivery: optimized protocol for production of high titer lentiviral vectors.

    PubMed

    Hewinson, James; Paton, Julian F R; Kasparov, Sergey

    2013-01-01

    HIV-derived lentiviral vectors (LVV) are among the most commonly used gene delivery vehicles. Their production in high quantities, which enables concentration of viral particles to high titers, is important for their successful application in both biomedical research and gene therapy. LVV are produced by co-transfection of three or more plasmids into a packaging cell line followed by several purification and concentration steps. Protocols currently in circulation differ from each other but the direct comparison of their efficacy based on the published information is extremely difficult because more than one variable may be changed and essential information may be omitted. We systematically evaluated three protocols and found that one single modification described here, using FuGene(®) 6 in the co-transfection step, increase LVV output almost 20 times as compared to the most commonly used calcium phosphate (CaPO4) transfection technique. Unexpectedly FuGene(®) 6 was also much more efficient than another widely used reagent, Superfect. Dependent on requirements, this permits a dramatic downscaling of the packaging stage of viral production, and/or super-concentration of LVV to achieve stronger expression. For example we were able to prepare ∼25 μL of high titer LVV suitable for injections into rodent brain using a single T75 cm(2) cell culture flask of packaging cells. The same output would require up to 20 times more packaging cells and reagents following conventional protocols. We illustrate the potential of our approach using transfection of primary neuronal cultures with LVV expressing an optogenetic actuator channelrhodopsin-2. Our observations should help to achieve reproducible production of high titer LVV for experimental and potential therapeutic applications. PMID:23529421

  5. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively-parallel sequencing of transposon libraries (Tn-seq)

    PubMed Central

    Klein, Brian A.; Duncan, Margaret J.; Hu, Linden T.

    2016-01-01

    Summary Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify non-essential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures. PMID:25636611

  6. Reference genes selection and relative expression analysis from Shiraia sp. SUPER-H168 productive of hypocrellin.

    PubMed

    Deng, Huaxiang; Gao, Ruijie; Liao, Xiangru; Cai, Yujie

    2016-04-10

    Shiraia bambusicola is an essential pharmaceutical fungus due to its production of hypocrellin with antiviral, antidepressant, and antiretroviral properties. Based on suitable reference gene (RG) normalization, gene expression analysis enables the exploitation of significant genes relative to hypocrellin biosynthesis by quantitative real-time polymerase chain reaction. We selected and assessed nine candidate RGs in the presence and absence of hypocrellin biosynthesis using GeNorm and NormFinder algorithms. After stepwise exclusion of unstable genes, GeNorm analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytochrome oxidase (CyO) as the most stable expression, while NormFinder determined 18S ribosomal RNA (18S rRNA) as the most appropriate candidate gene for normalization. Tubulin (Tub) was observed to be the least stable gene and should be avoided for relative expression analysis. We further analyzed relative expression levels of essential proteins correlative with hypocrellin biosynthesis, including polyketide synthase (PKS), O-methyltransferase (Omef), FAD/FMN-dependent oxidoreductase (FAD), and monooxygenase (Mono). Compared to PKS, Mono kept a similar expression pattern and simulated PKS expression, while FAD remained constantly expressed. Omef presented lower transcript levels and had no relation to PKS expression. These relative expression analyses will pave the way for further interpretation of the hypocrellin biosynthesis pathway. PMID:26779826

  7. Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region.

    PubMed

    Haramoto, Yoshikazu; Tanegashima, Kousuke; Onuma, Yasuko; Takahashi, Shuji; Sekizaki, Hiroyuki; Asashima, Makoto

    2004-01-01

    In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling. PMID:14697360

  8. Initial catabolism of aromatic biogenic amines by Pseudomonas aeruginosa PAO: pathway description, mapping of mutations, and cloning of essential genes.

    PubMed

    Cuskey, S M; Peccoraro, V; Olsen, R H

    1987-06-01

    Pseudomonas aeruginosa PAO1 was able to utilize several aromatic biogenic amines as sole sources of carbon or nitrogen. These included the phenethylamines tyramine and dopamine and the phenethanolamines octopamine, synephrine, and norepinephrine. Initial catabolism of the phenethylamines was mediated by a membrane-bound tyramine dehydrogenase which produced 4-hydroxyphenylacetaldehyde (4HPAL) with tyramine as the substrate. The enzyme was induced by growth with both classes of amines. Initial catabolism of octopamine (except when present as the sole source of carbon and nitrogen) was mediated by a soluble enzyme with activity against the phenethanolamines but not against tyramine or dopamine. The product of the reaction with octopamine as substrate was also 4HPAL. Addition of NAD to reaction mixtures yielded 4-hydroxyphenylacetic acid and NADH. These activities, octopamine hydrolyase and 4-HPAL dehydrogenase (measured as a combined activity, OCAH-4HPALDH), were only induced by growth with phenethanolamines. However, the combined activities were not observed in extracts from cells grown with octopamine as the sole source of carbon and nitrogen, suggesting that an alternate pathway is used under this growth condition. Two independently isolated mutant strains were unable to utilize tyramine as a sole source of carbon or nitrogen. These mutants were also unable to utilize dopamine but grew at wild-type rates on the phenethanolamines. The mutations were mapped at about 70 min on the PAO1 chromosome with the chromosome-mobilizing plasmid R68.45, and both were linked to the catA1, mtu-9002, tyu-9009, and puuE mutations. DNA complementing both of the mutations was cloned on a single BamHI fragment approximately 13.8 kilobase pairs in length. Analysis of a subcloned fragment showed that the two mutations were in different genes. PMID:3034855

  9. Initial catabolism of aromatic biogenic amines by Pseudomonas aeruginosa PAO: pathway description, mapping of mutations, and cloning of essential genes.

    PubMed Central

    Cuskey, S M; Peccoraro, V; Olsen, R H

    1987-01-01

    Pseudomonas aeruginosa PAO1 was able to utilize several aromatic biogenic amines as sole sources of carbon or nitrogen. These included the phenethylamines tyramine and dopamine and the phenethanolamines octopamine, synephrine, and norepinephrine. Initial catabolism of the phenethylamines was mediated by a membrane-bound tyramine dehydrogenase which produced 4-hydroxyphenylacetaldehyde (4HPAL) with tyramine as the substrate. The enzyme was induced by growth with both classes of amines. Initial catabolism of octopamine (except when present as the sole source of carbon and nitrogen) was mediated by a soluble enzyme with activity against the phenethanolamines but not against tyramine or dopamine. The product of the reaction with octopamine as substrate was also 4HPAL. Addition of NAD to reaction mixtures yielded 4-hydroxyphenylacetic acid and NADH. These activities, octopamine hydrolyase and 4-HPAL dehydrogenase (measured as a combined activity, OCAH-4HPALDH), were only induced by growth with phenethanolamines. However, the combined activities were not observed in extracts from cells grown with octopamine as the sole source of carbon and nitrogen, suggesting that an alternate pathway is used under this growth condition. Two independently isolated mutant strains were unable to utilize tyramine as a sole source of carbon or nitrogen. These mutants were also unable to utilize dopamine but grew at wild-type rates on the phenethanolamines. The mutations were mapped at about 70 min on the PAO1 chromosome with the chromosome-mobilizing plasmid R68.45, and both were linked to the catA1, mtu-9002, tyu-9009, and puuE mutations. DNA complementing both of the mutations was cloned on a single BamHI fragment approximately 13.8 kilobase pairs in length. Analysis of a subcloned fragment showed that the two mutations were in different genes. PMID:3034855

  10. Recombinant protein production by transient gene transfer into Mammalian cells.

    PubMed

    Geisse, Sabine; Fux, Cornelia

    2009-01-01

    The timely availability of recombinant proteins in sufficient quantity and of validated quality is of utmost importance in driving drug discovery and the development of low molecular weight compounds, as well as for biotherapeutics. Transient gene expression (TGE) in mammalian cells has emerged as a promising technology for protein generation over the past decade as TGE meets all the prerequisites with respect to quantity and quality of the product as well as cost-effectiveness and speed of the process. Optimized protocols have been developed for both HEK293 and CHO cell lines which allow protein production at any desired scale up to >100 l and in milligram to gram quantities. Along with an overview on current scientific and technological knowledge, detailed protocols for expression of recombinant proteins on small, medium, and large scale are discussed in the following chapter. PMID:19892175

  11. rFTR1 is Required for Pathogenesis, and appears to be an Essential Gene, of Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Rhizopus oryzae is a multinucleated fungus responsible for the majority of cases of mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iron-limited environments. We sought to disrupt the gene to define its role in virulence. METHODS: ...

  12. Contact Toxicity and Repellency of the Essential Oil of Dictamnus dasycarpus Roots from China against Two Stored-Product Insects.

    PubMed

    Yang, Kai; Guo, Shan-Shan; Geng, Zhu-Feng; Du, Shu-Shan; Wang, Cheng-Fang; Deng, Zhi-Wei

    2015-06-01

    During our screening program for new agrochemicals from traditional medicinal herbs, Dictamnus dasycarpus was found to possess contact toxicity and repellent activity against the cigarette beetle Lasioderma serricorne and the booklouse Liposcelis bostrychophila. The essential oil obtained by hydrodistillation of D. dasycarpus roots was characterized by GC-FID and GC/MS analyses, and the main components identified were syn-7-hydroxy-7-anisylnorbornene (1, 49.9%), 1,3,4,5,6,7-hexahydro-2H-inden-2-one (2, 11.6%), 5,6-diethenyl-1-methylcyclohexene (3, 7.4%), and 3,9-dimethyltricyclo[4.2.1.1(2,5)]dec-3-en-9-ol (4, 6.35%). The D. dasycarpus root oil exhibited strong contact toxicity against L. serricorne adults and L. bostrychophila, with LD50 values of 12.4 μg/adult and 27.2 μg/cm(2), respectively. Moreover, the essential oil also showed strong repellency against both stored-product insects tested. PMID:26080743

  13. Vitreoscilla hemoglobin gene ( vgb) improves lutein production in Chlorella vulgaris

    NASA Astrophysics Data System (ADS)

    Ma, Ruijuan; Lin, Xiangzhi

    2014-03-01

    Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene ( vgb) into Chlorella vulgaris by Agrobacterium tumefaciens -mediated transformation (ATMT). PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.

  14. Electron transfer to nitrogenase in Klebsiella pneumoniae. nifF gene cloned and the gene product, a flavodoxin, purified.

    PubMed

    Deistung, J; Cannon, F C; Cannon, M C; Hill, S; Thorneley, R N

    1985-11-01

    The nifF gene of Klebsiella pneumoniae was cloned into a multicopy plasmid in order to construct a strain that synthesizes and retains an elevated concentration of the gene product relative to the wild-type strain. Characterization of the isolated flavodoxin, which serves as an electron donor to nitrogenase, shows unambiguously that it is the product of the nifF gene. PMID:3907625

  15. Gas-inducible product gene expression in bioreactors.

    PubMed

    Weber, Wilfried; Rimann, Markus; de Glutz, François-Nicolas; Weber, Eric; Memmert, Klaus; Fussenegger, Martin

    2005-05-01

    Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals. PMID:15885616

  16. Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female gametophyte and the maternal sporophyte

    PubMed Central

    Johnston, Amal J; Meier, Patrick; Gheyselinck, Jacqueline; Wuest, Samuel EJ; Federer, Michael; Schlagenhauf, Edith; Becker, Jrg D; Grossniklaus, Ueli

    2007-01-01

    Background The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis. Results We identified 1,260 genes as embryo sac expressed by analyzing both our dataset and a recently reported dataset, obtained by a similar approach, using three statistical procedures. Spatial expression of nine genes (for instance a central cell expressed trithorax-like gene, an egg cell expressed gene encoding a kinase, and a synergid expressed gene encoding a permease) validated our approach. We analyzed mutants in five of the newly identified genes that exhibited developmental anomalies during reproductive development. A total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac, at levels that were twofold higher than in the wild type. Conclusion Identification of embryo sac expressed genes establishes a basis for the functional dissection of embryo sac development and function. Sporophytic gain of expression in mutants lacking an embryo sac suggests that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac. PMID:17915010

  17. Antifungal activity and inhibition of fumonisin production by Rosmarinus officinalis L. essential oil in Fusarium verticillioides (Sacc.) Nirenberg.

    PubMed

    da Silva Bomfim, Natalia; Nakassugi, Lydiana Polis; Faggion Pinheiro Oliveira, Jessica; Kohiyama, Cassia Yumie; Mossini, Simone Aparecida Galerani; Grespan, Renata; Nerilo, Samuel Botião; Mallmann, Carlos Augusto; Alves Abreu Filho, Benicio; Machinski, Miguel

    2015-01-01

    The chemical composition of Rosmarinus officinalis L. essential oil (REO) was analysed by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The main compounds of the REO were 1.8 cineole (52.2%), camphor (15.2%) and α-pinene (12.4%). The mycelial growth of Fusarium verticillioides (Sacc.) Nirenberg was reduced significantly by 150 μg/mL of REO. Significant microscopic morphological changes were visualised, such as the rupture of the cell wall and the leakage of cytoplasm at 300 μg/mL of REO. At lower concentrations of REO, the effects on the production of ergosterol and the biomass of mycelium varied, as did the effects on the production of fumonisins, but at ≥300 μg/mL of REO, these processes were significantly inhibited, showing the effectiveness of the REO as an antifungal agent. The results suggested that the REO acts against F. verticillioides by disrupting the cell wall and causing the loss of cellular components, subsequently inhibiting the production of fumonisins and ergosterol. PMID:25053064

  18. Essential and toxic heavy metals in cereals and agricultural products marketed in Kermanshah, Iran, and human health risk assessment.

    PubMed

    Pirsaheb, Meghdad; Fattahi, Nazir; Sharafi, Kiomars; Khamotian, Razieh; Atafar, Zahra

    2016-03-01

    Levels of some essential and toxic heavy metals such as lead, cadmium, chromium, nickel, zinc and copper in cereals and agricultural products obtained from the markets in Kermanshah city, west Iran, were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The average concentrations for lead and cadmium in some cereals were higher than the maximum levels set by the Codex Alimentarius. A potential human health risk assessment was conducted by calculating estimated weekly intake (EWI) of the metals from eating cereals and comparison of these values with provisional tolerable weekly intake (PTWI) values. In combination with recent cereal consumption data, the EWIs of heavy metals were calculated for the Kermanshah population. EWI data for the studied metals through cereal consumption were lower than the PTWI values. Cr, Ni, Zn and Cu levels in all samples analysed were within the ranges reported for similar cereals from various parts of the world. PMID:26465977

  19. Early sporulation gene spo0F: nucleotide sequence and analysis of gene product.

    PubMed Central

    Shimotsu, H; Kawamura, F; Kobayashi, Y; Saito, H

    1983-01-01

    We have determined the sequence of a 1,162-base-pair DNA fragment containing a spo0F gene which is required for an early stage of sporulation in Bacillus subtilis. The sequence has only one long open reading frame consisting of 173 codons, which has been confirmed to be the spo0F cistron by DNA-mediated transformation and in vitro transcription. In UV-irradiated "maxicells" containing pUBSF13, the plasmid that carries cloned spo0F gene, we have observed the synthesis of a 20-kilodalton polypeptide that is absent from cells carrying a vector plasmid pUB110. The molecular weight of this protein is in agreement with the calculated molecular weight of the spo0F gene product (Mr, 19,065). The putative promoter sequences of spo0F gene were 5' T-A-T-A-A-T 3' at -10 and 5' T-T-G-A-T-T 3' at -35. An octamer sequence, 5' A-A-A-G-G-A-G-G 3', situated 8 base pairs prior to the initiation codon was found to be perfectly complementary with the 3' end of 16S ribosomal RNA. This result offers additional evidence for the proposal by Rabinowitz's group that an extensive mRNA-rRNA interaction is a requirement for efficient translation by B. subtilis ribosomes. Images PMID:6402773

  20. Functional annotation of human cytomegalovirus gene products: an update

    PubMed Central

    Van Damme, Ellen; Van Loock, Marnix

    2014-01-01

    Human cytomegalovirus is an opportunistic double-stranded DNA virus with one of the largest viral genomes known. The 235 kB genome is divided in a unique long (UL) and a unique short (US) region which are flanked by terminal and internal repeats. The expression of HCMV genes is highly complex and involves the production of protein coding transcripts, polyadenylated long non-coding RNAs, polyadenylated anti-sense transcripts and a variety of non-polyadenylated RNAs such as microRNAs. Although the function of many of these transcripts is unknown, they are suggested to play a direct or regulatory role in the delicately orchestrated processes that ensure HCMV replication and life-long persistence. This review focuses on annotating the complete viral genome based on three sources of information. First, previous reviews were used as a template for the functional keywords to ensure continuity; second, the Uniprot database was used to further enrich the functional database; and finally, the literature was manually curated for novel functions of HCMV gene products. Novel discoveries were discussed in light of the viral life cycle. This functional annotation highlights still poorly understood regions of the genome but more importantly it can give insight in functional clusters and/or may be helpful in the analysis of future transcriptomics and proteomics studies. PMID:24904534

  1. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells’ Transcription Factors

    PubMed Central

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

  2. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

    PubMed

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

  3. Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development

    PubMed Central

    José-Edwards, Diana S.; Oda-Ishii, Izumi; Nibu, Yutaka; Di Gregorio, Anna

    2013-01-01

    T-box genes are potent regulators of mesoderm development in many metazoans. In chordate embryos, the T-box transcription factor Brachyury (Bra) is required for specification and differentiation of the notochord. In some chordates, including the ascidian Ciona, members of the Tbx2 subfamily of T-box genes are also expressed in this tissue; however, their regulatory relationships with Bra and their contributions to the development of the notochord remain uncharacterized. We determined that the notochord expression of Ciona Tbx2/3 (Ci-Tbx2/3) requires Ci-Bra, and identified a Ci-Tbx2/3 notochord CRM that necessitates multiple Ci-Bra binding sites for its activity. Expression of mutant forms of Ci-Tbx2/3 in the developing notochord revealed a role for this transcription factor primarily in convergent extension. Through microarray screens, we uncovered numerous Ci-Tbx2/3 targets, some of which overlap with known Ci-Bra-downstream notochord genes. Among the Ci-Tbx2/3 notochord targets are evolutionarily conserved genes, including caspases, lineage-specific genes, such as Noto4, and newly identified genes, such as MLKL. This work sheds light on a large section of the notochord regulatory circuitry controlled by T-box factors, and reveals new components of the complement of genes required for the proper formation of this structure. PMID:23674602

  4. The Podospora rmp1 gene implicated in nucleus-mitochondria cross-talk encodes an essential protein whose subcellular location is developmentally regulated.

    PubMed Central

    Contamine, Véronique; Zickler, Denise; Picard, Marguerite

    2004-01-01

    It has been previously reported that, at the time of death, the Podospora anserina AS1-4 mutant strains accumulate specific deleted forms of the mitochondrial genome and that their life spans depend on two natural alleles (variants) of the rmp1 gene: AS1-4 rmp1-2 strains exhibit life spans strikingly longer than those of AS1-4 rmp1-1. Here, we show that rmp1 is an essential gene. In silico analyses of eight rmp1 natural alleles present in Podospora isolates and of the putative homologs of this orphan gene in other filamentous fungi suggest that rmp1 evolves rapidly. The RMP1 protein is localized in the mitochondrial and/or the cytosolic compartment, depending on cell type and developmental stage. Strains producing RMP1 without its mitochondrial targeting peptide are viable but exhibit vegetative and sexual defects. PMID:15020413

  5. The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7.

    PubMed

    Mith, Hasika; Clinquart, Antoine; Zhiri, Abdesselam; Daube, Georges; Delcenserie, Véronique

    2015-01-01

    The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics. PMID:25790499

  6. Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

    PubMed Central

    2012-01-01

    Background Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). Results Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. Conclusions Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies. PMID:22857656

  7. Repellency of an over-the-counter essential oil product in China against workers of red imported fire ants.

    PubMed

    Chen, Jian

    2009-01-28

    Repellency of an over-the-counter essential oil product from China, and its major components against workers of red imported fire ants, Solenopsis invicta Buren, was evaluated using an ant digging bioassay. Three concentrations (1.0, 10.0, and 100.0 mg/kg in sand) of the product were tested. At 100.0 mg/kg, the digging suppress index (DSI) was 1.0 +/- 0.00 (mean +/- SE) for all six test colonies, indicating this product produced a complete digging suppression; at 10 mg/kg, DSI was 0.22 +/- 0.089 to 0.75 +/- 0.12 and significant repellency occurred against five of six colonies; and at 1.0 mg/kg, DSI was 0.21 +/- 0.091 to 0.38 +/- 0.14 and significant repellency occurred against four of six colonies. The chemical components of this product were analyzed using gas chromatography-mass spectrometry (GC-MS). Camphor, eucalyptol, eugenol, menthol, methyl salicylate, and phenylethanol were identified. A digging bioassay was also conducted on each of those identified compounds at concentrations of 1.0, 10.0, and 100.0 mg/kg. Based on pooled data from three colonies, each component significantly suppressed the digging behavior at 100 mg/kg. Eugenol, menthol, and methyl salicylate significantly suppressed the digging at 10 mg/kg. At 1.0 and 10.0 mg/kg, DSI for eucalyptol was -0.039 +/- 0.032 and -0.050 +/- 0.021, respectively. The negative DSI indicated a digging facilitation. However, only at 10.0 mg/kg, was such facilitation statistically significant. PMID:19123820

  8. Cotranscription of genes encoding indoleacetic acid production in Pseudomonas syringae subsp. savastanoi.

    PubMed

    Palm, C J; Gaffney, T; Kosuge, T

    1989-02-01

    Indoleacetic acid (IAA) production by the plant pathogen Pseudomonas syringae subsp. savastanoi is essential for tumor formation on olive and oleander. The bacterium produces IAA from tryptophan in reactions catalyzed by tryptophan monooxygenase and indoleacetamide hydrolase. The genetic determinants are, respectively, iaaM and iaaH. In oleander isolates, the genes encoding the IAA biosynthetic enzymes are located on a plasmid; in olive isolates, the genes occur on the chromosome. The IAA genes from the oleander isolate strain EW2009 are located within a 4-kilobase (kb) segment of the 52-kb plasmid pIAA1. Escherichia coli strains harboring a recombinant plasmid, pCJP3, which contains this 4-kb fragment, excreted IAA into culture media, and crude cell extracts had both tryptophan monooxygenase and indoleacetamide hydrolase activity. In vitro coupled transcription-translation of pCJP3 demonstrated that this fragment coded for proteins of 62 and 47 kilodaltons which correspond to tryptophan monooxygenase and indoleacetamide hydrolase, respectively. Expression of these genes was dependent upon a vector promoter in pCJP3. However, in the absence of a vector promoter, E. coli containing recombinant plasmids with additional pIAA1 DNA in front of iaaM had high levels of tryptophan monooxygenase. Northern (RNA) hybridization experiments verified that iaaM and iaaH are cotranscribed as a portion of a ca. 4- to 5-kb transcript in vivo. Southern hybridization experiments with IAA plasmids from different oleander strains of P. syringae subsp. savastanoi revealed that all IAA plasmids contained a region of at least 10 kb of homology, with the IAA genes at one end. Repetitive DNA and a copy of IS51 were found at the end of this region of homology. PMID:2644217

  9. Essential Tremor

    MedlinePLUS

    MENU Return to Web version Essential Tremor Overview What is essential tremor? Essential tremor, sometimes called benign (non-cancerous) or familial tremor, is an uncontrollable shaking, most often in ...

  10. The Polycomb-group gene Rae28 sustains Nkx2.5/Csx expression and is essential for cardiac morphogenesis

    PubMed Central

    Shirai, Manabu; Osugi, Tomoaki; Koga, Hideyuki; Kaji, Yoshikazu; Takimoto, Eiki; Komuro, Issei; Hara, Junichi; Miwa, Takeshi; Yamauchi-Takihara, Keiko; Takihara, Yoshihiro

    2002-01-01

    The Polycomb-group (PcG) gene Rae28 is a mammalian homologue of the Drosophila gene polyhomeotic. PcG genes are known to maintain transcription states, once initiated, probably by regulating chromatin structure. Since homozygous Rae28-deficient (Rae28–/–) mice displayed cardiac anomalies similar to congenital heart diseases in humans, we examined the role of Rae28 in cardiac morphogenesis at the molecular level. In Rae28–/– embryos, expression of the cardiac selector gene Nkx2.5/Csx (Nkx2.5) was initiated properly but was not sufficiently sustained later in development. This impaired expression of Nkx2.5 in the maintenance phase proved to have a crucial effect on cardiac morphogenesis, as demonstrated by the results of a genetic complementation experiment in which the cardiac anomalies were suppressed by overexpression of human NKX2.5/CSX1 in Rae28–/– embryos. Ubiquitous expression of exogenous Rae28 likewise restored the impaired Nkx2.5 expression in Rae28–/– embryos, further supporting the notion that Rae28 sustains Nkx2.5 expression in cardiomyocytes. Thus, our data show that a mammalian PcG gene can play a key role in organogenesis by helping to maintain the expression of a selector gene. PMID:12122109

  11. Functional circadian clock genes are essential for the overwintering diapause of the Northern house mosquito, Culex pipiens

    PubMed Central

    Meuti, Megan E.; Stone, Mary; Ikeno, Tomoko; Denlinger, David L.

    2015-01-01

    The short day lengths of late summer are used to program the overwintering adult diapause (dormancy) of the Northern house mosquito, Culex pipiens. Here, we investigated the role of clock genes in initiating this diapause and asked whether the circadian cycling of clock gene expression persists during diapause. We provide evidence that the major circadian clock genes continue to cycle throughout diapause and after diapause has been terminated. RNA interference (RNAi) was used to knock down the core circadian clock genes and to then assess the impact of the various clock genes on the ability of females to enter diapause. RNAi directed against negative circadian regulators (period, timeless and cryptochrome2) caused females that were reared under diapause-inducing, short day conditions to avert diapause. In contrast, knocking down the circadian-associated gene pigment dispersing factor caused females that were reared under diapause-averting, long day conditions to enter a diapause-like state. Our results implicate the circadian clock in the initiation of diapause in C. pipiens. PMID:25653422

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

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

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

  13. Associations between human aldosterone synthase CYP11B2 (-344T/C) gene polymorphism and antihypertensive response to valsartan in Chinese patients with essential hypertension

    PubMed Central

    Ji, Xu; Qi, Hua; Li, Dong-Bao; Liu, Rong-Kun; Zheng, Yang; Chen, Hai-Ling; Guo, Jin-Cheng

    2015-01-01

    Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is associated with essential hypertension. But if the genetic variations in aldosterone synthesis could influence the antihypertensive response to Valsartan is not clear. A Chinese sample of 502 persons (217 women) was studied, which was divided into the hypertensive group (EH) of 345 persons and the normotensive group (NB) of 157 persons. Subjects were genotyped through the use of the polymerase chain reaction for the diallelic polymorphisms in CYP11B2. 98 persons of the essential hypertension group received 4 weeks therapy with valsartan. Blood pressure, 24-hour ambulatory blood pressure, biochemical index were also determined. The frequency of CC+CT genotypes in hypertensive group was significantly higher than that in normotensive group (P<0.05), the frequency of C allele of gene CYP11B2 (-344T/C) in hypertensive group was significantly higher than that in normotensive group (P<0.01). The descending values of SBP (systolic blood pressure), DBP (diastolic blood pressure), MAP (mean arterial pressure), 24 h SBP (mean SBP of 24 hours), 24 h DBP (mean DBP of 24 hours), 24 h MAP (mean arterial pressure of 24 hours) of CC+CT genotype group were significantly higher than those of the TT genotype group (P<0.05). The aldosterone synthase CYP11B2 (-344T/C) gene polymorphism is associated with essential hypertension in Chinese. And the aldosterone synthase CYP11B2 (-344T/C) gene polymorphism may be the predictor of the antihypertensive response to Valsartan. PMID:25785110

  14. Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

    SciTech Connect

    Kondou, Youhei; Kitazawa, Daisuke; Takeda, Shigeki; Yamashita, Eiki; Mizuguchi, Mineyuki; Kawano, Keiichi; Tsukihara, Tomitake

    2005-01-01

    Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

  15. Inhibitory effects of geranium essential oil and its major component, citronellol, on degranulation and cytokine production by mast cells.

    PubMed

    Kobayashi, Yuko; Sato, Harumi; Yorita, Mika; Nakayama, Hiroto; Miyazato, Hironari; Sugimoto, Keiichiro; Jippo, Tomoko

    2016-06-01

    We investigated the effects of geranium essential oil (GEO) on anaphylaxis. GEO can exert antioxidant and anti-inflammatory effects, but its roles in allergic reactions are incompletely understood. Here, we used mouse cells to show that GEO inhibited the degranulation of cultured mast cells (CMCs). Citronellol is the major component of GEO and inhibited CMC degranulation. The l-enantiomer of citronellol more effectively suppressed CMC degranulation than did d-citronellol. We also examined whether citronellol could inhibit the immunoglobulin (Ig) E-induced production of tumor necrosis factor (TNF)-α. Treatment with various concentrations of citronellol before CMC activation with IgE significantly inhibited the induction of TNF-α in a dose-dependent manner. Mechanistically, citronellol suppressed the phosphorylation of mitogen-activated protein kinase (ERK), which is critical for ERK activation and the production of inflammatory cytokines in mast cells. These findings suggest that citronellol may represent a candidate compound for the effective treatment of allergic diseases. PMID:26927807

  16. Extraction and refining of essential oil from Australian tea tree, Melaleuca alterfornia, and the antimicrobial activity in cosmetic products

    NASA Astrophysics Data System (ADS)

    Huynh, Q.; Phan, T. D.; Thieu, V. Q. Q.; Tran, S. T.; Do, S. H.

    2012-03-01

    Tea tree oil (TTO) comes from the leaves of Melaleuca alternifornia that belongs to the myrtle family (Myrtaceae). It is one of the most powerful immune system stimulants and sorts out most viral, bacterial and fungal infections in a snap, while it is great to heal wounds and acnes. In Vietnam, Melaleuca trees can grow on acid land that stretches in a large portion of lands in the Mekong Delta region. So, there are some Melaleuca plantations developed under the Vietnamese government plans of increasing plantation forests now. However, TTO contains various amounts of 1,8-cineole that causes skin irritant. So TTO purification is very necessary. In this study, the purification of TTO that meet International Standard ISO 4730 was carried out via two steps. The first step is steam distillation to obtain crude TTO (terpinen-4-ol 35% v/v) and the average productivity is among 2.37% (v/wet-wt) or 1.23% (v/dry-wt). In the second step, the cleaned TTO is collected by vacuum distillation column and extraction yield of the whole process is about 0.3% (w/w). Besides, high concentration essential oil was applied in the cosmetic products to increase its commercial value.

  17. Vertical stratification of matrix production is essential for physical integrity and architecture of macrocolony biofilms of Escherichia coli.

    PubMed

    Serra, Diego O; Klauck, Gisela; Hengge, Regine

    2015-12-01

    Bacterial macrocolony biofilms grow into intricate three-dimensional structures that depend on self-produced extracellular polymers conferring protection, cohesion and elasticity to the biofilm. In Escherichia coli, synthesis of this matrix - consisting of amyloid curli fibres and cellulose - requires CsgD, a transcription factor regulated by the stationary phase sigma factor RpoS, and occurs in the nutrient-deprived cells of the upper layer of macrocolonies. Is this asymmetric matrix distribution functionally important or is it just a fortuitous by-product of an unavoidable nutrient gradient? In order to address this question, the RpoS-dependent csgD promoter was replaced by a vegetative promoter. This re-wiring of csgD led to CsgD and matrix production in both strata of macrocolonies, with the lower layer transforming into a rigid 'base plate' of growing yet curli-connected cells. As a result, the two strata broke apart followed by desiccation and exfoliation of the top layer. By contrast, matrix-free cells at the bottom of wild-type macrocolonies maintain colony contact with the humid agar support by flexibly filling the space that opens up under buckling areas of the macrocolony. Precisely regulated stratification in matrix-free and matrix-producing cell layers is thus essential for the physical integrity and architecture of E. coli macrocolony biofilms. PMID:26234179

  18. Transition to farming more likely for small, conservative groups with property rights, but increased productivity is not essential

    PubMed Central

    Gallagher, Elizabeth M.; Shennan, Stephen J.; Thomas, Mark G.

    2015-01-01

    Theories for the origins of agriculture are still debated, with a range of different explanations offered. Computational models can be used to test these theories and explore new hypotheses; Bowles and Choi [Bowles S, Choi J-K (2013) Proc Natl Acad Sci USA 110(22):8830–8835] have developed one such model. Their model shows the coevolution of farming and farming-friendly property rights, and by including climate variability, replicates the timings for the emergence of these events seen in the archaeological record. Because the processes modeled occurred a long time ago, it can be difficult to justify exact parameter values; hence, we propose a fitting to idealized outcomes (FIO) method to explore the model’s parameter space in more detail. We have replicated the model of Bowles and Choi, and used the FIO method to identify complexities and interactions of the model previously unidentified. Our results indicate that the key parameters for the emergence of farming are group structuring, group size, conservatism, and farming-friendly property rights (lending further support to Bowles and Choi’s original proposal). We also find that although advantageous, it is not essential that farming productivity be greater than foraging productivity for farming to emerge. In addition, we highlight how model behaviors can be missed when gauging parameter sensitivity via a fix-all-but-one variation approach. PMID:26578766

  19. Transition to farming more likely for small, conservative groups with property rights, but increased productivity is not essential.

    PubMed

    Gallagher, Elizabeth M; Shennan, Stephen J; Thomas, Mark G

    2015-11-17

    Theories for the origins of agriculture are still debated, with a range of different explanations offered. Computational models can be used to test these theories and explore new hypotheses; Bowles and Choi [Bowles S, Choi J-K (2013) Proc Natl Acad Sci USA 110(22):8830-8835] have developed one such model. Their model shows the coevolution of farming and farming-friendly property rights, and by including climate variability, replicates the timings for the emergence of these events seen in the archaeological record. Because the processes modeled occurred a long time ago, it can be difficult to justify exact parameter values; hence, we propose a fitting to idealized outcomes (FIO) method to explore the model's parameter space in more detail. We have replicated the model of Bowles and Choi, and used the FIO method to identify complexities and interactions of the model previously unidentified. Our results indicate that the key parameters for the emergence of farming are group structuring, group size, conservatism, and farming-friendly property rights (lending further support to Bowles and Choi's original proposal). We also find that although advantageous, it is not essential that farming productivity be greater than foraging productivity for farming to emerge. In addition, we highlight how model behaviors can be missed when gauging parameter sensitivity via a fix-all-but-one variation approach. PMID:26578766

  20. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2014-08-05

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  1. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    SciTech Connect

    Wohlbach, Dana J.; Gasch, Audrey P.

    2015-09-29

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  2. P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Mutations Affecting Embryonic Pns Development

    PubMed Central

    Salzberg, A.; Prokopenko, S. N.; He, Y.; Tsai, P.; Pal, M.; Maroy, P.; Glover, D. M.; Deak, P.; Bellen, H. J.

    1997-01-01

    To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P-element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens. PMID:9409832

  3. Fumigant Toxicity of Essential Oils from Basil and Spearmint Against Two Major Pyralid Pests of Stored Products.

    PubMed

    Eliopoulos, P A; Hassiotis, C N; Andreadis, S S; Porichi, A-E E

    2015-04-01

    The fumigant activity of essential oil vapors distilled from sweet basil Ocimum basilicum L. and spearmint Mentha spicata L. (Lamiaceae) were tested against two major stored products pests Ephestia kuehniella (Zeller) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). Various oil doses (0.5, 2.5, 5, 50, 250, 500, 1,000, and 1,500 µl/liter air), for an exposure period of 24 h, were tested. The essential oils were subjected to gas chromatography-mass spectrometry analysis and revealed that the major compounds were for spearmint oil carvone (67.1%) and limonene (+1,8 cineole; 14.3%) and for basil oil linalool (45.9%), 1,8 cineole (16.7%) and eugenol (10.3%). Apart from a few exceptions, no significant differences in insecticidal action were observed between basil and spearmint oil. Both oils were highly effective against adult moths, given that notable mortality (>80%) was recorded after exposure to low doses such as 2.5 µl/liter. Noteworthy, egg mortality was also recorded, reaching 73-79% for basil and 56-60% for spearmint. Toxicity data indicated that larvae and pupae were the most tolerant stages in all cases. Larval mortality never exceeded 21 and 18%, for basil and spearmint, respectively, irrespective of moth species. Basil and spearmint oils displayed mortalities as high as 38 and 28% in pupae. Lethal doses (LD50 and LD99) values were estimated via probit analysis. Developmental stage proved to be a significant factor, whereas the effect of oil species on insect mortality was insignificant. With the exception of adult individuals, basil and spearmint oils did not show satisfactory overall insecticidal activity against E. kuehniella and P. interpunctella. PMID:26470193

  4. The effect of alpha-interferon on bone marrow megakaryocytes and platelet production rate in essential thrombocythemia

    SciTech Connect

    Wadenvik, H.; Kutti, J.; Ridell, B.; Revesz, P.; Jacobsson, S.; Magnusson, B.; Westin, J.; Vilen, L. )

    1991-05-15

    In 10 patients with previously untreated essential thrombocythemia (ET), by using {sup 111}In-labeled platelets and megakaryocyte morphometry, the relation between platelet production rate and bone marrow megakaryocytes was evaluated before and during alpha-2b-interferon (IFN) therapy. A highly significant decrease in platelet count occurred during IFN therapy; the platelet counts, at baseline and after 2 and 6 months of IFN therapy, were 1,102 +/- 345 x 10(9)/L, 524 +/- 169 x 10(9)/L (P less than .0001), and 476 +/- 139 x 10(9)/L (P less than .0001), respectively. The decrement in platelet count was mainly a result of diminished platelet production rate, which at baseline and after 2 and 6 months of IFN therapy was 89 +/- 30 x 10(10) platelets/d, 53 +/- 18 x 10(10) platelets/d (P = .0033), and 45 +/- 20 x 10(10) platelets/d (P less than .0001), respectively. Also, a slight shortening of platelet mean life-span (MLS) was observed in response to IFN treatment; platelet MLS was 7.96 +/- 0.69 days at baseline and 6.68 +/- 1.30 days (P = .012) after 6 months of IFN therapy. IFN induced a significant decrease in bone marrow megakaryocyte volume; both megakaryocyte nuclear and cytoplasmatic volumes were affected. The mean megakaryocyte volume was 372 +/- 126 x 10(2) pL/microL at baseline and 278 +/- 147 x 10(2) pL/microL (P = .049) after 6 months of IFN therapy. However, the number of megakaryocytes did not show any significant change in response to IFN. It is concluded that alpha-IFN reduces platelet production rate and the peripheral platelet count in ET mainly through an anti-proliferative action on the megakaryocytes and to a considerably lesser degree by a shortening of platelet MLS.

  5. HIF-1α Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in Pseudomonas aeruginosa Keratitis

    PubMed Central

    Berger, Elizabeth A.; McClellan, Sharon A.; Vistisen, Kerry S.; Hazlett, Linda D.

    2013-01-01

    Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production. PMID:23874197

  6. Identification and characterization of duck plague virus glycoprotein C gene and gene product

    PubMed Central

    2010-01-01

    Background Viral envelope proteins have been proposed to play significant roles in the process of viral infection. Results In this study, an envelope protein gene, gC (NCBI GenBank accession no. EU076811), was expressed and characterized from duck plague virus (DPV), a member of the family herpesviridae. The gene encodes a protein of 432 amino acids with a predicted molecular mass of 45 kDa. Sequence comparisons, multiple alignments and phylogenetic analysis showed that DPV gC has several features common to other identified herpesvirus gC, and was genetically close to the gallid herpervirus. Antibodies raised in rabbits against the pET32a-gC recombinant protein expressed in Escherichia coli BL21 (DE3) recognized a 45-KDa DPV-specific protein from infected duck embryo fibroblast (DEF) cells. Transcriptional and expression analysis, using real-time fluorescent quantitative PCR (FQ-PCR) and Western blot detection, revealed that the transcripts encoding DPV gC and the protein itself appeared late during infection of DEF cells. Immunofluorescence localization further demonstrated that the gC protein exhibited substantial cytoplasm fluorescence in DPV-infected DEF cells. Conclusions In this work, the DPV gC protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gC product for the first time. These properties of the gC protein provided a prerequisite for further functional analysis of this gene. PMID:21110887

  7. Identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis B virus gene X.

    PubMed Central

    Twu, J S; Rosen, C A; Haseltine, W A; Robinson, W S

    1989-01-01

    Hepatitis B virus (HBV) X-gene product activates transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). To identify a cis-acting regulatory sequence within the HIV-1 LTR which is responsive to the HBV X-gene trans-activating function, we examined the effects of HBV X-gene expression in cells with a series of LTR/CAT deletion mutants. A region of the HIV-1 LTR containing the previously identified kappa B-like enhancer element was found to be responsive to HBV X-gene activation, and this effect was independent of, and additive with, the effect of the HIV-1 tat-III protein on CAT expression. Since kappa B-like enhancer sequences are known to regulate transcription of a variety of viruses and cellular genes, our results suggest that the X gene could activate such a gene during HBV infection and replication. Images PMID:2724417

  8. Melav2, an elav-like gene, is essential for spermatid differentiation in the flatworm Macrostomum lignano

    PubMed Central

    2009-01-01

    Background Failure of sperm differentiation is one of the major causes of male sterility. During spermiogenesis, spermatids undergo a complex metamorphosis, including chromatin condensation and cell elongation. Although the resulting sperm morphology and property can vary depending on the species, these processes are fundamental in many organisms. Studying genes involved in such processes can thus provide important information for a better understanding of spermatogenesis, which might be universally applied to many other organisms. Results In a screen for genes that have gonad-specific expression we isolated an elav-like gene, melav2, from Macrostomum lignano, containing the three RNA recognition motifs characteristic of elav-like genes. We found that melav2 mRNA was expressed exclusively in the testis, as opposed to the known elav genes, which are expressed in the nervous system. The RNAi phenotype of melav2 was characterized by an aberrant spermatid morphology, where sperm elongation often failed, and an empty seminal vesicle. Melav2 RNAi treated worms were thus male-sterile. Further analysis revealed that in melav2 RNAi treated worms precocious chromatin condensation occurred during spermatid differentiation, resulting in an abnormally tightly condensed chromatin and large vacuoles in round spermatids. In addition, immunostaining using an early-spermatid specific antibody revealed that melav2 RNAi treated worms had a larger amount of signal positive cells, suggesting that many cells failed the transition from early spermatid stage. Conclusion We characterize a new function for elav-like genes, showing that melav2 plays a crucial role during spermatid differentiation, especially in the regulation of chromatin condensation and/or cell elongation. PMID:19995429

  9. Significance of Calcium Binding, Tyrosine Phosphorylation, and Lysine Trimethylation for the Essential Function of Calmodulin in Vertebrate Cells Analyzed in a Novel Gene Replacement System*

    PubMed Central

    Panina, Svetlana; Stephan, Alexander; la Cour, Jonas M.; Jacobsen, Kivin; Kallerup, Line K.; Bumbuleviciute, Rasita; Knudsen, Kristoffer V. K.; Snchez-Gonzlez, Pablo; Villalobo, Antonio; Olesen, Uffe H.; Berchtold, Martin W.

    2012-01-01

    Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr99/Tyr138 or trimethylated at Lys115 survived and grew well. CaM mutated at both Ca2+ binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM. PMID:22493455

  10. Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance in Arabidopsis[W

    PubMed Central

    Attaran, Elham; Zeier, Tatiana E.; Griebel, Thomas; Zeier, Jürgen

    2009-01-01

    Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway. PMID:19329558

  11. A novel cis-acting element is essential for cytokine-mediated transcriptional induction of the serum amyloid A gene in nonhepatic cells.

    PubMed Central

    Ray, A; Ray, B K

    1996-01-01

    Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions -280 and 224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells. PMID:8657133

  12. MADS Domain Transcription Factors Mediate Short-Range DNA Looping That Is Essential for Target Gene Expression in Arabidopsis[W

    PubMed Central

    Mendes, Marta Adelina; Guerra, Rosalinda Fiorella; Berns, Markus Christian; Manzo, Carlo; Masiero, Simona; Finzi, Laura; Kater, Martin M.; Colombo, Lucia

    2013-01-01

    MADS domain transcription factors are key regulators of eukaryotic development. In plants, the homeotic MIKC MADS factors that regulate floral organ identity have been studied in great detail. Based on genetic and protein–protein interaction studies, a floral quartet model was proposed that describes how these MADS domain proteins assemble into higher order complexes to regulate their target genes. However, despite the attractiveness of this model and its general acceptance in the literature, solid in vivo proof has never been provided. To gain deeper insight into the mechanisms of transcriptional regulation by MADS domain factors, we studied how SEEDSTICK (STK) and SEPALLATA3 (SEP3) directly regulate the expression of the reproductive meristem gene family transcription factor–encoding gene VERDANDI (VDD). Our data show that STK-SEP3 dimers can induce loop formation in the VDD promoter by binding to two nearby CC(A/T)6GG (CArG) boxes and that this is essential for promoter activity. Our in vivo data show that the size and position of this loop, determined by the choice of CArG element usage, is essential for correct expression. Our studies provide solid in vivo evidence for the floral quartet model. PMID:23847151

  13. Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

    PubMed Central

    Romanczuk, H; Wormington, W M

    1989-01-01

    Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication. Images PMID:2540419

  14. Association of Matrix Metalloproteinase-9 Gene −1562C/T Polymorphism with Essential Hypertension: A Systematic Review and Meta-Analysis Article

    PubMed Central

    YANG, Wenchao; LU, Jiaojiao; YANG, Liu; ZHANG, Jinjin

    2015-01-01

    Background: Matrix metalloproteinase-9 (MMP-9) gene −1562C/T polymorphism could regulate its expression level and thus affected people’s predisposition to essential hypertension. However, related studies yielded inconsistent or contradictory results. Methods: To evaluate the association of MMP-9 −1562C/T polymorphism with essential hypertension, we performed a meta-analysis by combining all available independent case-control studies (n=6).A systematic literature search of PubMed, Web of Science, Scopus and CNKI (Chinese National Knowledge Infrastructure) databases was conducted by two researchers independently for all relevant articles published before March 2015. Results: MMP-9 −1562C/T polymorphism was associated with essential hypertension under the allelic model (T vs.C, OR=1.36, 95% CI=1.17–1.59, P<0.0001). Subsequent sensitivity analysis confirmed the stability of the results. Such association was also observed in the dominant (TT+CT vs. CC, OR=1.30, 95% CI=1.10–1.54, P=0.002) and co-dominant (CT vs. TT+CC, OR=1.27, 95% CI=1.05–1.53, P=0.01) models but not in the recessive model (TT vs. CT+CC, OR=1.30, 95% CI=0.50–3.36, P=0.59). Conclusion: MMP-9 −1562C/T polymorphism was associated with the risk of essential hypertension. PMID:26744701

  15. Production and clinical development of nanoparticles for gene delivery

    PubMed Central

    Chen, Jie; Guo, Zhaopei; Tian, Huayu; Chen, Xuesi

    2016-01-01

    Gene therapy is a promising strategy for specific treatment of numerous gene-associated human diseases by intentionally altering the gene expression in pathological cells. A successful clinical application of gene-based therapy depends on an efficient gene delivery system. Many efforts have been attempted to improve the safety and efficiency of gene-based therapies. Nanoparticles have been proved to be the most promising vehicles for clinical gene therapy due to their tunable size, shape, surface, and biological behaviors. In this review, the clinical development of nanoparticles for gene delivery will be particularly highlighted. Several promising candidates, which are closest to clinical applications, will be briefly reviewed. Then, the recent developments of nanoparticles for clinical gene therapy will be identified and summarized. Finally, the development of nanoparticles for clinical gene delivery in future will be prospected. PMID:27088105

  16. G9a is essential for epigenetic silencing of K(+) channel genes in acute-to-chronic pain transition.

    PubMed

    Laumet, Geoffroy; Garriga, Judit; Chen, Shao-Rui; Zhang, Yuhao; Li, De-Pei; Smith, Trevor M; Dong, Yingchun; Jelinek, Jaroslav; Cesaroni, Matteo; Issa, Jean-Pierre; Pan, Hui-Lin

    2015-12-01

    Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K(+) channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. We found that nerve injury increased dimethylation of Lys9 on histone H3 (H3K9me2) at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect levels of DNA methylation on these genes in DRGs. Nerve injury increased activity of euchromatic histone-lysine N-methyltransferase-2 (G9a), histone deacetylases and enhancer of zeste homolog-2 (EZH2), but only G9a inhibition consistently restored K(+) channel expression. Selective knockout of the gene encoding G9a in DRG neurons completely blocked K(+) channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K(+) channels but also normalized 638 genes down- or upregulated by nerve injury. Thus G9a has a dominant function in transcriptional repression of K(+) channels and in acute-to-chronic pain transition after nerve injury. PMID:26551542

  17. Genetic variation in glutathione S-transferase genes and risk of nonfatal cerebral stroke in patients suffering from essential hypertension.

    PubMed

    Polonikov, Alexey; Vialykh, Ekaterina; Vasil'eva, Oksana; Bulgakova, Irina; Bushueva, Olga; Illig, Thomas; Solodilova, Maria

    2012-07-01

    Oxidative stress resulting from an increased amount of reactive oxygen species and an imbalance between oxidants and antioxidants has been implicated in pathogenesis of cerebral stroke. The purpose of this study was to investigate the relationship between common polymorphisms of glutathione S-transferase M1, T1, and P1 genes and risk of stroke in hypertensive individuals. A total of 667 unrelated Russian individuals with hypertension, including 306 hypertensives who suffered from cerebral stroke and 361 hypertensives who did not have cerebrovascular accidents, were recruited for the study. The deletion polymorphisms of GSTM1 and GSTT1 genes and polymorphism Ile105Val of the GSTP1 gene were genotyped by a multiplex polymerase chain reaction and restriction analyses, respectively. No differences in GSTM1 and GSTP1 genotype distributions between the cases and controls have been observed. The null GSTT1 genotype was found to be associated with increased risk of cerebral stroke after Bonferroni correction and adjusting for confounding variables such as gender, blood pressure, body mass index, and antihypertensive medication use (odds ratio 1.51 95 % CI 1.09-2.07, P = 0.01). The present study was the first to show the association of null genotype of the GSTT1 gene with increased risk of cerebral stroke. PMID:22528457

  18. Two linked genes encoding a secreted effector and a membrane protein are essential for Ustilago maydis-induced tumour formation.

    PubMed

    Doehlemann, Gunther; Reissmann, Stefanie; Assmann, Daniela; Fleckenstein, Martin; Kahmann, Regine

    2011-08-01

    Ustilago maydis is a biotrophic fungal pathogen that colonizes living tissue of its host plant maize. Based on transcriptional upregulation during biotrophic development we identified the pit (proteins important for tumours) cluster, a novel gene cluster comprising four genes of which two are predicted to encode secreted effectors. Disruption of the gene cluster abolishes U. maydis-induced tumour formation and this phenotype can be caused by deleting either pit1 encoding a transmembrane protein or pit2 encoding a secreted protein. Pit1 localizes to the fungal plasma membrane at hyphal tips, endosomes and vacuoles while Pit2 is secreted to the biotrophic interface. Both Δpit1 and Δpit2 mutants are able to penetrate maize epidermis and grow intracellularly at sites of infection but fail to spread in the infected leaf. Microarray analysis shows an indistinguishable response of the plant to infection by Δpit1 and Δpit2 mutant strains. Transcriptional activation of maize defence genes in infections with Δpit1/2 mutant strains indicates that the mutants have a defect in suppressing plant immune responses. Our results suggest that the activity of Pit1 and Pit2 during tumour formation might be functionally linked and we discuss possibilities for a putative functional connection of the two proteins. PMID:21692877

  19. Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP.

    PubMed

    Wu, K; Malik, K; Tian, L; Hu, M; Martin, T; Foster, E; Brown, D; Miki, B

    2001-07-01

    Cryptic gene regulatory elements are sequences that are inactive at their native locations in the genome but have the ability to become functional when positioned adjacent to genes. We have recently isolated such a cryptic sequence from tobacco, tCUP, that can act as a promoter. A 135-bp fragment spanning extending from position -197 to -62, relative to the transcription start site, was found to promote GUS expression in all of the major organs of transgenic Arabidopsis plants. Furthermore, this 135-bp fragment complemented the -46 minimal promoter of CaMV 35S and conferred constitutive expression on transgenic Arabidopsis plants. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from tobacco leaves interact with the 135-bp fragment. tCUP has a core promoter that lacks the TATA consensus sequence but addition of a TATA-box sequence increased the core promoter activity by three-fold. The sequence surrounding the transcription start site of tCUP has sequence similarity with the initiator element (Inr), and deletion of this sequence significantly reduced promoter activity, suggesting that an essential Inr element may exist in the tCUP core promoter. Fusion of the GCC-box enhancer element from pathogenesis-related genes to the core promoter elevated tCUP core promoter activity. Our study indicates that cryptic promoters are similar in composition and organization to promoters associated with expressed genes and that their promoter elements can be combined to create composite promoters that are fully functional. This data provides direct evidence that the expression pattern of plant genes can be influenced by cryptic gene regulatory elements when they are brought into juxtaposition with genes through DNA rearrangements. PMID:11523793

  20. Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus.

    PubMed Central

    de Lencastre, H; Tomasz, A

    1994-01-01

    A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance. Images PMID:7872753

  1. Effect of gibberellic acid and calliterpenone on plant growth attributes, trichomes, essential oil biosynthesis and pathway gene expression in differential manner in Mentha arvensis L.

    PubMed

    Bose, Subir K; Yadav, Ritesh Kumar; Mishra, Smrati; Sangwan, Rajender S; Singh, A K; Mishra, B; Srivastava, A K; Sangwan, Neelam S

    2013-05-01

    Extensive research is going on throughout the world to find out new molecules from natural sources to be used as plant growth promoter. Mentha arvensis L. is the main source of menthol rich essential oil used commercially in various food, pharmaceutical and other preparations. Experiments were conducted on field grown plants for understanding the effect of calliterpenone (CA), a stereo-isomer of abbeokutone, in comparison to gibberellic acid (GA3) on growth attributes, trichomes, essential oil biosynthesis and expression of some oil biosynthetic pathway genes. The exogenous application of CA (1 μM, 10 μM and 100 μM) was found to be better in improving plant biomass and stolon yield, leaf area, branching and leaf stem ratio than with counterpart GA3 at the same concentrations. CA treated plants showed higher glandular trichome number, density and diameter and also correlated with enhanced oil biogenetic capacity as revealed by feeding labeled (14)C-sucrose for 72 h to excised shoots. Semi-quantitative PCR analysis of key pathway genes revealed differential up regulation under CA treatments. Transcript level of menthol dehydrogenase/menthone reductase was found highly up regulated in CA treated plants with increased content of menthone and menthol in oil. These findings demonstrate that CA positively regulated the yields by enhanced branching and higher density of trichomes resulting into higher accumulation of essential oil. The results suggest CA as a novel plant derived diterpenoid with growth promoting action and opens up new possibilities for improving the crop yields and essential oil biosynthesis in qualitative and quantitative manner. PMID:23514759

  2. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  3. No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

    PubMed

    Luderer, R; Rivas, S; Nürnberger, T; Mattei, B; Van den Hooven, H W; Van der Hoorn, R A; Romeis, T; Wehrfritz, J M; Blume, B; Nennstiel, D; Zuidema, D; Vervoort, J; De Lorenzo, G; Jones, J D; De Wit, P J; Joosten, M H

    2001-07-01

    The gene-for-gene model postulates that for every gene determining resistance