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Sample records for essential gene products

  1. Metabolites production improvement by identifying minimal genomes and essential genes using flux balance analysis.

    PubMed

    Salleh, Abdul Hakim Mohamed; Mohamad, Mohd Saberi; Deris, Safaai; Illias, Rosli Md

    2015-01-01

    With the advancement in metabolic engineering technologies, reconstruction of the genome of host organisms to achieve desired phenotypes can be made. However, due to the complexity and size of the genome scale metabolic network, significant components tend to be invisible. We proposed an approach to improve metabolite production that consists of two steps. First, we find the essential genes and identify the minimal genome by a single gene deletion process using Flux Balance Analysis (FBA) and second by identifying the significant pathway for the metabolite production using gene expression data. A genome scale model of Saccharomyces cerevisiae for production of vanillin and acetate is used to test this approach. The result has shown the reliability of this approach to find essential genes, reduce genome size and identify production pathway that can further optimise the production yield. The identified genes and pathways can be extendable to other applications especially in strain optimisation. PMID:26489144

  2. 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. 65:1627–1635, 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

  3. Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius

    PubMed Central

    El Khoury, Rachelle; Atoui, Ali; Verheecke, Carol; Maroun, Richard; El Khoury, Andre; Mathieu, Florence

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O.As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 °C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O.As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium. PMID:27548221

  4. Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius.

    PubMed

    El Khoury, Rachelle; Atoui, Ali; Verheecke, Carol; Maroun, Richard; El Khoury, Andre; Mathieu, Florence

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O.As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 °C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O.As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium. PMID:27548221

  5. EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

    PubMed

    Dertli, Enes; Mayer, Melinda J; Colquhoun, Ian J; Narbad, Arjan

    2016-07-01

    Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression. PMID:26401596

  6. 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.

  7. 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

  8. Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus.

    PubMed

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

    2012-05-01

    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 H(2)O(2), 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

  9. 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

  10. A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products.

    PubMed Central

    Huang, J; Liang, T J

    1993-01-01

    Many viruses possess complex mechanisms involving multiple gene products and cis-regulatory elements in order to achieve a fine control of their gene expression at both transcriptional and posttranscriptional levels. Hepatitis B virus (HBV) and retroviruses share many structural and functional similarities. In this study, by genetic and biochemical analyses, we have demonstrated the existence of a novel genetic element within the HBV genome which is essential for high-level expression of viral gene products. This element is located 3' to the envelope coding region. We have shown that this genetic element is cis acting at the posttranscriptional level and that its function is exerted at the level of RNA processing as part of transcribed sequences. This RNA element is also functional in the context of a heterologous gene. Similar to the function of Rev-Rev response element interaction of human immunodeficiency virus type 1, this element appears to inhibit the splicing process and facilitate the transport and utilization of HBV transcripts. Images PMID:8246965

  11. The yeast NOP4 gene product is an essential nucleolar protein required for pre-rRNA processing and accumulation of 60S ribosomal subunits.

    PubMed Central

    Sun, C; Woolford, J L

    1994-01-01

    The Saccharomyces cerevisiae NOP4 gene was isolated by screening a lambda gt11 yeast genomic DNA library with a monoclonal antibody against a yeast nucleolar protein. NOP4 encodes a 78 kDa protein that contains two prototypical RNA recognition motifs (RRMs) flanking an imperfect RRM lacking characteristic RNP1 and RNP2 motifs. In addition, there is a fourth incomplete RRM. NOP4 is a single copy essential gene present on chromosome XVI, between RAD1 and PEP4. To examine the function of Nop4p, we constructed a conditional null allele of NOP4 by placing this gene under the control of the glucose-repressible GAL1 promoter. When cells are shifted from galactose-containing medium to glucose-containing medium, NOP4 transcription is terminated, Nop4 protein is depleted and cell growth is impaired. Nop4 protein depletion results in diminished accumulation of 60S ribosomal subunits, assignable to a defect in ribosome biogenesis arising from a lack of production of mature 25S rRNA from 27S precursor rRNA. Images PMID:8039505

  12. 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.

  13. The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

    PubMed Central

    Waterham, H R; de Vries, Y; Russel, K A; Xie, W; Veenhuis, M; Cregg, J M

    1996-01-01

    We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1. PMID:8628321

  14. 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

  15. The essential gene set of a photosynthetic organism

    PubMed Central

    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.

    2015-01-01

    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 tRNALeu, 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

  16. 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

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

    PubMed Central

    Wang, Tim; Birsoy, Kıvanç; 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

  18. Comparative analysis of essential genes in prokaryotic genomic islands

    PubMed Central

    Zhang, Xi; Peng, Chong; Zhang, Ge; Gao, Feng

    2015-01-01

    Essential genes are thought to encode proteins that carry out the basic functions to sustain a cellular life, and genomic islands (GIs) usually contain clusters of horizontally transferred genes. It has been assumed that essential genes are not likely to be located in GIs, but systematical analysis of essential genes in GIs has not been explored before. Here, we have analyzed the essential genes in 28 prokaryotes by statistical method and reached a conclusion that essential genes in GIs are significantly fewer than those outside GIs. The function of 362 essential genes found in GIs has been explored further by BLAST against the Virulence Factor Database (VFDB) and the phage/prophage sequence database of PHAge Search Tool (PHAST). Consequently, 64 and 60 eligible essential genes are found to share the sequence similarity with the virulence factors and phage/prophages-related genes, respectively. Meanwhile, we find several toxin-related proteins and repressors encoded by these essential genes in GIs. The comparative analysis of essential genes in genomic islands will not only shed new light on the development of the prediction algorithm of essential genes, but also give a clue to detect the functionality of essential genes in genomic islands. PMID:26223387

  19. Exploring the relationship between fractal features and bacterial essential genes

    NASA Astrophysics Data System (ADS)

    Yong-Ming, Yu; Li-Cai, Yang; Qian, Zhou; Lu-Lu, Zhao; Zhi-Ping, Liu

    2016-06-01

    Essential genes are indispensable for the survival of an organism in optimal conditions. Rapid and accurate identifications of new essential genes are of great theoretical and practical significance. Exploring features with predictive power is fundamental for this. Here, we calculate six fractal features from primary gene and protein sequences and then explore their relationship with gene essentiality by statistical analysis and machine learning-based methods. The models are applied to all the currently available identified genes in 27 bacteria from the database of essential genes (DEG). It is found that the fractal features of essential genes generally differ from those of non-essential genes. The fractal features are used to ascertain the parameters of two machine learning classifiers: Naïve Bayes and Random Forest. The area under the curve (AUC) of both classifiers show that each fractal feature is satisfactorily discriminative between essential genes and non-essential genes individually. And, although significant correlations exist among fractal features, gene essentiality can also be reliably predicted by various combinations of them. Thus, the fractal features analyzed in our study can be used not only to construct a good essentiality classifier alone, but also to be significant contributors for computational tools identifying essential genes. Project supported by the Shandong Provincial Natural Science Foundation, China (Grant No. ZR2014FM022).

  20. How to identify essential genes from molecular networks?

    PubMed Central

    del Rio, Gabriel; Koschützki, Dirk; Coello, Gerardo

    2009-01-01

    Background The prediction of essential genes from molecular networks is a way to test the understanding of essentiality in the context of what is known about the network. However, the current knowledge on molecular network structures is incomplete yet, and consequently the strategies aimed to predict essential genes are prone to uncertain predictions. We propose that simultaneously evaluating different network structures and different algorithms representing gene essentiality (centrality measures) may identify essential genes in networks in a reliable fashion. Results By simultaneously analyzing 16 different centrality measures on 18 different reconstructed metabolic networks for Saccharomyces cerevisiae, we show that no single centrality measure identifies essential genes from these networks in a statistically significant way; however, the combination of at least 2 centrality measures achieves a reliable prediction of most but not all of the essential genes. No improvement is achieved in the prediction of essential genes when 3 or 4 centrality measures were combined. Conclusion The method reported here describes a reliable procedure to predict essential genes from molecular networks. Our results show that essential genes may be predicted only by combining centrality measures, revealing the complex nature of the function of essential genes. PMID:19822021

  1. In silico network topology-based prediction of gene essentiality

    NASA Astrophysics Data System (ADS)

    da Silva, João Paulo Müller; Acencio, Marcio Luis; Mombach, José Carlos Merino; Vieira, Renata; da Silva, José Camargo; Lemke, Ney; Sinigaglia, Marialva

    2008-02-01

    The identification of genes essential for survival is important for the understanding of the minimal requirements for cellular life and for drug design. As experimental studies with the purpose of building a catalog of essential genes for a given organism are time-consuming and laborious, a computational approach which could predict gene essentiality with high accuracy would be of great value. We present here a novel computational approach, called NTPGE (Network Topology-based Prediction of Gene Essentiality), that relies on the network topology features of a gene to estimate its essentiality. The first step of NTPGE is to construct the integrated molecular network for a given organism comprising protein physical, metabolic and transcriptional regulation interactions. The second step consists in training a decision-tree-based machine-learning algorithm on known essential and non-essential genes of the organism of interest, considering as learning attributes the network topology information for each of these genes. Finally, the decision-tree classifier generated is applied to the set of genes of this organism to estimate essentiality for each gene. We applied the NTPGE approach for discovering the essential genes in Escherichia coli and then assessed its performance.

  2. High Confidence Prediction of Essential Genes in Burkholderia Cenocepacia

    PubMed Central

    Juhas, Mario; Stark, Manuel; von Mering, Christian; Lumjiaktase, Puthapoom; Crook, Derrick W.; Valvano, Miguel A.; Eberl, Leo

    2012-01-01

    Background Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex. Methodology/Principal Findings We hypothesize that essential genes may be highly conserved within a group of evolutionary closely related organisms. Using a bioinformatics approach we determined that the core genome of the order Burkholderiales consists of 649 genes. All but two of these identified genes were located on chromosome 1 of Burkholderia cenocepacia. Although many of the 649 core genes of Burkholderiales have been shown to be essential in other bacteria, we were also able to identify a number of novel essential genes present mainly, or exclusively, within this order. The essentiality of some of the core genes, including the known essential genes infB, gyrB, ubiB, and valS, as well as the so far uncharacterized genes BCAL1882, BCAL2769, BCAL3142 and BCAL3369 has been confirmed experimentally in B. cenocepacia. Conclusions/Significance We report on the identification of essential genes using a novel bioinformatics strategy and provide bioinformatics and experimental evidence that the large majority of the identified genes are indeed essential. The essential genes identified here may represent valuable targets for the development of novel antimicrobials and their detailed study may shed new light on the functions required to support life. PMID:22768221

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

    SciTech Connect

    Salem, Tamer Z.; 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.

  4. 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

  5. Essential genes as antimicrobial targets and cornerstones of synthetic biology.

    PubMed

    Juhas, Mario; Eberl, Leo; Church, George M

    2012-11-01

    Essential genes are absolutely required for the survival of any living entity. Investigation of essential genes is therefore expected to advance tremendously our understanding of the universal principles of life. Determination of a minimal set of essential genes needed to sustain life also plays an important role in the emerging field of synthetic biology, whose goals include creation of a stringently controlled minimal cell with predesigned phenotypic traits. In addition, due to their indispensability for survival of bacteria, genes encoding essential cellular functions have great potential in medicine as promising targets for the development of novel antimicrobials. Here, we review recent advances in the investigation of essential genes, with emphasis on the practical applications in medicine and synthetic biology. PMID:22951051

  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. Genome Scanning in Haemophilus influenzae for Identification of Essential Genes

    PubMed Central

    Reich, Karl A.; Chovan, Linda; Hessler, Paul

    1999-01-01

    We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented. PMID:10438768

  9. 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

  10. Redundancy complicates the definition of essential genes for vaccinia virus.

    PubMed

    Dobson, Bianca M; Tscharke, David C

    2015-11-01

    Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants. PMID:26290187

  11. 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.

  12. 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

  13. 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

  14. Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Stolz, L E; Huynh, C V; Thorner, J; York, J D

    1998-01-01

    We recently demonstrated that the S. cerevisiae INP51 locus (YIL002c) encodes an inositol polyphosphate 5-phosphatase. Here we describe two related yeast loci, INP52 (YNL106c) and INP53 (YOR109w). Like Inp51p, the primary structures of Inp52p and Inp53p resemble the mammalian synaptic vesicle-associated protein, synaptojanin, and contain a carboxy-terminal catalytic domain and an amino-terminal SAC1-like segment. Inp51p (108 kD), Inp52p (136 kD) and Inp53p (124 kD) are membrane-associated. Single null mutants (inp51, inp52, or inp53) are viable. Both inp51 inp52 and inp52 inp53 double mutants display compromised cell growth, whereas an inp51 inp53 double mutant does not. An inp51 inp52 inp53 triple mutant is inviable on standard medium, but can grow weakly on media supplemented with an osmotic stabilizer (1 M sorbitol). An inp51 mutation, and to a lesser degree an inp52 mutation, confers cold-resistant growth in a strain background that cannot grow at temperatures below 15 degrees. Analysis of inositol metabolites in vivo showed measurable accumulation of phosphatidylinositol 4,5-bisphosphate in the inp51 mutant. Electron microscopy revealed plasma membrane invaginations and cell wall thickening in double mutants and the triple mutant grown in sorbitol-containing medium. A fluorescent dye that detects endocytic and vacuolar membranes suggests that the vacuole is highly fragmented in inp51 inp52 double mutants. Our observations indicate that Inp51p, Inp52p, and Inp53p have distinct functions and that substrates and/or products of inositol polyphosphate 5-phosphatases may have roles in vesicle trafficking, membrane structure, and/or cell wall formation. PMID:9560389

  15. 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

  16. Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

    PubMed

    Mori, Hirotada; Baba, Tomoya; Yokoyama, Katsushi; Takeuchi, Rikiya; Nomura, Wataru; Makishi, Kazuichi; Otsuka, Yuta; Dose, Hitomi; Wanner, Barry L

    2015-01-01

    Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein. PMID:25636612

  17. Highly parallel identification of essential genes in cancer cells.

    PubMed

    Luo, Biao; Cheung, Hiu Wing; Subramanian, Aravind; Sharifnia, Tanaz; Okamoto, Michael; Yang, Xiaoping; Hinkle, Greg; Boehm, Jesse S; Beroukhim, Rameen; Weir, Barbara A; Mermel, Craig; Barbie, David A; Awad, Tarif; Zhou, Xiaochuan; Nguyen, Tuyen; Piqani, Bruno; Li, Cheng; Golub, Todd R; Meyerson, Matthew; Hacohen, Nir; Hahn, William C; Lander, Eric S; Sabatini, David M; Root, David E

    2008-12-23

    More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process. PMID:19091943

  18. 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

  19. 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

  20. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families

    PubMed Central

    Vyas, Valmik K.; Barrasa, M. Inmaculada; Fink, Gerald R.

    2015-01-01

    Candida albicans is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe a C. albicans CRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of Candida and provides a new window into the biology of this pathogen. PMID:25977940

  1. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication.

    PubMed Central

    Evans, E; Traktman, P

    1987-01-01

    We have identified a gene encoded by vaccinia virus which is essential for DNA replication. The gene, located in the HindIII D fragment of the viral genome, is transcribed early after infection into two transcripts of 3.0 and 3.7 kilobases which share a 3' terminus. The lesions of three temperature-sensitive DNA replication mutants with defects in this gene have been localized by marker rescue with progressively smaller DNA fragments. We have determined by hybrid selection that the gene encodes an 82-kilodalton protein. An antibody has been prepared against this polypeptide and used to quantitate expression of the protein after infection with wild-type virus or with a viral mutant whose lesion maps within this gene. The temporal pattern of expression in the mutant is unaffected, but the product encoded by the mutant is significantly more thermolabile than the wild-type protein. Images PMID:3041037

  2. 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

  3. PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

    PubMed Central

    O'Connor, J P; Peebles, C L

    1992-01-01

    We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed. Images PMID:1508188

  4. Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi

    PubMed Central

    2009-01-01

    Background Wolbachia (wBm) is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium. Results wBm protein sequences were aligned using BLAST to the Database of Essential Genes (DEG) version 5.2, a collection of 5,260 experimentally identified essential genes in 15 bacterial strains. A confidence score, the Multiple Hit Score (MHS), was developed to predict each wBm gene's essentiality based on the top alignments to essential genes in each bacterial strain. This method was validated using a jackknife methodology to test the ability to recover known essential genes in a control genome. A second estimation of essentiality, the Gene Conservation Score (GCS), was calculated on the basis of phyletic conservation of genes across Wolbachia's parent order Rickettsiales. Clusters of orthologous genes were predicted within the 27 currently available complete genomes. Druggability of wBm proteins was predicted by alignment to a database of protein targets of known compounds. Conclusion Ranking wBm genes by either MHS or GCS predicts and prioritizes potentially essential genes. Comparison of the MHS to GCS produces quadrants representing four types of predictions: those with high confidence of essentiality by both methods (245 genes), those highly conserved across Rickettsiales (299 genes), those similar to distant essential genes (8 genes), and those with low confidence of essentiality (253 genes). These data facilitate selection of wBm genes

  5. 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...

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

    PubMed

    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

  7. 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

  8. A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

    PubMed

    Peters, Jason M; Colavin, Alexandre; Shi, Handuo; Czarny, Tomasz L; Larson, Matthew H; Wong, Spencer; Hawkins, John S; Lu, Candy H S; Koo, Byoung-Mo; Marta, Elizabeth; Shiver, Anthony L; Whitehead, Evan H; Weissman, Jonathan S; Brown, Eric D; Qi, Lei S; Huang, Kerwyn Casey; Gross, Carol A

    2016-06-01

    Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis. PMID:27238023

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes.

    PubMed

    Morgens, David W; Deans, Richard M; Li, Amy; Bassik, Michael C

    2016-06-01

    We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology. PMID:27159373

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

    PubMed

    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

  17. 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

  18. The rpoE gene of Escherichia coli, which encodes sigma E, is essential for bacterial growth at high temperature.

    PubMed Central

    Hiratsu, K; Amemura, M; Nashimoto, H; Shinagawa, H; Makino, K

    1995-01-01

    In vitro transcription analysis has shown that only RNA polymerase containing an alternative sigma subunit, sigma E, activates transcription from one of the rpoH promoters and the htrA promoter. The location of the rpoE gene encoding sigma E on the Escherichia coli chromosome has recently been established, but no rpoE mutant has yet become available for phenotypic testing. We cloned the rpoE gene from the lambda-ordered clones of the E. coli genome and confirmed that the reconstituted RNA polymerase containing the gene product (E sigma E) can transcribe htrA in vitro. We constructed an rpoE-defective strain by gene disruption using the cloned rpoE gene. We demonstrate that expression of htrA is completely dependent on the rpoE gene in vivo and that the rpoE gene is essential for bacterial growth at high temperature. PMID:7751307

  19. 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

  20. Hox11 paralogous genes are essential for metanephric kidney induction.

    PubMed

    Wellik, Deneen M; Hawkes, Patrick J; Capecchi, Mario R

    2002-06-01

    The mammalian Hox complex is divided into four linkage groups containing 13 sets of paralogous genes. These paralogous genes have retained functional redundancy during evolution. For this reason, loss of only one or two Hox genes within a paralogous group often results in incompletely penetrant phenotypes which are difficult to interpret by molecular analysis. For example, mice individually mutant for Hoxa11 or Hoxd11 show no discernible kidney abnormalities. Hoxa11/Hoxd11 double mutants, however, demonstrate hypoplasia of the kidneys. As described in this study, removal of the last Hox11 paralogous member, Hoxc11, results in the complete loss of metanephric kidney induction. In these triple mutants, the metanephric blastema condenses, and expression of early patterning genes, Pax2 and Wt1, is unperturbed. Eya1 expression is also intact. Six2 expression, however, is absent, as is expression of the inducing growth factor, Gdnf. In the absence of Gdnf, ureteric bud formation is not initiated. Molecular analysis of this phenotype demonstrates that Hox11 control of early metanephric induction is accomplished by the interaction of Hox11 genes with the pax-eya-six regulatory cascade, a pathway that may be used by Hox genes more generally for the induction of multiple structures along the anteroposterior axis. PMID:12050119

  1. 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

  2. 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

  3. 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

  4. An ACC Oxidase Gene Essential for Cucumber Carpel Development.

    PubMed

    Chen, Huiming; Sun, Jinjing; Li, Shuai; Cui, Qingzhi; Zhang, Huimin; Xin, Fengjiao; Wang, Huaisong; Lin, Tao; Gao, Dongli; Wang, Shenhao; Li, Xia; Wang, Donghui; Zhang, Zhonghua; Xu, Zhihong; Huang, Sanwen

    2016-09-01

    Sex determination in plants gives rise to unisexual flowers that facilitate outcrossing and enhance genetic diversity. In cucumber and melon, ethylene promotes carpel development and arrests stamen development. Five sex-determination genes have been identified, including four encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase that catalyzes the rate-limiting step in ethylene biosynthesis, and a transcription factor gene CmWIP1 that corresponds to the Mendelian locus gynoecious in melon and is a negative regulator of femaleness. ACC oxidase (ACO) converts ACC into ethylene; however, it remains elusive which ACO gene in the cucumber genome is critical for sex determination and how CmWIP1 represses development of female flowers. In this study, we discovered that mutation in an ACO gene, CsACO2, confers androecy in cucumber that bears only male flowers. The mutation disrupts the enzymatic activity of CsACO2, resulting in 50% less ethylene emission from shoot tips. CsACO2 was expressed in the carpel primordia and its expression overlapped with that of CsACS11 in female flowers at key stages for sex determination, presumably providing sufficient ethylene required for proper CsACS2 expression. CmACO3, the ortholog of CsACO2, showed a similar expression pattern in the carpel region, suggesting a conserved function of CsACO2/CmACO3. We demonstrated that CsWIP1, the ortholog of CmWIP1, could directly bind the promoter of CsACO2 and repress its expression. Taken together, we propose a presumably conserved regulatory module consisting of WIP1 transcription factor and ACO controls unisexual flower development in cucumber and melon. PMID:27403533

  5. 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

  6. 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

  7. 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

  8. Unknown unknowns: essential genes in quest for function.

    PubMed

    Danchin, Antoine; Fang, Gang

    2016-09-01

    The experimental design of a minimal synthetic genome revealed the presence of a large number of genes without ascribed function, in part because the abstract laws of life must be implemented within ad hoc material contraptions. Creating a function needs recruitment of some pre-existing structure and this reveals kludges in their set-up and history. Here, we show that looking for functions as an engineer would help in discovery of a significant number of those, proposed together with conceptual handles allowing investigators to pursue this endeavour in other contexts. PMID:27435445

  9. [Essential genes, minimal genome and synthetic cell of bacteria: a review].

    PubMed

    Qiu, Dongru

    2012-05-01

    Single-cell prokaryotes represent a simple and primitive cellular life form. The identification of the essential genes of bacteria and the minimal genome for the free-living cellular life could provide insights into the origin, evolution, and essence of life forms. The principles, methodology, and recent progresses in the identification of essential genes and minimal genome and the creation of synthetic cells are reviewed and particularly the strategies for creating the minimal genome and the potential applications are introduced. PMID:22916492

  10. Steroid Hormone Signaling Is Essential for Pheromone Production and Oenocyte Survival.

    PubMed

    Chiang, Yin Ning; Tan, Kah Junn; Chung, Henry; Lavrynenko, Oksana; Shevchenko, Andrej; Yew, Joanne Y

    2016-06-01

    Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. In Drosophila, the composition of the information-rich lipid profile is dynamic and changes over the lifetime of an individual. However, the molecular basis of this change is not well understood. To identify genes that control cuticular lipid production in Drosophila, we performed a RNA interference screen and used Direct Analysis in Real Time and gas chromatography mass spectrometry to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, we characterized a gene which we name spidey, and which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood. PMID:27333054

  11. Steroid Hormone Signaling Is Essential for Pheromone Production and Oenocyte Survival

    PubMed Central

    Chiang, Yin Ning; Tan, Kah Junn; Shevchenko, Andrej

    2016-01-01

    Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. In Drosophila, the composition of the information-rich lipid profile is dynamic and changes over the lifetime of an individual. However, the molecular basis of this change is not well understood. To identify genes that control cuticular lipid production in Drosophila, we performed a RNA interference screen and used Direct Analysis in Real Time and gas chromatography mass spectrometry to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, we characterized a gene which we name spidey, and which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood. PMID:27333054

  12. Flux balance analysis predicts essential genes in clear cell renal cell carcinoma metabolism

    PubMed Central

    Gatto, Francesco; Miess, Heike; Schulze, Almut; Nielsen, Jens

    2015-01-01

    Flux balance analysis is the only modelling approach that is capable of producing genome-wide predictions of gene essentiality that may aid to unveil metabolic liabilities in cancer. Nevertheless, a systemic validation of gene essentiality predictions by flux balance analysis is currently missing. Here, we critically evaluated the accuracy of flux balance analysis in two cancer types, clear cell renal cell carcinoma (ccRCC) and prostate adenocarcinoma, by comparison with large-scale experiments of gene essentiality in vitro. We found that in ccRCC, but not in prostate adenocarcinoma, flux balance analysis could predict essential metabolic genes beyond random expectation. Five of the identified metabolic genes, AGPAT6, GALT, GCLC, GSS, and RRM2B, were predicted to be dispensable in normal cell metabolism. Hence, targeting these genes may selectively prevent ccRCC growth. Based on our analysis, we discuss the benefits and limitations of flux balance analysis for gene essentiality predictions in cancer metabolism, and its use for exposing metabolic liabilities in ccRCC, whose emergent metabolic network enforces outstanding anabolic requirements for cellular proliferation. PMID:26040780

  13. A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis Elegans

    PubMed Central

    Bucher, E. A.; Greenwald, I.

    1991-01-01

    We have devised a simple genetic mosaic screen, which circumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific early blastomeres, suggesting that many zygotic genes contribute to specific early processes. We discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome. PMID:2071016

  14. 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.

  15. 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

  16. Relationship between EPHX2 gene polymorphisms and essential hypertension in Uygur, Kazakh, and Han.

    PubMed

    Zhu, X L; Wang, L; Wang, Z; Chen, S Z; Zhang, W Q; Ma, M M

    2015-01-01

    We investigated the association between rs751141 polymorphisms in the EPHX2 gene and essential hypertension in Uygur, Kazakh, and Han subjects in Xinjiang, China. A total of 302 essential hypertensive patients in Uygur, 267 in Kazakh, and 368 in Han, as well as 323 normotensive controls in Uygur, 284 in Kazakh, and 348 in Han were enrolled in this study. The TaqMan assay was used to detect the rs751141 G/A gene polymorphism in EPHX2. The rs751141 G/A genotype frequencies for the GA+AA genotypes were 40.2% in essential hypertensive subjects and 52.0% in control subjects in the Han population. The frequencies were significantly different between the 2 Han groups (P < 0.01). The rs751141G/A gene polymorphism showed no significant difference between essential hypertensive patients and normotensive controls in Kazakh and Uygur (all P > 0.05). Essential hypertension in Xinjiang was associated with the rs751141 G/A allele gene polymorphism in EPHX2 in Han subjects but not in Kazakh and Uygur subjects. The rs751141 allele gene polymorphism may be an independent protective factor against essential hypertension in the Han population. PMID:25966114

  17. From essential to persistent genes: a functional approach to constructing synthetic life

    PubMed Central

    Acevedo-Rocha, Carlos G.; Fang, Gang; Schmidt, Markus; Ussery, David W.; Danchin, Antoine

    2013-01-01

    A central undertaking in synthetic biology (SB) is the quest for the ‘minimal genome’. However, ‘minimal sets’ of essential genes are strongly context-dependent and, in all prokaryotic genomes sequenced to date, not a single protein-coding gene is entirely conserved. Furthermore, a lack of consensus in the field as to what attributes make a gene truly essential adds another aspect of variation. Thus, a universal minimal genome remains elusive. Here, as an alternative to defining a minimal genome, we propose that the concept of gene persistence can be used to classify genes needed for robust long-term survival. Persistent genes, although not ubiquitous, are conserved in a majority of genomes, tend to be expressed at high levels, and are frequently located on the leading DNA strand. These criteria impose constraints on genome organization, and these are important considerations for engineering cells and for creating cellular life-like forms in SB. PMID:23219343

  18. Role of Azotobacter vinelandii mucA and mucC Gene Products in Alginate Production

    PubMed Central

    Núñez, Cinthia; León, Renato; Guzmán, Josefina; Espín, Guadalupe; Soberón-Chávez, Gloria

    2000-01-01

    Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor ςE regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the ςE 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

  19. Bioconverted Products of Essential Fatty Acids as Potential Antimicrobial Agents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This review deals with the recent findings on the microbial conversion of essential fatty acids (EFAs) through Pseudomonas aeruginosa PR3 NRRL-B-18602, and the antimicrobial properties of bioconverted essential fatty acids, with particular emphasis on n-3 or n-6 fatty acids. The first section deals...

  20. Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS

    PubMed Central

    Wong, Yee-Chin; Abd El Ghany, Moataz; Naeem, Raeece; Lee, Kok-Wei; Tan, Yung-Chie; Pain, Arnab; Nathan, Sheila

    2016-01-01

    Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence. PMID:27597847

  1. Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS.

    PubMed

    Wong, Yee-Chin; Abd El Ghany, Moataz; Naeem, Raeece; Lee, Kok-Wei; Tan, Yung-Chie; Pain, Arnab; Nathan, Sheila

    2016-01-01

    Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence. PMID:27597847

  2. 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

  3. 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

  4. Polymorphisms in the promoter region of catalase gene and essential hypertension.

    PubMed

    Zhou, Xiao Feng; Cui, Jing; DeStefano, Anita L; Chazaro, Irmarie; Farrer, Lindsay A; Manolis, Athanasios J; Gavras, Haralambos; Baldwin, Clinton T

    2005-01-01

    Genetic variations that predispose individuals to complex disorders, such as essential hypertension, may be found in gene coding regions, intronic regions or in gene promoter regions. Most studies have focused on gene variations that result in amino acid substitutions because they result in different isoforms of the protein, presumably resulting in differences in protein properties. Less attention has been placed on the role of intronic or promoter mutations. In this report, we examined two single nucleotide polymorphisms (SNPs) in the catalase (CAT) gene prompter region in a cohort of hypertensive Caucasians and African Americans with a Mass Spec based Homogenous MassEXTEND assay. We found an association when a specific combination of the two promoter SNPs was examined in Caucasians. No association was observed in African Americans. Our data suggest that genetic variations in the promoter region of catalase gene influence the susceptibility to essential hypertension. In addition, the genetic factors that contribute to hypertension maybe different between ethnic groups. PMID:15735318

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

    PubMed Central

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

    2015-01-01

    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

  6. 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; Döring, 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

  7. 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

  8. The Identification of Transposon-Tagged Mutations in Essential Genes That Affect Cell Morphology in Saccharomyces Cerevisiae

    PubMed Central

    Chun, K. T.; Goebl, M. G.

    1996-01-01

    The yeast Saccharomyces cerevisiae reproduces by budding, and many genes are required for proper bud development. Mutations in some of these genes cause cells to die with an unusual terminal morphology--elongated or otherwise aberrantly shaped buds. To gain insight into bud development, we set out to identify novel genes that encode proteins required for proper bud morphogenesis. Previous studies screened collections of conditional mutations to identify genes required for essential functions, including bud formation. However, genes that are not susceptible to the generation of mutations that cause a conditional phenotype will not be identified in such screens. To identify a more comprehensive collection of mutants, we used transposon mutagenesis to generate a large collection of lethal disruption mutations. This collection was used to identify 209 mutants with disruptions that cause an aberrant terminal bud morphology. The disruption mutations in 33 of these mutants identify three previously uncharacterized genes as essential, and the mutant phenotypes suggest roles for their products in bud morphogenesis. PMID:8770583

  9. 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

  10. Comparison of inherently essential genes of Porphyromonas gingivalis identified in two transposon-sequencing libraries.

    PubMed

    Hutcherson, J A; Gogeneni, H; Yoder-Himes, D; Hendrickson, E L; Hackett, M; Whiteley, M; Lamont, R J; Scott, D A

    2016-08-01

    Porphyromonas gingivalis is a Gram-negative anaerobe and keystone periodontal pathogen. A mariner transposon insertion mutant library has recently been used to define 463 genes as putatively essential for the in vitro growth of P. gingivalis ATCC 33277 in planktonic culture (Library 1). We have independently generated a transposon insertion mutant library (Library 2) for the same P. gingivalis strain and herein compare genes that are putatively essential for in vitro growth in complex media, as defined by both libraries. In all, 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in fundamentally important metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis, and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat-shock protein homologues, sigma factors, enzymes with proteolytic activity, and the majority of sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon-sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets. PMID:26358096

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

    PubMed

    Engebrecht, J; Silverman, M

    1984-07-01

    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. PMID:6377310

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

    PubMed Central

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

    2014-01-01

    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 Rpgril1 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

  13. Disruption of Individual Members of Arabidopsis Syntaxin Gene Families Indicates Each Has Essential Functions

    PubMed Central

    Sanderfoot, Anton A.; Pilgrim, Marsha; Adam, Luc; Raikhel, Natasha V.

    2001-01-01

    Syntaxins are a large group of proteins found in all eukaryotes involved in the fusion of transport vesicles to target membranes. Twenty-four syntaxins grouped into 10 gene families are found in the model plant Arabidopsis thaliana, each group containing one to five paralogous members. The Arabidopsis SYP2 and SYP4 gene families contain three members each that share 60 to 80% protein sequence identity. Gene disruptions of the yeast (Saccharomyces cerevisiae) orthologs of the SYP2 and SYP4 gene families (Pep12p and Tlg2p, respectively) indicate that these syntaxins are not essential for growth in yeast. However, we have isolated and characterized gene disruptions in two genes from each family, finding that disruption of individual syntaxins from these families is lethal in the male gametophyte of Arabidopsis. Complementation of the syp21-1 gene disruption with its cognate transgene indicated that the lethality is linked to the loss of the single syntaxin gene. Thus, it is clear that each syntaxin in the SYP2 and SYP4 families serves an essential nonredundant function. PMID:11251103

  14. The Candida albicans KRE9 gene is required for cell wall β-1,6-glucan synthesis and is essential for growth on glucose

    PubMed Central

    Lussier, Marc; Sdicu, Anne-Marie; Shahinian, Serge; Bussey, Howard

    1998-01-01

    We have isolated CaKRE9, a gene from Candida albicans, that is a functional homologue of the Saccharomyces cerevisiae KRE9 gene involved in β-1,6-glucan synthesis. Disruption of the CaKRE9 gene in C. albicans shows that CaKre9p is required for the synthesis or assembly of this fungal polymer. Homozygous null disruptants of CaKRE9 grow poorly on galactose and fail to form hyphae in serum, and, in growth medium containing glucose, the gene is essential. Thus, the CaKRE9 gene product is a potentially useful candidate as a target for fungal-specific drugs. PMID:9707560

  15. 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

  16. 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

  17. 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

  18. Differential expressions of essential and nonessential alpha-tubulin genes in Schizosaccharomyces pombe.

    PubMed

    Adachi, Y; Toda, T; Niwa, O; Yanagida, M

    1986-06-01

    The fission yeast Schizosaccharomyces pombe has two alpha-tubulin genes and one beta-tubulin gene. Gene disruption experiments showed that the alpha 1-tubulin gene (NDA2) is essential whereas the alpha 2 gene is dispensable. The alpha 2-disrupted cells missing alpha 2 transcript and alpha 2 polypeptide could grow and sporulate normally. The alpha 2 gene, however, was expressed in the wild type and the alpha 1 mutant. Alpha 2-Tubulin was distinguished as an electrophoretic band and was assembled into microtubules. The alpha 2-disrupted cells had an increased sensitivity to an antimicrotubule drug thiabendazole, and the alpha 1(cold-sensitive [cs]) alpha 2 (disrupted) cells became not only cs but also temperature sensitive. Northern blot experiments indicated that alpha 2 transcription was minor and constitutive whereas alpha 1 transcription was major and modulated, depending on the gene copy number of the alpha 2 gene. The amounts of alpha 1 and alpha 2 polypeptides estimated by beta-galactosidase activities of the lacZ-fused genes integrated on the chromosome and by intensities of the electrophoretic bands in crude tubulin fractions, however, were comparable, indicating that alpha 2 tubulin is not a minor subtype. We assume that the cells of Schizosaccharomyces pombe have no excess tubulin pool. alpha 1 mutants would then be blocked in the cell cycle because only half the amount of functional alpha-tubulin required for growth can be produced by the alpha 2 gene. On the other hand, the alpha 2-disrupted cells became viable because the synthesis of alpha 1 tubulin was increased by transcriptional or translational modulation or both. The real cause for essential alpha 1 and dispensable alpha 2 genes seems to be in their regulatory sequences instead of the coding sequences. PMID:3785193

  19. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development.

    PubMed

    Robledo, Raymond F; Rajan, Lakshmi; Li, Xue; Lufkin, Thomas

    2002-05-01

    Dlx homeobox genes are mammalian homologs of the Drosophila Distal-less (Dll) gene. The Dlx/Dll gene family is of ancient origin and appears to play a role in appendage development in essentially all species in which it has been identified. In Drosophila, Dll is expressed in the distal portion of the developing appendages and is critical for the development of distal structures. In addition, human Dlx5 and Dlx6 homeobox genes have been identified as possible candidate genes for the autosomal dominant form of the split-hand/split-foot malformation (SHFM), a heterogeneous limb disorder characterized by missing central digits and claw-like distal extremities. Targeted inactivation of Dlx5 and Dlx6 genes in mice results in severe craniofacial, axial, and appendicular skeletal abnormalities, leading to perinatal lethality. For the first time, Dlx/Dll gene products are shown to be critical regulators of mammalian limb development, as combined loss-of-function mutations phenocopy SHFM. Furthermore, spatiotemporal-specific transgenic overexpression of Dlx5, in the apical ectodermal ridge of Dlx5/6 null mice can fully rescue Dlx/Dll function in limb outgrowth. PMID:12000792

  20. 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

  1. 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

  2. Systematic exploration of essential yeast gene function with temperature-sensitive mutants

    PubMed Central

    Li, Zhijian; Vizeacoumar, Franco J; Bahr, Sondra; Li, Jingjing; Warringer, Jonas; Vizeacoumar, Frederick S; Min, Renqiang; VanderSluis, Benjamin; Bellay, Jeremy; DeVit, Michael; Fleming, James A; Stephens, Andrew; Haase, Julian; Lin, Zhen-Yuan; Baryshnikova, Anastasia; Lu, Hong; Yan, Zhun; Jin, Ke; Barker, Sarah; Datti, Alessandro; Giaever, Guri; Nislow, Corey; Bulawa, Chris; Myers, Chad L; Costanzo, Michael; Gingras, Anne-Claude; Zhang, Zhaolei; Blomberg, Anders; Bloom, Kerry; Andrews, Brenda; Boone, Charles

    2012-01-01

    Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes. PMID:21441928

  3. 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

  4. 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

  5. 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.

  6. Preferential DNA repair of 3-alkyladenine sites in essential and nonessential genes of human astrocytes

    SciTech Connect

    Lapcevich, R.K.; Weiss, R.B.; Gallagher, P.E. )

    1991-03-11

    In recent years, numbers of studies examining excision rates of DNA damaged lesions in defined, subgenomic sequences have shown that DNA repair is not a uniform process throughout the genome. Here, the authors report data on the preferential, in vivo DNA repair of alkylation-induced lesions within specific DNA sequences of essential and nonessential genes. The formation and rate of removal of 3-alkyladenine were studied in these DNA fragments following treatment of human astrocytes with dimethyl sulfate. The distribution and quantitation of this damaged lesion in the isolated DNA from these cells were determined by a polymerase chain reaction assay. The results indicate that alkyladenines are more efficiently repaired in DNA fragments of essential genes than in comparable fragments of nonessential genes. In subsequent experiments, the repair rate of 3-alkyladenine was examined in DNA isolated from alkylation-treated human astrocytes, grown in serum-free medium to inhibit proliferation. The rate of repair of alkylation-induced lesions in essential and nonessential gene fragments also differed in actively growing and quiescent human astrocytes. The results of this study indicate that transcription plays an important role in the efficient removal of 3-alkyladenine by DNA repair systems.

  7. An Essential Interrelationship: Healthy Self-Esteem and Productive Creativity.

    ERIC Educational Resources Information Center

    Yau, Cecilia

    1991-01-01

    This paper defines the term "self-image" or "self-esteem" and defines the term "creativity" from psychoanalytic and humanist interpretations. It then proposes the theory that a positive self-image enhances the possibilities for creative productivity or lifestyle. Practical implications for child rearing are offered. (JDD)

  8. 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

  9. 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

  10. 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

  11. Transcriptional activation by EBV nuclear antigen 1 is essential for the expression of EBV's transforming genes

    PubMed Central

    Altmann, Markus; Pich, Dagmar; Ruiss, Romana; Wang, Jindong; Sugden, Bill; Hammerschmidt, Wolfgang

    2006-01-01

    EBV is a paradigm for human tumor viruses because, although it infects most people benignly, it also can cause a variety of cancers. Both in vivo and in vitro, EBV infects B lymphocytes in G0, induces them to become blasts, and can maintain their proliferation in cell culture or in vivo as tumors. How EBV succeeds in these contrasting cellular environments in expressing its genes that control the host has not been explained. We have genetically dissected the EBV nuclear antigen 1 (EBNA1) gene that is required for replication of the viral genome, to elucidate its possible role in the transcription of viral genes. Strikingly, EBNA1 is essential to drive transcription of EBV's transforming genes after infection of primary B lymphocytes. PMID:16966603

  12. 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

    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

  13. 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 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 novel mediator of DR benefits with broad implications for clinical translation. PMID:25542313

  14. 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

  15. A gene homologous to beta-type carbonic anhydrase is essential for the growth of Corynebacterium glutamicum under atmospheric conditions.

    PubMed

    Mitsuhashi, S; Ohnishi, J; Hayashi, M; Ikeda, M

    2004-02-01

    Carbonic anhydrase catalyzes the interconversion of CO(2) and bicarbonate. We focused on this enzyme in the amino acid-producing organism Corynebacterium glutamicum in order to assess the availability of bicarbonate for carboxylation reactions essential to growth and for those required for L-lysine overproduction. A whole-genome sequence revealed two genes encoding putative beta-type and gamma-type carbonic anhydrases in C. glutamicum. These genes encode polypeptides containing zinc ligands strictly conserved in each type of carbonic anhydrase and were designated bca and gca, respectively. Internal deletion of the chromosomal bca gene resulted in a phenotype showing severely reduced growth under atmospheric conditions (0.04% CO(2)) on both complete and minimal media. The growth defect of the Delta bca strain was restored under elevated CO(2) conditions (5% CO(2)). Introduction of the red alga Porphyridium purpureum carbonic anhydrase gene ( pca) could compensate for the bca deletion, allowing normal growth under an atmospheric level of CO(2). In contrast, the Delta gca strain behaved identically to the wild-type strain with respect to growth, irrespective of the CO(2) conditions. Attempts to increase the dosage of bca, gca, and pca in the defined L-lysine-producing strain C. glutamicum AHD-2 led to no discernable effects on growth and production. Northern blot analysis indicated that the bca transcript in strain AHD-2 and another L-lysine producer, C. glutamicum B-6, was present at a much higher level than in the wild-type strain, particularly during exponential growth phases. These results indicate that: (1) the bca product is essential to achieving normal growth under ordinary atmospheric conditions, and this effect is most likely due to the bca product's ability to maintain favorable intracellular bicarbonate/CO(2) levels, and (2) the expression of bca is induced during exponential growth phases and also in the case of L-lysine overproduction, both of which are

  16. COLT-Cancer: functional genetic screening resource for essential genes in human cancer cell lines

    PubMed Central

    Koh, Judice L. Y.; Brown, Kevin R.; Sayad, Azin; Kasimer, Dahlia; Ketela, Troy; Moffat, Jason

    2012-01-01

    Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation and provide a ‘functional genetic’ map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting approximately 16 000 human genes and a newly developed scoring approach, we identified essential gene profiles in more than 70 breast, pancreatic and ovarian cancer cell lines. We developed a web-accessible database system for capturing information from each step in our standardized screening pipeline and a gene-centric search tool for exploring shRNA activities within a given cell line or across multiple cell lines. The database consists of a laboratory information and management system for tracking each step of a pooled shRNA screen as well as a web interface for querying and visualization of shRNA and gene-level performance across multiple cancer cell lines. COLT-Cancer Version 1.0 is currently accessible at http://colt.ccbr.utoronto.ca/cancer. PMID:22102578

  17. 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

  18. 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

  19. Horizontal gene transfer: essentiality and evolvability in prokaryotes, and roles in evolutionary transitions.

    PubMed

    Koonin, Eugene V

    2016-01-01

    The wide spread of gene exchange and loss in the prokaryotic world has prompted the concept of 'lateral genomics' to the point of an outright denial of the relevance of phylogenetic trees for evolution. However, the pronounced coherence congruence of the topologies of numerous gene trees, particularly those for (nearly) universal genes, translates into the notion of a statistical tree of life (STOL), which reflects a central trend of vertical evolution. The STOL can be employed as a framework for reconstruction of the evolutionary processes in the prokaryotic world. Quantitatively, however, horizontal gene transfer (HGT) dominates microbial evolution, with the rate of gene gain and loss being comparable to the rate of point mutations and much greater than the duplication rate. Theoretical models of evolution suggest that HGT is essential for the survival of microbial populations that otherwise deteriorate due to the Muller's ratchet effect. Apparently, at least some bacteria and archaea evolved dedicated vehicles for gene transfer that evolved from selfish elements such as plasmids and viruses. Recent phylogenomic analyses suggest that episodes of massive HGT were pivotal for the emergence of major groups of organisms such as multiple archaeal phyla as well as eukaryotes. Similar analyses appear to indicate that, in addition to donating hundreds of genes to the emerging eukaryotic lineage, mitochondrial endosymbiosis severely curtailed HGT. These results shed new light on the routes of evolutionary transitions, but caution is due given the inherent uncertainty of deep phylogenies. PMID:27508073

  20. Horizontal gene transfer: essentiality and evolvability in prokaryotes, and roles in evolutionary transitions

    PubMed Central

    Koonin, Eugene V.

    2016-01-01

    The wide spread of gene exchange and loss in the prokaryotic world has prompted the concept of ‘lateral genomics’ to the point of an outright denial of the relevance of phylogenetic trees for evolution. However, the pronounced coherence congruence of the topologies of numerous gene trees, particularly those for (nearly) universal genes, translates into the notion of a statistical tree of life (STOL), which reflects a central trend of vertical evolution. The STOL can be employed as a framework for reconstruction of the evolutionary processes in the prokaryotic world. Quantitatively, however, horizontal gene transfer (HGT) dominates microbial evolution, with the rate of gene gain and loss being comparable to the rate of point mutations and much greater than the duplication rate. Theoretical models of evolution suggest that HGT is essential for the survival of microbial populations that otherwise deteriorate due to the Muller’s ratchet effect. Apparently, at least some bacteria and archaea evolved dedicated vehicles for gene transfer that evolved from selfish elements such as plasmids and viruses. Recent phylogenomic analyses suggest that episodes of massive HGT were pivotal for the emergence of major groups of organisms such as multiple archaeal phyla as well as eukaryotes. Similar analyses appear to indicate that, in addition to donating hundreds of genes to the emerging eukaryotic lineage, mitochondrial endosymbiosis severely curtailed HGT. These results shed new light on the routes of evolutionary transitions, but caution is due given the inherent uncertainty of deep phylogenies. PMID:27508073

  1. Identification and characterization of Helicobacter pylori genes essential for gastric colonization.

    PubMed

    Kavermann, Holger; Burns, Brendan P; Angermuller, Katrin; Odenbreit, Stefan; Fischer, Wolfgang; Melchers, Klaus; Haas, Rainer

    2003-04-01

    Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections. PMID:12668646

  2. Normalization of transposon-mutant library sequencing datasets to improve identification of conditionally essential genes.

    PubMed

    DeJesus, Michael A; Ioerger, Thomas R

    2016-06-01

    Sequencing of transposon-mutant libraries using next-generation sequencing (TnSeq) has become a popular method for determining which genes and non-coding regions are essential for growth under various conditions in bacteria. For methods that rely on quantitative comparison of counts of reads at transposon insertion sites, proper normalization of TnSeq datasets is vitally important. Real TnSeq datasets are often noisy and exhibit a significant skew that can be dominated by high counts at a small number of sites (often for non-biological reasons). If two datasets that are not appropriately normalized are compared, it might cause the artifactual appearance of Differentially Essential (DE) genes in a statistical test, constituting type I errors (false positives). In this paper, we propose a novel method for normalization of TnSeq datasets that corrects for the skew of read-count distributions by fitting them to a Beta-Geometric distribution. We show that this read-count correction procedure reduces the number of false positives when comparing replicate datasets grown under the same conditions (for which no genuine differences in essentiality are expected). We compare these results to results obtained with other normalization procedures, and show that it results in greater reduction in the number of false positives. In addition we investigate the effects of normalization on the detection of DE genes. PMID:26932272

  3. 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

  4. 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

  5. Reversible suppression of an essential gene in adult mice using transgenic RNA interference

    PubMed Central

    McJunkin, Katherine; Mazurek, Anthony; Premsrirut, Prem K.; Zuber, Johannes; Dow, Lukas E.; Simon, Janelle; Stillman, Bruce; Lowe, Scott W.

    2011-01-01

    RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes. We set out to directly target cell proliferation by screening an RNAi library against DNA replication factors and identified multiple shRNAs against Replication Protein A, subunit 3 (RPA3). We generated transgenic mice with TRE-driven Rpa3 shRNAs whose expression enforced a reversible cell cycle arrest. In adult mice, the block in cell proliferation caused rapid atrophy of the intestinal epithelium which led to weight loss and lethality within 8–11 d of shRNA induction. Upon shRNA withdrawal, villus atrophy and weight loss were fully reversible. Thus, shRpa3 transgenic mice provide an interesting tool to study tissue maintenance and regeneration. Overall, we have established a robust system that serves the purpose of temperature-sensitive alleles in other model organisms, enabling inducible and reversible suppression of essential genes in a mammalian system. PMID:21482754

  6. A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

    PubMed

    Sidik, Saima M; Huet, Diego; Ganesan, Suresh M; Huynh, My-Hang; Wang, Tim; Nasamu, Armiyaw S; Thiru, Prathapan; Saeij, Jeroen P J; Carruthers, Vern B; Niles, Jacquin C; Lourido, Sebastian

    2016-09-01

    Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions. PMID:27594426

  7. 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

  8. SUVH2 and SUVH9 Couple Two Essential Steps for Transcriptional Gene Silencing in Arabidopsis.

    PubMed

    Jing, Yuqing; Sun, Han; Yuan, Wei; Wang, Yue; Li, Qi; Liu, Yannan; Li, Yan; Qian, Weiqiang

    2016-08-01

    In Arabidopsis, an RNA-directed DNA methylation pathway (RdDM) is responsible for de novo establishment of DNA methylation and contributes to transcriptional gene silencing. Recently, the microrchidia (MORC)-type ATPases were shown to play essential roles in enforcing transcriptional gene silencing of a subset of genes and transposons by regulating the formation of higher-order chromatin architecture. However, how MORC proteins cooperate with the RdDM pathway components to regulate gene expression remains largely unclear. In this study, SUVH9 and MORC6 were identified from a screening of suppressors of idm1, which is a mutant defective in active DNA demethylation. SUVH9 and MORC6 are required for silencing of two reporter genes and some endogenous genes without enhancing DNA methylation levels. SUVH9, one of SU(VAR)3-9 homologs involved in RdDM, directly interacts with MORC6 and its two close homologs, MORC1 and MORC2. Similar to MORC6, SUVH9 and its homolog SUVH2 are required for heterochromatin condensation and formation of 3D chromatin architecture at SDC and Solo-LTR loci. We propose that SUVH2 and SUVH9 bind to the methylated DNA and facilitate the recruitment of a chromatin-remodeling complex to the target loci in association with MORC proteins. PMID:27216319

  9. Essential contribution of IRF3 to intestinal homeostasis and microbiota-mediated Tslp gene induction.

    PubMed

    Negishi, Hideo; Miki, Shoji; Sarashina, Hana; Taguchi-Atarashi, Naoko; Nakajima, Akira; Matsuki, Kosuke; Endo, Nobuyasu; Yanai, Hideyuki; Nishio, Junko; Honda, Kenya; Taniguchi, Tadatsugu

    2012-12-18

    The large intestinal epithelial cells and immune cells are exposed to a variety of molecules derived from commensal microbiota that can activate innate receptors, such as Toll-like receptors (TLRs) and retinoic acid-inducible gene-I-like receptors (RLRs). Although the activation of these receptors is known to be critical for homeostasis of the large intestine, the underlying gene regulatory mechanisms are not well understood. Here, we show that IFN regulatory factor (IRF)3 is critical for the suppression of dextran sulfate sodium-induced colitis. IRF3-deficient mice exhibited lethal defects in the inflammatory and recovery phases of the colitis, accompanied by marked defects in the gene induction for thymic stromal lymphopoietin (TSLP), a cytokine known to be essential for protection of the large intestine. We further provide evidence that DNA and RNA of the large intestinal contents are critical for Tslp gene induction via IRF3 activation by cytosolic nucleic acid receptors. We also demonstrate that IRF3 indeed activates the gene promoter of Tslp via IRF-binding sequences. This newly identified intestinal gene regulatory mechanism, wherein IRF3 activated by microbiota-derived nucleic acids plays a critical role in intestinal homeostasis, may have clinical implication in colonic inflammatory disorders. PMID:23213237

  10. 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

  11. Systematic Analysis of Essential Genes Reveals New Regulators of G protein Signaling

    PubMed Central

    Cappell, Steven D.; Baker, Rachael; Skowyra, Dorota; Dohlman, Henrik G.

    2010-01-01

    SUMMARY The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly-represented. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways. PMID:20542006

  12. 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

  13. A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli.

    PubMed Central

    Wang, X D; de Boer, P A; Rothfield, L I

    1991-01-01

    Cell division in Escherichia coli requires the products of the ftsQ, ftsA and ftsZ genes. It is not known how the cell regulates the cellular concentrations of these essential elements of the division system. We describe here a factor that activates cell division by specifically increasing transcription from one of the two promoters that lie immediately upstream of the ftsQAZ gene cluster. The trans-acting factor is the product of the sdiA gene, which was isolated on the basis of its ability to suppress the division inhibitory effect of the MinC/MinD division inhibitor. In addition, the sdiA gene product suppressed the action of other chromosomally encoded division inhibitors, induced minicell formation in wild type cells, and restored division activity to an ftsZ temperature-sensitive mutant grown under nonpermissive conditions. All of these properties were explained by the ability of the sdiA gene product specifically to increase transcription of the ftsQAZ gene cluster, resulting in an increase in cellular concentration of the FtsZ protein. The sdiA gene product is the first factor thus far identified that specifically regulates expression of this key group of cell division genes. Images PMID:1915297

  14. The pyrH Gene of Vibrio vulnificus Is an Essential In Vivo Survival Factor▿

    PubMed Central

    Lee, Shee Eun; Kim, Soo Young; Kim, Choon Mee; Kim, Mi-Kwang; Kim, Young Ran; Jeong, Kwangjoon; Ryu, Hwa-Ja; Lee, Youn Suhk; Chung, Sun Sik; Choy, Hyon E.; Rhee, Joon Haeng

    2007-01-01

    We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines. PMID:17371864

  15. 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

  16. 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

  17. Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans

    PubMed Central

    Rao, Anita U.; Cerqueira, Gustavo C.; Mitreva, Makedonka; El-Sayed, Najib M.; Krause, Michael; Hamza, Iqbal

    2010-01-01

    Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme—a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 µM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA–mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival. PMID:20686661

  18. 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

  19. Point Mutation in Essential Genes with Loss or Mutation of the Second Allele

    PubMed Central

    Beck-Engeser, Gabriele B.; Monach, Paul A.; Mumberg, Dominik; Yang, Farley; Wanderling, Sherry; Schreiber, Karin; Espinosa, Rafael; Le Beau, Michelle M.; Meredith, Stephen C.; Schreiber, Hans

    2001-01-01

    Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4+ T cell–recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light–induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4+ T cell–recognized antigen. Thus, for both L9 and L26 genes, we observe “two hit” kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth. PMID:11489948

  20. Identification of candidate genes for familial early-onset essential tremor.

    PubMed

    Liu, Xinmin; Hernandez, Nora; Kisselev, Sergey; Floratos, Aris; Sawle, Ashley; Ionita-Laza, Iuliana; Ottman, Ruth; Louis, Elan D; Clark, Lorraine N

    2016-07-01

    Essential tremor (ET) is one of the most common causes of tremor in humans. Despite its high heritability and prevalence, few susceptibility genes for ET have been identified. To identify ET genes, whole-exome sequencing was performed in 37 early-onset ET families with an autosomal-dominant inheritance pattern. We identified candidate genes for follow-up functional studies in five ET families. In two independent families, we identified variants predicted to affect function in the nitric oxide (NO) synthase 3 gene (NOS3) that cosegregated with disease. NOS3 is highly expressed in the central nervous system (including cerebellum), neurons and endothelial cells, and is one of three enzymes that converts l-arginine to the neurotransmitter NO. In one family, a heterozygous variant, c.46G>A (p.(Gly16Ser)), in NOS3, was identified in three affected ET cases and was absent in an unaffected family member; and in a second family, a heterozygous variant, c.164C>T (p.(Pro55Leu)), was identified in three affected ET cases (dizygotic twins and their mother). Both variants result in amino-acid substitutions of highly conserved amino-acid residues that are predicted to be deleterious and damaging by in silico analysis. In three independent families, variants predicted to affect function were also identified in other genes, including KCNS2 (KV9.2), HAPLN4 (BRAL2) and USP46. These genes are highly expressed in the cerebellum and Purkinje cells, and influence function of the gamma-amino butyric acid (GABA)-ergic system. This is in concordance with recent evidence that the pathophysiological process in ET involves cerebellar dysfunction and possibly cerebellar degeneration with a reduction in Purkinje cells, and a decrease in GABA-ergic tone. PMID:26508575

  1. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides.

    PubMed

    Mitsuhashi, Satoshi

    2014-04-01

    Amino acids play important roles in both human and animal nutrition and in the maintenance of health. Here, amino acids are classified into three groups: first, essential amino acids, which are essential to nutrition; second, functional amino acids, recently found to be important in the promotion of physiological functions; and third, dipeptides, which are used to resolve problematic features of specific free amino acids, such as their instability or insolubility. This review focusses on recent researches concerning the microbial production of essential amino acids (lysine and methionine), functional amino acids (histidine and ornithine), and a dipeptide (L-alanyl-L-glutamine). PMID:24679256

  2. Chemical Composition and Bioactivity of Essential Oil of Atalantia guillauminii against Three Species Stored Product Insects.

    PubMed

    Yang, Kai; You, Chun-Xue; Wang, Cheng-Fang; Lei, Ning; Guo, Shan-Shan; Geng, Zhu-Feng; Du, Shu-Shan; Ma, Ping; Deng, Zhi-Wei

    2015-01-01

    The toxic and repellent activities of the essential oil extracted from the leaves of Atalantia guillauminii Swingle were evaluated against three stored product insects, red flour beetles (Tribolium castaneum), cigarette beetles (Lasioderma serricorne) and booklice (Liposcelis bostrychophila). The essential oil obtained by hydrodistillation was investigated by GC-MS. The main constituents of the essential oil were β-thujene (27.18%), elemicin (15.03%), eudesma-3, 7(11)-diene (9.64%), followed by (-)-4-terpeniol (6.70%) and spathulenol (5.25%). The crude oil showed remarkable contact toxicity against T. castaneum, L. serricorne adults and L. bostrychophila with LD50 values of 17.11, 24.07 µg/adult and 55.83 µg/cm(2) respectively and it also displayed strong fumigant toxicity against T. castaneum, L. serricorne adults with LC50 values of 17.60 and 12.06 mg/L respectively, while weak fumigant toxicity against L. bostrychophila with a LC50 value of 16.75 mg/L. Moreover, the essential oil also exhibited the same level repellency against the three stored product insects, relative to the positive control, DEET. At the same concentrations, the essential oil was more repellent to T. castaneum than to L. serricorne. Thus, the essential oil of A. guillauminii may be potential to be developed as a new natural fumigant/repellent in the control of stored product insects. PMID:26369599

  3. 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

  4. Production of transgenic rice with agronomically useful genes: an assessment.

    PubMed

    Giri, C C; Vijaya Laxmi, G

    2000-12-01

    Rice is the most important food crop in tropical and subtropical regions of the world. Yield enhancement to increase rice production is one of the essential strategies to meet the demand for food of the growing population. Both abiotic and biotic features limit adversely the productivity of rice growing areas. Conventional breeding has been an effective means for developing high yielding varieties, however; it is associated with its own limitations. It is envisaged that recent trends in biotechnology can contribute to the agronomic improvement of rice in terms of yield and nutritional quality as a supplement to traditional breeding methods. Genetic transformation of rice has demonstrated numerous important opportunities resulting in the genetic improvement of existing elite rice varieties and production of new plant types. Significant advances have been made in the genetic engineering of rice since the first transgenic rice plant production in the late 1980s. Several gene transfer protocols have been employed successfully for the introduction of foreign genes to rice. In more than 60 rice cultivars belonging to indica, japonica, javanica, and elite African cultivars, the protocol has been standardized for transgenic rice production. Selection and use of appropriate promoters, selectable markers, and reporter genes has been helpful for development of efficient protocols for transgenic rice in a number of rice cultivars. The present review is an attempt to assess the current state of development in transgenic rice for the transfer of agronomically useful genes, emphasizing the application and future prospects of transgenic rice production for the genetic improvement of this food crop. PMID:14538093

  5. A proposed essential gene discovery pipeline: a Campylobacter jejuni case study.

    PubMed

    Reuter, Mark; Gaskin, Duncan J H; Metris, Aline

    2015-01-01

    Genes required for an organism's growth and survival are termed essential and represent potential intervention targets. Following in the footsteps of the genomics era, the "next-gen" genomic era provides vast amounts of genetic information. Sequencing of a representative bacterial pathogen genome has been superseded by sequencing of whole strain collections, whether from environmental or clinical sources (Harris et al., Science 327:469-474, 2010; Lewis et al., J Hosp Infect 75:37-41, 2010; Beres et al., Proc Natl Acad Sci U S A 107:4371-4376, 2010; Qi et al., PLoS Pathog 5:e1000580, 2009; He et al., Proc Natl Acad Sci U S A 107:7527-7532, 2010; Barrick et al., Nature 461:1243-1247, 2009; Sheppard et al., Mol Ecol 22:1051-1064, 2013). However, the challenge of using this information to gain biological insight remains. Nonetheless, this information, in combination with experimental data from the literature, can serve as the framework for gaining a better understanding of an organism's biology. Generic metabolic pathways have long been known, and a number of websites (e.g., KEGG and BioCyc) attempt to map information from genome annotation to metabolic pathways (Kanehisa et al., Nucleic Acids Res 40:D109-D114, 2010; Karp et al., Nucleic Acids Res 33:6083-6089, 2005). Extending this analysis to incorporate metabolic flux models further allows in silico prediction of potential essential genes. Such efforts are of value, either to highlight novel generic antimicrobials or to seek novel treatments for non-paradigm organisms. Such in silico approaches are attractive as they can highlight pathways and genes that would otherwise only be identified by costly and time-consuming laboratory methods. PMID:25636619

  6. TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis

    PubMed Central

    Grive, Kathryn J.; Gustafson, Eric A.; Seymour, Kimberly A.; Baddoo, Melody; Schorl, Christoph; Golnoski, Kayla; Rajkovic, Aleksandar; Brodsky, Alexander S.; Freiman, Richard N.

    2016-01-01

    TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women. PMID:27341508

  7. ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis

    PubMed Central

    Abderrahmani, Amar; Cheviet, Séverine; Ferdaoussi, Mourad; Coppola, Thierry; Waeber, Gérard; Regazzi, Romano

    2006-01-01

    The GTPases Rab3a and Rab27a and their effectors Granuphilin/Slp4 and Noc2 are essential regulators of neuroendocrine secretion. Chronic exposure of pancreatic β-cells to supraphysiological glucose levels decreased selectively the expression of these proteins. This glucotoxic effect was mimicked by cAMP-raising agents and blocked by PKA inhibitors. We demonstrate that the transcriptional repressor ICER, which is induced in a PKA-dependent manner by chronic hyperglycemia and cAMP-raising agents, is responsible for the decline of the four genes. ICER overexpression diminished the level of Granuphilin, Noc2, Rab3a and Rab27a by binding to cAMP responsive elements located in the promoters of these genes and inhibited exocytosis of β-cells in response to secretagogues. Moreover, the loss in the expression of the genes of the secretory machinery caused by glucose and cAMP-raising agents was prevented by an antisense construct that reduces ICER levels. We propose that induction of inappropriate ICER levels lead to defects in the secretory process of pancreatic β-cells possibly contributing, in conjunction with other known deleterious effects of hyperglycemia, to defective insulin release in type 2 diabetes. PMID:16498408

  8. ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis.

    PubMed

    Abderrahmani, Amar; Cheviet, Séverine; Ferdaoussi, Mourad; Coppola, Thierry; Waeber, Gérard; Regazzi, Romano

    2006-03-01

    The GTPases Rab3a and Rab27a and their effectors Granuphilin/Slp4 and Noc2 are essential regulators of neuroendocrine secretion. Chronic exposure of pancreatic beta-cells to supraphysiological glucose levels decreased selectively the expression of these proteins. This glucotoxic effect was mimicked by cAMP-raising agents and blocked by PKA inhibitors. We demonstrate that the transcriptional repressor ICER, which is induced in a PKA-dependent manner by chronic hyperglycemia and cAMP-raising agents, is responsible for the decline of the four genes. ICER overexpression diminished the level of Granuphilin, Noc2, Rab3a and Rab27a by binding to cAMP responsive elements located in the promoters of these genes and inhibited exocytosis of beta-cells in response to secretagogues. Moreover, the loss in the expression of the genes of the secretory machinery caused by glucose and cAMP-raising agents was prevented by an antisense construct that reduces ICER levels. We propose that induction of inappropriate ICER levels lead to defects in the secretory process of pancreatic beta-cells possibly contributing, in conjunction with other known deleterious effects of hyperglycemia, to defective insulin release in type 2 diabetes. PMID:16498408

  9. 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

  10. 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

  11. Gene PA2449 Is Essential for Glycine Metabolism and Pyocyanin Biosynthesis in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lundgren, Benjamin R.; Thornton, William; Dornan, Mark H.; Villegas-Peñaranda, Luis Roberto; Boddy, Christopher N.

    2013-01-01

    Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria. PMID:23457254

  12. Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops in Chlamydomonas[W

    PubMed Central

    Ramundo, Silvia; Rahire, Michèle; Schaad, Olivier; Rochaix, Jean-David

    2013-01-01

    Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry. PMID:23292734

  13. 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

  14. Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets

    PubMed Central

    Kumar, Sanjay; Chaudhary, Kshitiz; Foster, Jeremy M.; Novelli, Jacopo F.; Zhang, Yinhua; Wang, Shiliang; Spiro, David; Ghedin, Elodie; Carlow, Clotilde K. S.

    2007-01-01

    We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression. PMID:18000556

  15. 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

  16. 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

  17. 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

  18. 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

  19. The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense.

    PubMed

    Capewell, Paul; Clucas, Caroline; DeJesus, Eric; Kieft, Rudo; Hajduk, Stephen; Veitch, Nicola; Steketee, Pieter C; Cooper, Anneli; Weir, William; MacLeod, Annette

    2013-01-01

    Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense. PMID:24098129

  20. The TgsGP Gene Is Essential for Resistance to Human Serum in Trypanosoma brucei gambiense

    PubMed Central

    DeJesus, Eric; Kieft, Rudo; Hajduk, Stephen; Veitch, Nicola; Steketee, Pieter C.; Cooper, Anneli; Weir, William; MacLeod, Annette

    2013-01-01

    Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense. PMID:24098129

  1. If I am only my genes, what am I? Genetic essentialism and a Jewish response.

    PubMed

    Wolpe, Paul Root

    1997-09-01

    With the advent of the Genetic Age comes a unique new set of problems and ethical decisions. There is a tendency to take the scientific developments presented by modern genetics at face value, as if the science itself were value-neutral and not influenced by cultural and religious images. One example of the fallout of the Genetic Age is the development of a "genetic self," the idea that our essential selfhood lies in our genes. It is important to understand the assumptions of the Genetic Age, the development of genetic selfhood, and the broader cultural trends and assumptions that underlie modern genetic thinking. It is equally important, however, to shape a reaction to the concept of a genetic self. Judaism has long carried on a unique discussion about the nature of selfhood in different times and places and about the relation of the corporeal self to the essential self. Insights from Judaism therefore may help to craft a reaction to the modern genetic self that incorporates the best of modern genetics as well as the integrity of a more transcendent selfhood. PMID:11660355

  2. 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...

  3. 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

  4. 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

  5. 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

  6. 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

  7. Identification of a Gene Essential for the First Committed Step in the Biosynthesis of Bacteriochlorophyll c*

    PubMed Central

    Liu, Zhenfeng; Bryant, Donald A.

    2011-01-01

    Bacteriochlorophylls (BChls) c, d, and e are the major chlorophylls in chlorosomes, which are the largest and one of the most efficient antennae produced by chlorophototrophic organisms. In the biosynthesis of these three BChls, a C-132-methylcarboxyl group found in all other chlorophylls (Chls) must be removed. This reaction is postulated to be the first committed step in the synthesis of these BChls. Analyses of gene neighborhoods of (B)Chl biosynthesis genes and distribution patterns in organisms producing chlorosomes helped to identify a gene (bciC) that appeared to be a good candidate to produce the enzyme involved in this biochemical reaction. To confirm that this was the case, a deletion mutant of an open reading frame orthologous to bciC, CT1077, was constructed in Chlorobaculum tepidum, a genetically tractible green sulfur bacterium. The CT1077 deletion mutant was unable to synthesize BChl c but still synthesized BChl a and Chl a. The deletion mutant accumulated large amounts of various (bacterio)pheophorbides, all of which still had C-132-methylcarboxyl groups. A C. tepidum strain in which CT1077 was replaced by an orthologous gene, Cabther_B0031 from “Candidatus Chloracidobacterium thermophilum” was constructed. Although the product of Cabther_B0031 was only 28% identical to the product of CT1077, this strain synthesized BChl c, BChl a, and Chl a in amounts similar to wild-type C. tepidum cells. To indicate their roles in the first committed step of BChl c, d, and e biosynthesis, open reading frames CT1077 and Cabther_B0031 have been redesignated bciC. The potential mechanism by which BciC removes the C-132-methylcarboxyl moiety of chlorophyllide a is discussed. PMID:21550979

  8. Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing.

    PubMed Central

    Jackson, S P; Lossky, M; Beggs, J D

    1988-01-01

    Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36 degrees C), suggesting that the RNA8 gene encodes a component of the splicing machinery. The RNA8 gene was cloned by complementation of the temperature-sensitive growth defect of an rna8-1 mutant strain. Integrative transformation and gene disruption experiments confirmed the identity of the cloned DNA and demonstrated that the RNA8 gene encodes an essential function. The RNA8 gene was shown to be represented once per S. cerevisiae haploid genome and to encode a low-abundance transcript of approximately 7.4 kilobases. By using antisera raised against beta-galactosidase-RNA8 fusion proteins, the RNA8 gene product was identified in S. cerevisiae cell extracts as a low-abundance protein of approximately 260 kilodaltons. Immunodepletion of the RNA8 protein specifically abolished the activity of S. cerevisiae in vitro splicing extracts, confirming that RNA8 plays an essential role in splicing. Images PMID:2835658

  9. Getting essential health products to their end users: Subsidize, but how much?

    PubMed Central

    Dupas, Pascaline

    2015-01-01

    Although coverage rates and health outcomes are improving, many poor people around the world still do not benefit from essential health products. An estimated two-thirds of child deaths could be prevented with increased coverage of products such as vaccines, point-of-use water treatment, iron fortification, and insecticide-treated bednets. What limits the flow of products from the producer's laboratory bench to the end users, and what can be done about it? Recent empirical research suggests a crucial role for heavy subsidies. PMID:25214612

  10. SuhB Is a Regulator of Multiple Virulence Genes and Essential for Pathogenesis of Pseudomonas aeruginosa

    PubMed Central

    Li, Kewei; Xu, Chang; Jin, Yongxin; Sun, Ziyu; Liu, Chang; Shi, Jing; Chen, Gukui; Chen, Ronghao; Jin, Shouguang; Wu, Weihui

    2013-01-01

    ABSTRACT During initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis. Pseudomonas aeruginosa has evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened a P. aeruginosa transposon (Tn) insertion library and found suhB to be an essential gene for the T3SS gene expression. The expression of suhB was upregulated in a mouse acute lung infection model, and loss of suhB resulted in avirulence. Suppression of T3SS gene expression in the suhB mutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated that suhB mutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that an in vivo-inducible gene, suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis of P. aeruginosa. PMID:24169572

  11. 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

  12. 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

  13. Association between G-217A polymorphism in the AGT gene and essential hypertension: a meta-analysis.

    PubMed

    Yao, R; Du, Y Y; Zhang, Y Z; Chen, Q H; Zhao, L S; Li, L

    2015-01-01

    Numerous studies have evaluated the association between the angiotensinogen (AGT) G-217A gene polymorphism and essential hypertension risk. However, the results have been inconsistent. We examined whether the AGT G-217A gene polymorphism confers essential hypertension risk by conducting a meta-analysis. We conducted a literature search of the Google Scholar, PubMed, and China National Knowledge Infrastructure databases for relevant studies that examined the G-217A polymorphism and risk of essential hypertension. Statistical analyses were carried out using Stata 12.0 to combine all relevant studies. Crude odds ratios (ORs) with 95% confidence intervals (95%CIs) were calculated to estimate the strength of this association. A total of 2017 patients with psoriasis and 1708 controls from 7 comparative studies were included in this meta-analysis. We found a significant association between the AGT G-217A gene polymorphism and the risk of essential hypertension (AA vs GG: OR = 2.52, 95%CI = 1.68-3.78; AA vs GA: OR = 2.26, 95%CI = 1.48-3.45; dominant model: OR = 0.38, 95%CI = 0.26-0.57; recessive model: OR = 1.20, 95%CI = 1.03-1.39). Further stratified analyses were conducted by ethnicity and sample size and produced similar results. No evidence of publication bias was found. This meta-analysis confirms that the AGT G-217A gene polymorphism is associated with essential hypertension susceptibility. PMID:26125750

  14. 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

  15. A fungal conserved gene from the basidiomycete Hebeloma cylindrosporum is essential for efficient ectomycorrhiza formation.

    PubMed

    Doré, Jeanne; Marmeisse, Roland; Combier, Jean-Philippe; Gay, Gilles

    2014-10-01

    We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter(-1), a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter(-1). Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability. PMID:24918768

  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. 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

  18. 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

  19. 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

  20. The histone-like protein Hlp is essential for growth of Streptococcus pyogenes: comparison of genetic approaches to study essential genes.

    PubMed

    Bugrysheva, Julia V; Froehlich, Barbara J; Freiberg, Jeffrey A; Scott, June R

    2011-07-01

    Selection of possible targets for vaccine and drug development requires an understanding of the physiology of bacterial pathogens, for which the ability to manipulate expression of essential genes is critical. For Streptococcus pyogenes (the group A streptococcus [GAS]), an important human pathogen, the lack of genetic tools for such studies has seriously hampered research. To address this problem, we characterized variants of the inducible Ptet cassette, in both sense and antisense contexts, as tools to regulate transcription from GAS genes. We found that although the three-operator Ptet construct [Ptet(O)3] had low uninduced expression, its induction level was low, while the two-operator construct [Ptet(O)2] was inducible to a high level but showed significant constitutive expression. Use of Ptet(O)3 in the chromosome allowed us to demonstrate previously that RNases J1 and J2 are required for growth of GAS. Here we report that the uninduced level from the chromosomally inserted Ptet(O)2 construct was too high for us to observe differential growth. For the highly expressed histone-like protein (Hlp) of GAS, neither chromosomal insertion of Ptet(O)2 or Ptet(O)3 nor their use on a high-copy-number plasmid to produce antisense RNA specific to hlp resulted in adequate differential expression. However, by replacing the ribosome binding site of hlp with an engineered riboswitch to control translation of Hlp, we demonstrated for the first time that this protein is essential for GAS growth. PMID:21531823

  1. mamO and mamE genes are essential for magnetosome crystal biomineralization in Magnetospirillum gryphiswaldense MSR-1.

    PubMed

    Yang, Wei; Li, Ruiguo; Peng, Tao; Zhang, Yang; Jiang, Wei; Li, Ying; Li, Jilun

    2010-10-01

    Four non-magnetic mutants of Magnetospirillum gryphiswaldense strain MSR-1 were isolated by transposon mutagenesis and found to contain interruption of either the mamO or mamE gene within the mamAB operon. Studies indicated that mamO and mamE genes are essential for magnetosome crystal biomineralization in MSR-1. This is the first report of a single gene (mamO or mamE) whose mutation affects crystal biomineralization in MSR-1. PMID:20674739

  2. 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

  3. 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

  4. 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

  5. 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

  6. Antibacterial Activity of Carum copticum Essential Oil Against Escherichia Coli O157:H7 in Meat: Stx Genes Expression.

    PubMed

    Mahmoudzadeh, Maryam; Hosseini, Hedayat; Nasrollahzadeh, Javad; Khaneghah, Amin Mousavi; Rismanchi, Marjan; Chaves, Rafael Djalma; Shahraz, Farzaneh; Azizkhani, Maryam; Mahmoudzadeh, Leila; Haslberger, Alexander G

    2016-08-01

    This work were aimed to (a) determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Carum copticum essential oil (EO) against Escherichia. coli O157:H7 in vitro Trypticase Soy Broth, (TSB) and in ground beef; (b) evaluation of the effect of sub-inhibitory concentrations (sub-MICs) of EO on the growth of bacterium in TSB over 72 h (at 35 °C) and ground beef over 9 days (at 4 °C); and (c) investigation of gene expression involved in Shiga toxins production using relative quantitative real-time PCR method. The MIC in broth and ground beef medium were determined as 0.05 (v/v) and 1.75 % (v/w), respectively. In comparison with control cultures, the EO concentration of 0.03 % in broth caused reduction of colony counting as 1.93, 1.79, and 2.62 log10 CFU ml(-1) after 24, 48, and 72 h at 35 °C, and similarly EO (0.75 %) in ground beef resulted to reduction of colony counting as 1.03, 0.92, 1.48, and 2.12 log10 CFU g (-1) after 2, 5, 7, and 9 days at 4 °C, respectively. An increase and decrease in gene expression were observed as result of EO addition (0.03 %) to broth and (0.5 %) to ground beef was noticed, respectively. PMID:27155845

  7. 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

  8. 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

  9. The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes.

    PubMed Central

    Ross, T M; Pettiford, S M; Green, P L

    1996-01-01

    The mechanism of human T-cell leukemia virus (HTLV)-mediated transformation and induction of malignancy is unknown; however, several studies have implicated the viral gene product, Tax. Conclusive evidence for the role of Tax in the HTLV malignant process has been impeded by the inability to mutate tax in the context of an infectious virus and dissociate viral replication from cellular transformation. To circumvent this problem we constructed a mutant of HTLV type 2 (HTLV-2) that replicates by a Tax-independent mechanism. For these studies, the Tax response element in the viral long terminal repeat was replaced with the cytomegalovirus immediate-early promoter enhancer (C-enh). Transcription of the chimeric HTLV-2 (HTLVC-enh) was efficiently directed by this heterologous promoter. Also, the chimeric virus transformed primary human T lymphocytes with an efficiency similar to that of wild-type HTLV-2. A tax-knockout virus, termed HTLVC-enhDeltaTax, was constructed to directly assess the importance of Tax in cellular transformation. Transfection and infection studies indicated that HTLVC-enhDeltaTax was replication competent; however, HTLVC-enhDeltaTax failed to transform primary human T lymphocytes. We conclude that Tax is essential for HTLV-mediated transformation of human T lymphocytes. Furthermore, this chimeric HTLV, that replicates in the absence of Tax, should facilitate studies to determine the precise mechanism of T-lymphocyte transformation by HTLV. PMID:8764028

  10. 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

  11. Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

    PubMed Central

    Odo, Chioma; DuPont, Herbert L.

    2016-01-01

    ABSTRACT Clostridium difficile infection (CDI) is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients. C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr) quorum signaling system. Some C. difficile strains encode two Agr loci in their genomes, designated agr1 and agr2. The agr1 locus is present in all of the C. difficile strains sequenced to date, whereas the agr2 locus is present in a few strains. The functional roles of agr1 and agr2 in C. difficile toxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of both agr loci and examined the mutants for toxin production and virulence. The results showed that the agr1 mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-type agr1. Furthermore, the agr1 mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease. PMID:27531912

  12. 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

  13. The Coactivator SRC-1 is an Essential Coordinator of Hepatic Glucose Production

    PubMed Central

    Louet, Jean-Francois; Chopra, Atul R.; Sagen, Jorn V.; An, Jie; York, Brian; Tannour-Louet, Mounia; Saha, Pradip K.; Stevens, Robert D.; Wenner, Brett R.; Ilkayeva, Olga R.; Bain, James R.; Zhou, Suoling; DeMayo, Franco; Xu, Jianming; Newgard, Christopher B.; O'Malley, Bert W.

    2010-01-01

    Gluconeogenesis makes a major contribution to hepatic glucose production, a process critical for survival in mammals. In this study, we identify the p160 family member, SRC-1, as a key coordinator of the hepatic gluconeogenic program in vivo. SRC-1 null mice displayed hypoglycemia secondary to a deficit in hepatic glucose production. Selective re-expression of SRC-1 in the liver restored blood glucose levels to a normal range. SRC-1 was found induced upon fasting to coordinate in a cell-autonomous manner, the gene expression of rate-limiting enzymes of the gluconeogenic pathway. At the molecular level, the main role of SRC-1 was to modulate the expression and the activity of C/EBPα through a feed-forward loop in which SRC-1 used C/EBPα to transactivate pyruvate carboxylase, a crucial gene for initiation of the gluconeogenic program. We propose that SRC-1, acts as a novel and critical mediator of glucose homeostasis in the liver by adjusting the transcriptional activity of key genes involved in the hepatic glucose production machinery. PMID:21109193

  14. C. elegans patched-3 is an essential gene implicated in osmoregulation and requiring an intact permease transporter domain

    PubMed Central

    Soloviev, Alexander; Gallagher, Joseph; Marnef, Aline; Kuwabara, Patricia E.

    2011-01-01

    The nematode Caenorhabditis elegans has retained a rudimentary Hedgehog (Hh) signalling pathway; Hh and Smoothened (Smo) homologs are absent, but two highly related Patched gene homologs, ptc-1 and ptc-3, and 24 ptc-related (ptr) genes are present. We previously showed that ptc-1 is essential for germ line cytokinesis. Here, we report that ptc-3 is also an essential gene; the absence of ptc-3 results in a late embryonic lethality due to an apparent defect in osmoregulation. Rescue of a ptc-3 mutant with a ptc-3::gfp translational reporter reveals that ptc-3 is dynamically expressed in multiple tissues across development. Consistent with this pattern of expression, ptc-3(RNAi) reveals an additional postembryonic requirement for ptc-3 activity. Tissue-specific promoter studies indicate that hypodermal expression of ptc-3 is required for normal development. Missense changes in key residues of the sterol sensing domain (SSD) and the permease transporter domain GxxxD/E motif reveal that the transporter domain is essential for PTC-3 activity, whereas an intact SSD is dispensable. Taken together, our studies indicate that PTC proteins have retained essential roles in C. elegans that are independent of Smoothened (Smo). These observations reveal novel, and perhaps ancestral, roles for PTC proteins. PMID:21215260

  15. Effects of herbal essential oil mixture as a dietary supplement on egg production in quail.

    PubMed

    Çabuk, Metin; Eratak, Serdar; Alçicek, Ahmet; Bozkurt, Mehmet

    2014-01-01

    One hundred and eighty 7-week-old laying quail were fed various diets over a 12-week period. The diets included a control diet (without essential oil mixture (EOM) or antibiotics (ANTs)), a basal diet including EOM (24 mg/kg feed), and a basal diet including an ANT (avilamycin, 10 mg/kg feed). Each treatment comprised 4 replications with 4 cages (15 quail per cage), amounting to 60 quail per treatment group. Diets (in mash form) and water were provided for ad libitum consumption. EOM consisted of 6 different essential oils derived from the following herbs: oregano (Origanum sp.), laurel leaf (Laurus nobilis L.), sage leaf (Salvia triloba L.), myrtle leaf (Myrtus communis), fennel seeds (Foeniculum vulgare), and citrus peel (Citrus sp.). In comparison with the control diet, adding supplements such as EOM and ANTs to the basal diet increased egg production in quail (P < 0.001). However, egg production was similar between EOM and ANT treatment groups. Moreover, there were no differences between the treatment groups with regard to egg weight. Feed intake was not affected by EOM or ANT supplementation, whereas feed conversion ratio was significantly improved by EOM and ANT supplementation. Thus, we concluded that EOM has beneficial effects as a dietary supplement on egg production and feed conversion ratio. PMID:24587729

  16. Effects of Herbal Essential Oil Mixture as a Dietary Supplement on Egg Production in Quail

    PubMed Central

    Çabuk, Metin; Eratak, Serdar; Alçicek, Ahmet; Bozkurt, Mehmet

    2014-01-01

    One hundred and eighty 7-week-old laying quail were fed various diets over a 12-week period. The diets included a control diet (without essential oil mixture (EOM) or antibiotics (ANTs)), a basal diet including EOM (24 mg/kg feed), and a basal diet including an ANT (avilamycin, 10 mg/kg feed). Each treatment comprised 4 replications with 4 cages (15 quail per cage), amounting to 60 quail per treatment group. Diets (in mash form) and water were provided for ad libitum consumption. EOM consisted of 6 different essential oils derived from the following herbs: oregano (Origanum sp.), laurel leaf (Laurus nobilis L.), sage leaf (Salvia triloba L.), myrtle leaf (Myrtus communis), fennel seeds (Foeniculum vulgare), and citrus peel (Citrus sp.). In comparison with the control diet, adding supplements such as EOM and ANTs to the basal diet increased egg production in quail (P < 0.001). However, egg production was similar between EOM and ANT treatment groups. Moreover, there were no differences between the treatment groups with regard to egg weight. Feed intake was not affected by EOM or ANT supplementation, whereas feed conversion ratio was significantly improved by EOM and ANT supplementation. Thus, we concluded that EOM has beneficial effects as a dietary supplement on egg production and feed conversion ratio. PMID:24587729

  17. Efficacy of Cuminum cyminum essential oil on FUM1 gene expression of fumonisin-producing Fusarium verticillioides strains

    PubMed Central

    Khosravi, Ali Reza; Shokri, Hojjatollah; Mokhtari, Ali Reza

    2015-01-01

    Objectives: The purpose of this study was to evaluate the effect of Cuminum cyminum (C. cyminum) essential oil on the growth and FUM1 gene expression of fumonisin-producing Fusarium verticillioides (F. verticillioides) strains isolated from maize. Materials and Methods: All fungal strains were cultured on potato dextrose agar (PDA) slopes at 30°C for 7 days. The antifungal activity was evaluated by broth microdilution assay. One set of primers was F. verticillioides species specific, which selectively amplified the intergenic space region of rDNA. The other set of primers was specific to FUM1 gene region of fumonisin-producing F. verticillioides. FUM1 transcript levels were quantified using a reverse transcription-polymerase chain reaction (RT-PCR) protocol. Results: Minimum inhibitory concentration (MIC) values of C. cyminum oil against F. verticillioides strains varied from 0.195 to 0.781 µl.ml-1 (mean MIC value: 0.461 µl.ml-1) indicating 54.5% of the fungal strains inhibited at 0.390 µl.ml-1. PCR analysis of FUM1 gene expression revealed that DNA fragment of 183 bp was amplified in all the isolates of F. verticillioides before treatment with C. cyminum essential oil. Based on RT-PCR analyses, reduction in the expression of fumonisin biosynthetic genes was significant only for FUM1 gene (p<0.05), while no effect was observed on ITS gene. Conclusions: This study showed that all F. verticillioides isolates were susceptible to C. cyminum essential oil, indicating a significant reduction in the growth of fungal isolates. In addition, this oil completely inhibited the expression of FUM1 gene in concentrations dose-dependently. PMID:25767755

  18. 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

  19. 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

  20. 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.

  1. Generation of a complete single-gene knockout bacterial artificial chromosome library of cowpox virus and identification of its essential genes.

    PubMed

    Xu, Zhiyong; Zikos, Dimitrios; Osterrieder, Nikolaus; Tischer, B Karsten

    2014-01-01

    Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family. It infects a broad range of vertebrates and can cause zoonotic infections. CPXV has the largest genome among the orthopoxviruses and is therefore considered to have the most complete set of genes of all members of the genus. Since CPXV has also become a model for studying poxvirus genetics and pathogenesis, we created and characterized a complete set of single gene knockout bacterial artificial chromosome (BAC) clones of the CPXV strain Brighton Red. These mutants allow a systematic assessment of the contribution of single CPXV genes to the outcome of virus infection and replication, as well as to the virus host range. A full-length BAC clone of CPXV strain Brighton Red (pBRF) harboring the gene expressing the enhanced green fluorescent protein under the control of a viral late promoter was modified by introducing the mrfp1 gene encoding the monomeric red fluorescent protein driven by a synthetic early vaccinia virus promoter. Based on the modified BAC (pBRFseR), a library of targeted knockout mutants for each single viral open reading frame (ORF) was generated. Reconstitution of infectious virus was successful for 109 of the 183 mutant BAC clones, indicating that the deleted genes are not essential for virus replication. In contrast, 74 ORFs were identified as essential because no virus progeny was obtained upon transfection of the mutant BAC clones and in the presence of a helper virus. More than 70% of all late CPXV genes belonged to this latter group of essential genes. PMID:24155400

  2. 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

  3. Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

    PubMed Central

    Rempel, R E; Anderson, M K; Evans, E; Traktman, P

    1990-01-01

    Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection. Images PMID:2296077

  4. 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

  5. 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

  6. 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

  7. 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

  8. Enzymatic modification of palmarosa essential oil: chemical analysis and olfactory evaluation of acylated products.

    PubMed

    Ramilijaona, Jade; Raynaud, Elsa; Bouhlel, Charfeddine; Sarrazin, Elise; Fernandez, Xavier; Antoniotti, Sylvain

    2013-12-01

    We have developed an enzymatic protocol to modify the composition of palmarosa essential oil by acylation of its alcohol components by three different acyl donors at various rates. The resulting modified products were characterized by qualitative and quantitative analyses by gas chromatography, and their olfactory properties were evaluated by professional perfumers. We showed that our protocol resulted in two types of modifications of the olfactory properties. The first and most obvious effect observed was the decrease of the alcohol content, with the concomitant increase of the corresponding esters, along with their fruity notes (pear, most notably). The second and less obvious effect was the expression of notes from minor components ((E)-β-ocimene, linalool, β-caryophyllene, and farnesene), originally masked by the sweet-floral-rose odor of geraniol, present in 70% in the palmarosa essential oil used, and emergence of citrus, green, spicy and clove characters in the modified products. This methodology might be considered in the future as a sustainable route to new natural ingredients for the perfumer. PMID:24327448

  9. 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

  10. Novel Association of WNK4 Gene, Ala589Ser Polymorphism in Essential Hypertension, and Type 2 Diabetes Mellitus in Malaysia

    PubMed Central

    Ghodsian, Nooshin; Ismail, Patimah; Ahmadloo, Salma; Heidari, Farzad; Haghvirdizadeh, Polin; Ataollahi Eshkoor, Sima; Etemad, Ali

    2016-01-01

    With-no-lysine (K) Kinase-4 (WNK4) consisted of unique serine and threonine protein kinases, genetically associated with an autosomal dominant form of hypertension. Argumentative consequences have lately arisen on the association of specific single nucleotide polymorphisms of WNK4 gene and essential hypertension (EHT). The aim of this study was to determine the association of Ala589Ser polymorphism of WNK4 gene with essential hypertensive patients in Malaysia. WNK4 gene polymorphism was specified utilizing mutagenically separated polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method in 320 subjects including 163 cases and 157 controls. Close relation between Ala589Ser polymorphism and elevated systolic and diastolic blood pressure (SBP and DBP) was recognized. Sociodemographic factors including body mass index (BMI), age, the level of fasting blood sugar (FBS), low density lipoprotein (LDL), and triglyceride (TG) in the cases and healthy subjects exhibited strong differences (p < 0.05). The distribution of allele frequency and genotype of WNK4 gene Ala589Ser polymorphism showed significant differences (p < 0.05) between EHT subjects with or without type 2 diabetes mellitus (T2DM) and normotensive subjects, statistically. The WNK4 gene variation influences significantly blood pressure increase. Ala589Ser probably has effects on the enzymic activity leading to enhanced predisposition to the disorder. PMID:27314050

  11. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

    The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. omparisons of transcription initiation and termination site...

  12. 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

  13. 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

  14. 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

  15. Effect of Zingiber officinale essential oil on Fusarium verticillioides and fumonisin production.

    PubMed

    Yamamoto-Ribeiro, Milene Mayumi Garcia; Grespan, Renata; Kohiyama, Cássia Yumie; Ferreira, Flavio Dias; Mossini, Simone Aparecida Galerani; Silva, Expedito Leite; Filho, Benicio Alves de Abreu; Mikcha, Jane Martha Graton; Machinski, Miguel

    2013-12-01

    The antifungal activity of ginger essential oil (GEO; Zingiber officinale Roscoe) was evaluated against Fusarium verticillioides (Saccardo) Nirenberg. The minimum inhibitory concentration (MIC) of GEO was determined by micro-broth dilution. The effects of GEO on fumonisin and ergosterol production were evaluated at concentrations of 500-5000 μg/mL in liquid medium with a 5mm diameter mycelial disc of F. verticillioides. Gas chromatography-mass spectrometry showed that the predominant components of GEO were α-zingiberene (23.9%) and citral (21.7%). GEO exhibited inhibitory activity, with a MIC of 2500 μg/mL, and 4000 and 5000 μg/mL reduced ergosterol biosynthesis by 57% and 100%, respectively. The inhibitory effect on fumonisin B1 (FB1) and fumonisin B2 (FB2) production was significant at GEO concentrations of 4000 and 2000 μg/mL, respectively. Thus, the inhibition of fungal biomass and fumonisin production was dependent on the concentration of GEO. These results suggest that GEO was able to control the growth of F. verticillioides and subsequent fumonisin production. PMID:23871071

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Phyto-products may be essential for sustainability and implementation of phytoremediation.

    PubMed

    Bañuelos, G S

    2006-11-01

    Interest in selenium pollution and remediation technology has escalated during the past two decades. Although not known to be essential for plants, selenium is essential but could be toxic for humans and animals, depending on its concentration. A major selenium controversy in the 1980's emerged in California when the general public and scientific community became aware of selenium's potential as an environmental contaminant. After extensive research on several strategies to reduce loads of mobile Se for entering the agricultural ecosystem a plant-based technology, defined as 'phytoremediation' received increasing recognition, as a low-cost environmentally friendly approach for managing soluble Se in the soil and water environment. Successful long-term field remediation of Se by plants is, however, dependent upon acceptance and widespread use by growers, who are also concerned about potential commercial value from using the plant-based technology. Obtaining products with economic value from plants used in the cleanup of soil would certainly be an additional benefit to phytoremediation, which could help sustain its long-term use. PMID:16519976

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

    PubMed

    Werdin González, Jorge Omar; Gutiérrez, María Mercedes; Ferrero, Adriana Alicia; Fernández 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

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

    PubMed Central

    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.

    2015-01-01

    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 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 non-model systems. PMID:26680200

  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. 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

  6. 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

  7. ACE2 gene polymorphism and essential hypertension: an updated meta-analysis involving 11,051 subjects.

    PubMed

    Lu, Na; Yang, Yang; Wang, Yibo; Liu, Yan; Fu, Gang; Chen, Dongmei; Dai, Hui; Fan, Xiaohan; Hui, Rutai; Zheng, Yang

    2012-06-01

    The polymorphisms of angiotensin-converting enzyme 2 (ACE2) gene have been suggested to be linked to increase risk of essential hypertension in multiple populations. However, the results are still debatable. To assess the association between ACE2 G8970A genetic polymorphism and essential hypertension, we conducted a meta-analysis of case-control studies across different ethnicity. PubMed, Embase, CBM, Wanfang and VIP databases were searched, and a total of 11 separate studies in females and nine separate studies in males met the inclusion criteria. Because ACE2 is on the X chromosome, data for each sex were analyzed separately. The selected studies contained 7,251 (4,472 females/2,779 males) hypertensive patients and 3,800 (2,161 females/1,639 males) normotensive controls. A statistically significant association was observed between the G8970A gene polymorphism and essential hypertension risk in female hypertensive group in the recessive genetic model (AA vs. GG+GA: P = 0.03, OR = 1.15, 95% CI = 1.02-1.30, P(heterogeneity) = 0.40, I(2) = 5%, fixed-effects model). Although no association was shown between the frequency of the A allele and the genetic susceptibility to essential hypertension in all male patients (A Allele: P = 0.38, OR = 1.10, 95% CI = 0.89-1.38, P(heterogeneity) = 0.02, I(2) = 56%, random-effects model), we found that the relationship between carrier of A allele and the essential hypertension risk in Han-Chinese male patients subgroup (A Allele: P = 0.006, OR = 1.21, 95% CI = 1.06–1.38, P(heterogeneity) = 0.10, I(2) = 44%, fixed-effects model). The current meta-analysis provided solid evidence suggesting that ACE2 gene polymorphism G8790A was probably a genetic risk factor for essential hypertension across different ethnic populations in female subjects and in Han-Chinese male subjects. PMID:22297693

  8. An intein-mediated modulation of protein stability system and its application to study human cytomegalovirus essential gene function

    PubMed Central

    Pan, Deng; Xuan, Baoqin; Sun, Yamei; Huang, Shaowu; Xie, Maorong; Bai, Yadan; Xu, Wenjia; Qian, Zhikang

    2016-01-01

    Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems. To overcome this difficulty, we have established a novel approach, termed the intein-mediated modulation of protein stability (imPS), in which a destabilizing domain and part of a split intein are fused to the essential protein. The growth of the mutant virus can then be regulated by the degradation and splicing of the protein. We found that an ultrafast gp41-1 split intein was able to rescue or degrade the protein of interest (POI) by removing or adding a strong degron through protein splicing. As a result, the function of the POI was turned on or off during the process. Using HCMV essential gene IE1/IE2, we confirmed that imPS worked remarkably well in conditionally regulating protein stability during viral infection. This conditional approach is likely to be applicable for dissecting the gene functions of HCMV or other viruses. PMID:27188239

  9. 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

  10. The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability

    PubMed Central

    Nicola, André M; Andrade, Rosângela V; Dantas, Alessandra S; Andrade, Patrícia A; Arraes, Fabrício BM; Fernandes, Larissa; Silva-Pereira, Ildinete; Felipe, Maria Sueli S

    2008-01-01

    Background Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target. Results In this work, we decided to further study the Pbhsp90 gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a Pbhsp90 cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 μM, respectively. Conclusion The data confirm that the Pbhsp90 gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the P. brasiliensis yeast cells in a dose-responsive manner. PMID:18808717

  11. 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

  12. Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis.

    PubMed

    Fong, Jiunn C N; Syed, Khalid A; Klose, Karl E; Yildiz, Fitnat H

    2010-09-01

    Biofilm formation enhances the survival and persistence of the facultative human pathogen Vibrio cholerae in natural ecosystems and its transmission during seasonal cholera outbreaks. A major component of the V. cholerae biofilm matrix is the Vibrio polysaccharide (VPS), which is essential for development of three-dimensional biofilm structures. The vps genes are clustered in two regions, the vps-I cluster (vpsU, vpsA-K, VC0916-27) and the vps-II cluster (vpsL-Q, VC0934-39), separated by an intergenic region containing the rbm gene cluster that encodes biofilm matrix proteins. In-frame deletions of the vps clusters and genes encoding matrix proteins drastically altered biofilm formation phenotypes. To determine which genes within the vps gene clusters are required for biofilm formation and VPS synthesis, we generated in-frame deletion mutants for all the vps genes. Many of these mutants exhibited reduced capacity to produce VPS and biofilms. Infant mouse colonization assays revealed that mutants lacking either vps clusters or rbmA (encoding secreted matrix protein RbmA) exhibited a defect in intestinal colonization compared to the wild-type. Understanding the roles of the various vps gene products will aid in the biochemical characterization of the VPS biosynthetic pathway and elucidate how vps gene products contribute to VPS biosynthesis, biofilm formation and virulence in V. cholerae. PMID:20466768

  13. 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...

  14. Genetic Analysis of the Atrial Natriuretic Peptide Gene Polymorphisms among Essential Hypertensive Patients in Malaysia

    PubMed Central

    Ghodsian, Nooshin; Ismail, Patimah; Ahmadloo, Salma; Eskandarian, Narges; Etemad, Ali

    2016-01-01

    Background. Atrial natriuretic peptide (ANP) considerably influences blood pressure regulation through water and sodium homoeostasis. Several of the studies have utilized anonymous genetic polymorphic markers and made inconsequent claims about the ANP relevant disorders. Thus, we screened Insertion/Deletion (ID) and G191A polymorphisms of ANP to discover sequence variations with potential functional significance and to specify the linkage disequilibrium pattern between polymorphisms. The relationships of detected polymorphisms with EH with or without Type 2 Diabetes Mellitus (T2DM) status were tested subsequently. Method. ANP gene polymorphisms (I/D and A191G) were specified utilizing mutagenically separated Polymerase Chain Reaction (PCR) in 320 subjects including 163 EH case subjects and 157 controls. Result. This case-control study discovered a significant association between I/D polymorphisms of ANP gene in EH patient without T2DM. However, the study determined no association between G191A polymorphisms of ANP in EH with or without T2DM. In addition, sociodemographic factors in the case and healthy subjects exhibited strong differences (P < 0.05). Conclusion. As a risk factor, ANP gene polymorphisms may affect hypertension. Despite the small sample size in this study, it is the first research assessing the ANP gene polymorphisms in both EH and T2DM patients among Malaysian population. PMID:27413750

  15. 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

  16. The Arabidopsis HUELLENLOS Gene, Which Is Essential for Normal Ovule Development, Encodes a Mitochondrial Ribosomal Protein

    PubMed Central

    Skinner, Debra J.; Baker, Shawn C.; Meister, Robert J.; Broadhvest, Jean; Schneitz, Kay; Gasser, Charles S.

    2001-01-01

    The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning. PMID:11752383

  17. A genetic screen identifies genes essential for development of myelinated axons in zebrafish.

    PubMed

    Pogoda, Hans-Martin; Sternheim, Nitzan; Lyons, David A; Diamond, Brianne; Hawkins, Thomas A; Woods, Ian G; Bhatt, Dimple H; Franzini-Armstrong, Clara; Dominguez, Claudia; Arana, Naomi; Jacobs, Jennifer; Nix, Rebecca; Fetcho, Joseph R; Talbot, William S

    2006-10-01

    The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin. PMID:16875686

  18. BMP signaling is essential in neonatal surfactant production during respiratory adaptation.

    PubMed

    Luo, Yongfeng; Chen, Hui; Ren, Siying; Li, Nan; Mishina, Yuji; Shi, Wei

    2016-07-01

    Deficiency in pulmonary surfactant results in neonatal respiratory distress, and the known genetic mutations in key components of surfactant only account for a small number of cases. Therefore, determining the regulatory mechanisms of surfactant production and secretion, particularly during the transition from prenatal to neonatal stages, is essential for better understanding of the pathogenesis of human neonatal respiratory distress. We have observed significant increase of bone morphogenetic protein (BMP) signaling in neonatal mouse lungs immediately after birth. Using genetically manipulated mice, we then studied the relationship between BMP signaling and surfactant production in neonates. Blockade of endogenous BMP signaling by deleting Bmpr1a (Alk3) or Smad1 in embryonic day 18.5 in perinatal lung epithelial cells resulted in severe neonatal respiratory distress and death, accompanied by atelectasis in histopathology and significant reductions of surfactant protein B and C, as well as Abca3, whereas prenatal lung development was not significantly affected. We then identified a new BMP-Smad1 downstream target, Nfatc3, which is known as an important transcription activator for surfactant proteins and Abca3. Furthermore, activation of BMP signaling in cultured lung epithelial cells was able to promote endogenous Nfatc3 expression and also stimulate the activity of an Nfatc3 promoter that contains a Smad1-binding site. Therefore, our study suggests that the BMP-Alk3-Smad1-Nfatc3 regulatory loop plays an important role in enhancing surfactant production in neonates, possibly helping neonatal respiratory adaptation from prenatal amniotic fluid environment to neonatal air breathing. PMID:27190064

  19. Myxoma virus M063R is a host range gene essential for virus replication in rabbit cells.

    PubMed

    Barrett, John W; Shun Chang, Chew; Wang, Gen; Werden, Steven J; Shao, Zhuhong; Barrett, Catherine; Gao, Xiujuan; Belsito, Tara A; Villenevue, Danielle; McFadden, Grant

    2007-04-25

    The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells. PMID:17184804

  20. 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

  1. The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

    PubMed Central

    Wada, Y; Kitamoto, K; Kanbe, T; Tanaka, K; Anraku, Y

    1990-01-01

    The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions. Images PMID:2183024

  2. 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.

  3. 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

  4. SLC52A3, A Brown-Vialetto-van Laere syndrome candidate gene is essential for mouse development, but dispensable for motor neuron differentiation.

    PubMed

    Intoh, Atsushi; Suzuki, Naoki; Koszka, Kathryn; Eggan, Kevin

    2016-05-01

    Riboflavin, also known as vitamin B2, is essential for cellular reduction-oxidation reactions, but is not readily synthesized by mammalian cells. It has been proposed that riboflavin absorption occurs through solute carrier family 52 members (SLC52) A1, A2 and A3. These transporters are also candidate genes for the childhood onset-neural degenerative syndrome Brown-Vialetto-Van Laere (BVVL). Although riboflavin is an essential nutrient, why mutations in its transporters result in a neural cell-specific disorder remains unclear. Here, we provide evidence that Slc52a3 is the mouse ortholog of SLC52A3 and show that Slc52a3 deficiency results in early embryonic lethality. Loss of mutant embryos was associated with both defects in placental formation and increased rates of apoptosis in embryonic cells. In contrast, Slc52a3 -/- embryonic stem cell lines could be readily established and differentiated into motor neurons, suggesting that this transporter is dispensable for neural differentiation and short-term maintenance. Consistent with this finding, examination of Slc52a3 gene products in adult tissues revealed expression in the testis and intestine but little or none in the brain and spinal cord. Our results suggest that BVVL patients with SCL52A3 mutations may be good candidates for riboflavin replacement therapy and suggests that either the mutations these individuals carry are hypomorphic, or that in these cases alternative transporters act during human embryogenesis to allow full-term development. PMID:26976849

  5. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes.

    PubMed

    Evers, Bastiaan; Jastrzebski, Katarzyna; Heijmans, Jeroen P M; Grernrum, Wipawadee; Beijersbergen, Roderick L; Bernards, Rene

    2016-06-01

    High-throughput genetic screens have become essential tools for studying a wide variety of biological processes. Here we experimentally compare systems based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) or its transcriptionally repressive variant, CRISPR-interference (CRISPRi), with a traditional short hairpin RNA (shRNA)-based system for performing lethality screens. We find that the CRISPR technology performed best, with low noise, minimal off-target effects and consistent activity across reagents. PMID:27111720

  6. The UL47 gene of avian infectious laryngotracheitis virus is not essential for in vitro replication but is relevant for virulence in chickens.

    PubMed

    Helferich, Dorothee; Veits, Jutta; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

    2007-03-01

    The genome of infectious laryngotracheitis virus (ILTV) exhibits several differences from those of other avian and mammalian alphaherpesviruses. One of them is the translocation of the conserved UL47 gene from the unique long (UL) to the unique short (US) genome region, where UL47 is inserted upstream of the US4 gene homologue. As in other alphaherpesviruses, UL47 encodes a major tegument protein of ILTV particles, whereas the US4 gene product is a non-structural glycoprotein, gG, which is secreted from infected cells. For functional characterization, an ILTV recombinant was isolated in which US4 together with the 3'-terminal part of UL47 was replaced by a reporter gene cassette encoding green fluorescent protein. From this virus, UL47 and US4 single-gene deletion mutants without foreign sequences were derived and virus revertants were also generated. In vitro studies revealed that both genes were non-essential for ILTV replication in cultured cells. Whereas US4-negative ILTV exhibited no detectable growth defects, maximum virus titres of the double deletion mutant and of UL47-negative ILTV were reduced about 10-fold compared with those of wild-type virus and rescued virus. Experimental infection of chickens demonstrated that UL47-negative ILTV was significantly attenuated in vivo and was shed in reduced amounts, whereas wild-type and rescued viruses caused severe disease and high mortality rates. As all immunized animals were protected against subsequent challenge infection with virulent ILTV, the UL47 deletion mutant might be suitable as a live-virus vaccine. PMID:17325345

  7. 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

  8. 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.

  9. Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium

    PubMed Central

    Suzuki, Toshihiko; Kanagawa, Takahiro; Kamagata, Yoichi

    2002-01-01

    Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation. PMID:11772646

  10. The importance of bottlenecks in protein networks: correlation with gene essentiality and expression dynamics.

    PubMed

    Yu, Haiyuan; Kim, Philip M; Sprecher, Emmett; Trifonov, Valery; Gerstein, Mark

    2007-04-20

    It has been a long-standing goal in systems biology to find relations between the topological properties and functional features of protein networks. However, most of the focus in network studies has been on highly connected proteins ("hubs"). As a complementary notion, it is possible to define bottlenecks as proteins with a high betweenness centrality (i.e., network nodes that have many "shortest paths" going through them, analogous to major bridges and tunnels on a highway map). Bottlenecks are, in fact, key connector proteins with surprising functional and dynamic properties. In particular, they are more likely to be essential proteins. In fact, in regulatory and other directed networks, betweenness (i.e., "bottleneck-ness") is a much more significant indicator of essentiality than degree (i.e., "hub-ness"). Furthermore, bottlenecks correspond to the dynamic components of the interaction network-they are significantly less well coexpressed with their neighbors than non-bottlenecks, implying that expression dynamics is wired into the network topology. PMID:17447836