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Sample records for essential gene products

  1. Genes essential for morphological development and antibiotic production in Streptomyces coelicolor are targets of BldD during vegetative growth.

    PubMed

    den Hengst, Chris D; Tran, Ngat T; Bibb, Maureen J; Chandra, Govind; Leskiw, Brenda K; Buttner, Mark J

    2010-10-01

    BldD is a transcriptional regulator essential for morphological development and antibiotic production in Streptomyces coelicolor. Here we identify the BldD regulon by means of chromatin immunoprecipitation-microarray analysis (ChIP-chip). The BldD regulon encompasses ~167 transcriptional units, of which more than 20 are known to play important roles in development (e.g. bldA, bldC, bldH/adpA, bldM, bldN, ssgA, ssgB, ftsZ, whiB, whiG, smeA-ssfA) and/or secondary metabolism (e.g. nsdA, cvn9, bldA, bldC, leuA). Strikingly, 42 BldD target genes (~25% of the regulon) encode regulatory proteins, stressing the central, pleiotropic role of BldD. Almost all BldD binding sites identified by ChIP-chip are present in the promoters of the target genes. An exception is the tRNA gene bldA, where BldD binds within the region encoding the primary transcript, immediately downstream of the position corresponding to the processed, mature 3 end of the tRNA. Through gene overexpression, we identified a novel BldD target gene (cdgA) that influences differentiation and antibiotic production. cdgA encodes a GGDEF domain protein, implicating c-di-GMP in the regulation of Streptomyces development. Sequence analysis of the upstream regions of the complete regulon identified a 15 bp inverted repeat that functions as a high-affinity binding site for BldD, as was shown by electrophoretic mobility shift assays and DNase I footprinting analysis. High-scoring copies of the BldD binding site were found at relevant positions in the genomes of other bacteria containing a BldD homologue, suggesting the role of BldD is conserved in sporulating actinomycetes. PMID:20979333

  2. 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.

  3. Essential Genes Are More Evolutionarily Conserved Than Are Nonessential Genes

    E-print Network

    Jordan, King

    Essential Genes Are More Evolutionarily Conserved Than Are Nonessential Genes in Bacteria I. King The "knockout-rate" prediction holds that essential genes should be more evolutionarily conserved than are nonessential genes. This is because negative (purifying) selection acting on essential genes is expected

  4. 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

  5. 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 (?pesL) or pes1 (?pes1) 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 ?pesL 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

  6. Comparing the evolutionary conservation between human essential genes, human orthologs of mouse essential genes and human housekeeping genes.

    PubMed

    Lv, Wenhua; Zheng, Jiajia; Luan, Meiwei; Shi, Miao; Zhu, Hongjie; Zhang, Mingming; Lv, Hongchao; Shang, Zhenwei; Duan, Lian; Zhang, Ruijie; Jiang, Yongshuai

    2015-11-01

    Human housekeeping genes are often confused with essential human genes, and several studies regard both types of genes as having the same level of evolutionary conservation. However, this is not necessarily the case. To clarify this, we compared the differences between human housekeeping genes and essential human genes with respect to four aspects: the evolutionary rate (dN/dS), protein sequence identity, single-nucleotide polymorphism (SNP) density and level of linkage disequilibrium (LD). The results showed that housekeeping genes had lower evolutionary rates, higher sequence identities, lower SNP densities and higher levels of LD compared with essential genes. Together, these findings indicate that housekeeping and essential genes are two distinct types of genes, and that housekeeping genes have a higher level of evolutionary conservation. Therefore, we suggest that researchers should pay careful attention to the distinctions between housekeeping genes and essential genes. Moreover, it is still controversial whether we should substitute human orthologs of mouse essential genes for human essential genes. Therefore, we compared the evolutionary features between human orthologs of mouse essential genes and human housekeeping genes and we got inconsistent results in long-term and short-term evolutionary characteristics implying the irrationality of simply replacing human essential genes with human orthologs of mouse essential genes. PMID:25911641

  7. 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

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

    SciTech Connect

    McCarthy, Christina B.; Theilmann, David A.

    2008-05-25

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

  9. 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

  10. 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.

  11. 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

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

    PubMed Central

    Wang, Tim; Birsoy, K?vanç; 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

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

    PubMed

    Wang, Tim; Birsoy, K?vanç; 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

  14. Essential genes as antimicrobial targets and cornerstones of synthetic

    E-print Network

    Church, George M.

    Essential genes as antimicrobial targets and cornerstones of synthetic biology Mario Juhas1 , Leo Pasteur, Boston, MA 02115, USA Essential genes are absolutely required for the survival of any living entity. Investigation of essential genes is therefore expected to advance tremendously our under

  15. 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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    da Silva, Joăo 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.

  18. The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability

    PubMed Central

    1995-01-01

    Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins. PMID:7744953

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

    SciTech Connect

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

    2013-01-20

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

  20. Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici.

    PubMed

    Mavingui, P; Laeremans, T; Flores, M; Romero, D; Martínez-Romero, E; Palacios, R

    1998-06-01

    Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym. PMID:9603874

  1. Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

    PubMed Central

    Mavingui, Patrick; Laeremans, Toon; Flores, Margarita; Romero, David; Martínez-Romero, Esperanza; Palacios, Rafael

    1998-01-01

    Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym. PMID:9603874

  2. Why There Are No Essential Genes on Plasmids.

    PubMed

    Tazzyman, Samuel J; Bonhoeffer, Sebastian

    2015-12-01

    Mobile genetic elements such as plasmids are important for the evolution of prokaryotes. It has been suggested that there are differences between functions coded for by mobile genes and those in the "core" genome and that these differences can be seen between plasmids and chromosomes. In particular, it has been suggested that essential genes, such as those involved in the formation of structural proteins or in basic metabolic functions, are rarely located on plasmids. We model competition between genotypically varying bacteria within a single population to investigate whether selection favors a chromosomal location for essential genes. We find that in general, chromosomal locations for essential genes are indeed favored. This is because the inheritance of chromosomes is more stable than that for plasmids. We define the "degradation" rate as the rate at which chance genetic processes, for example, mutation, deletion, or translocation, render essential genes nonfunctioning. The only way in which plasmids can be a location for functioning essential genes is if chromosomal genes degrade faster than plasmid genes. If the two degradation rates are equal, or if plasmid genes degrade faster than chromosomal genes, functioning essential genes will be found only on chromosomes. PMID:25540453

  3. 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

  4. The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function.

    PubMed Central

    Zhu, L A; Weller, S K

    1992-01-01

    The UL5 protein of herpes simplex virus type 1, one component of the viral helicase-primase complex, contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases. Although this superfamily contains more than 20 members ranging from bacteria to mammalian cells and their viruses, the importance of these motifs has not been addressed experimentally for any one of them. In this study, we have examined the functional significance of these six motifs for the UL5 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. A transient replication complementation assay was used to test the effect of each mutation on the function of the UL5 protein in viral DNA replication. In this assay, a mutant UL5 protein expressed from an expression clone is used to complement a replication-deficient null mutant with a mutation in the UL5 gene for the amplification of herpes simplex virus origin-containing plasmids. Eight mutations in conserved regions and three similar mutations in nonconserved regions of the UL5 gene were analyzed, and the results indicate that all six conserved motifs are essential to the function of UL5 protein in viral DNA replication; on the other hand, mutations in nonconserved regions are tolerated. These data provide the first direct evidence for the importance of these conserved regions in any member of the superfamily of DNA and RNA helicases. In addition, three motif mutations were introduced into the viral genome, and the phenotypic analyses of these mutants are consistent with results from the transient replication complementation assay. The ability of these three mutant UL5 proteins to form specific interactions with other members of the helicase-primase complex, UL8 and UL52, indicates that the functional domains required for replication activity of UL5 are separable from domains responsible for protein-protein interactions. It is anticipated that this type of structure-function analysis will lead to the identification of protein domains that contribute not only to the enzymatic activities of helicase or primase but also to protein-protein interactions within members of the complex. Images PMID:1309257

  5. 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.

  6. Predicting bacterial essential genes using only sequence composition information.

    PubMed

    Ning, L W; Lin, H; Ding, H; Huang, J; Rao, N; Guo, F B

    2014-01-01

    Essential genes are those genes that are needed by organisms at any time and under any conditions. It is very important for us to identify essential genes from bacterial genomes because of their vital role in synthetic biology and biomedical practices. In this paper, we developed a support vector machine (SVM)-based method to predict essential genes of bacterial genomes using only compositional features. These features are all derived from the primary sequences, i.e., nucleotide sequences and protein sequences. After training on the multiple samplings of the labeled (essential or not essential) features using a library for SVM, we obtained an average area under the ROC curve (AUC) of about 0.82 in a 5-fold cross-validation for Escherichia coli and about 0.74 for Mycoplasma pulmonis. We further evaluated the performance of the method proposed using the dataset consisting of 16 bacterial genomes, and an average AUC of 0.76 was achieved. Based on this training dataset, a model for essential gene prediction was established. Another two independent genomes, Shewanella oneidensis RW1 and Salmonella enterica serovar Typhimurium SL1344 were used to evalutate the model. Results showed that the AUC sores were 0.77 and 0.81, respectively. For the convenience of the vast majority of experimental scientists, a web server has been constructed, which is freely available at http://cefg.uestc.edu.cn:9999/egp. PMID:25036505

  7. Hox11 paralogous genes are essential for metanephric kidney induction

    E-print Network

    Capecchi, Mario R.

    Hox11 paralogous genes are essential for metanephric kidney induction Deneen M. Wellik, Patrick J 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

  8. 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

  9. Functional Analysis of the Molecular Interactions of TATA Box-Containing Genes and Essential Genes

    PubMed Central

    Moon, Jisook

    2015-01-01

    Genes can be divided into TATA-containing genes and TATA-less genes according to the presence of TATA box elements at promoter regions. TATA-containing genes tend to be stress-responsive, whereas many TATA-less genes are known to be related to cell growth or “housekeeping” functions. In a previous study, we demonstrated that there are striking differences among four gene sets defined by the presence of TATA box (TATA-containing) and essentiality (TATA-less) with respect to number of associated transcription factors, amino acid usage, and functional annotation. Extending this research in yeast, we identified KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways that are statistically enriched in TATA-containing or TATA-less genes and evaluated the possibility that the enriched pathways are related to stress or growth as reflected by the individual functions of the genes involved. According to their enrichment for either of these two gene sets, we sorted KEGG pathways into TATA-containing-gene-enriched pathways (TEPs) and essential-gene-enriched pathways (EEPs). As expected, genes in TEPs and EEPs exhibited opposite results in terms of functional category, transcriptional regulation, codon adaptation index, and network properties, suggesting the possibility that the bipolar patterns in these pathways also contribute to the regulation of the stress response and to cell survival. Our findings provide the novel insight that significant enrichment of TATA-binding or TATA-less genes defines pathways as stress-responsive or growth-related. PMID:25789484

  10. 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

  11. 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 ? 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. 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

  15. Impacts of Gene Essentiality, Expression Pattern, and Gene Compactness on the Evolutionary Rate of Mammalian Proteins

    E-print Network

    Zhang, Jianzhi

    of mammalian protein evolution and to compare the rela- tive importance of these factors in yeasts and mammals Understanding the determinants of the rate of protein sequence evolution is of fundamental importance and a minor role of gene essentiality in determining the rate of protein sequence evolution. Whether

  16. Computational prediction of essential metabolic genes using constraint-based approaches.

    PubMed

    Basler, Georg

    2015-01-01

    In this chapter, we describe the application of constraint-based modeling to predict the impact of gene deletions on a metabolic phenotype. The metabolic reactions taking place inside cells form large networks, which have been reconstructed at a genome-scale for several organisms at increasing levels of detail. By integrating mathematical modeling techniques with biochemical principles, constraint-based approaches enable predictions of metabolite fluxes and growth under specific environmental conditions or for genetically modified microorganisms. Similar to the experimental knockout of a gene, predicting the essentiality of a metabolic gene for a phenotype further allows to generate hypotheses on its biological function and design of genetic engineering strategies for biotechnological applications. Here, we summarize the principles of constraint-based approaches and provide a detailed description of the procedure to predict the essentiality of metabolic genes with respect to a specific metabolic function. We exemplify the approach by predicting the essentiality of reactions in the citric acid cycle for the production of glucose from fatty acids. PMID:25636620

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

    PubMed Central

    Mihálik, Daniel; Kl?ová, Lenka; Ondrei?ková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; ?ertík, 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 ?-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 ?-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 ?-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

  18. Evolutionary conservation analysis between the essential and nonessential genes in bacterial genomes

    PubMed Central

    Luo, Hao; Gao, Feng; Lin, Yan

    2015-01-01

    Essential genes are thought to be critical for the survival of the organisms under certain circumstances, and the natural selection acting on essential genes is expected to be stricter than on nonessential ones. Up to now, essential genes have been identified in approximately thirty bacterial organisms by experimental methods. In this paper, we performed a comprehensive comparison between the essential and nonessential genes in the genomes of 23 bacterial species based on the Ka/Ks ratio, and found that essential genes are more evolutionarily conserved than nonessential genes in most of the bacteria examined. Furthermore, we also analyzed the conservation by functional clusters with the clusters of orthologous groups (COGs), and found that the essential genes in the functional categories of G (Carbohydrate transport and metabolism), H (Coenzyme transport and metabolism), I (Transcription), J (Translation, ribosomal structure and biogenesis), K (Lipid transport and metabolism) and L (Replication, recombination and repair) tend to be more evolutionarily conserved than the corresponding nonessential genes in bacteria. The results suggest that the essential genes in these subcategories are subject to stronger selective pressure than the nonessential genes, and therefore, provide more insights of the evolutionary conservation for the essential and nonessential genes in complex biological processes. PMID:26272053

  19. 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

  20. The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity.

    PubMed Central

    Doublet, P; van Heijenoort, J; Bohin, J P; Mengin-Lecreulx, D

    1993-01-01

    The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity. Images PMID:8098327

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

    PubMed Central

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

    2014-01-01

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

  2. 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

  3. 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

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

    PubMed Central

    Albuquerque, Joăo 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

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

    PubMed

    Albuquerque, Joăo 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

  6. An integrated machine-learning model to predict prokaryotic essential genes.

    PubMed

    Deng, Jingyuan

    2015-01-01

    Essential genes are indispensable for the target organism's survival. Large-scale identification and characterization of essential genes has shown to be beneficial in both fundamental biology and medicine fields. Current existing genome-scale experimental screenings of essential genes are time consuming and costly, also sometimes confer erroneous essential gene annotations. To circumvent these difficulties, many research groups turn to computational approaches as the alternative to identify essential genes. Here, we developed an integrative machine-learning based statistical framework to accurately predict essential genes in microorganisms. First we extracted a variety of relevant features derived from different aspects of an organism's genomic sequences. Then we selected a subset of features have high predictive power of gene essentiality through a carefully designed feature selection system. Using the selected features as input, we constructed an ensemble classifier and trained the model on a well-studied microorganism. After fine-tuning the model parameters in cross-validation, we tested the model on the other microorganism. We found that the tenfold cross-validation results within the same organism achieves a high predictive accuracy (AUC ~0.9), and cross-organism predictions between distant related organisms yield the AUC scores from 0.69 to 0.89, which significantly outperformed homology mapping. PMID:25636617

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

    PubMed Central

    2013-01-01

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

  8. 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 ?-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

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

    PubMed

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

    2015-08-15

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

  10. Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene

    E-print Network

    Zhang, Jianzhi

    Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene or essential residues is not always apparent via sequence alignments because these are limited by the depth of specific amino acid residues of proteins of known or unknown function. Here we describe an approach called

  11. 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

  12. 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

  13. Mining Association Rules in Analysis of Transcription Factors Essential to Gene Expressions

    E-print Network

    Gruenwald, Le

    Mining Association Rules in Analysis of Transcription Factors Essential to Gene Expressions Ruzhu classified into different families. This project reported the results and analysis of mining association and genes, designed and implemented an efficient algorithm of mining association rules for the analysis

  14. 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

  15. A statistical framework for improving genomic annotations of transposon mutagenesis (TM) assigned essential genes.

    PubMed

    Deng, Jingyuan

    2015-01-01

    Whole-genome transposon mutagenesis (TM) experiment followed by sequence-based identification of insertion sites is the most popular genome-wise experiment to identify essential genes in Prokaryota. However, due to the limitation of high-throughput technique, this approach yields substantial systematic biases resulting in the incorrect assignments of many essential genes. To obtain unbiased and accurate annotations of essential genes from TM experiments, we developed a novel Poisson model based statistical framework to refine these TM assignments. In the model, first we identified and incorporated several potential factors such as gene length and TM insertion information which may cause the TM assignment biases into the basic Poisson model. Then we calculated the conditional probability of an essential gene given the observed TM insertion number. By factorizing this probability through introducing a latent variable the real insertion number, we formalized the statistical framework. Through iteratively updating and optimizing model parameters to maximize the goodness-of-fit of the model to the observed TM insertion data, we finalized the model. Using this model, we are able to assign the probability score of essentiality to each individual gene given its TM assignment, which subsequently correct the experimental biases. To enable our model widely useable, we established a user-friendly Web-server that is accessible to the public: http://research.cchmc.org/essentialgene/. PMID:25636618

  16. 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

  17. 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

  18. 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

  19. 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

  20. The yield of essential oils in Melaleuca alternifolia (Myrtaceae) is regulated through transcript abundance of genes in the MEP pathway.

    PubMed

    Webb, Hamish; Lanfear, Robert; Hamill, John; Foley, William J; Külheim, Carsten

    2013-01-01

    Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway. PMID:23544156

  1. 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

  2. Identifying essential genes in bacterial metabolic networks with machine learning methods

    PubMed Central

    2010-01-01

    Background Identifying essential genes in bacteria supports to identify potential drug targets and an understanding of minimal requirements for a synthetic cell. However, experimentally assaying the essentiality of their coding genes is resource intensive and not feasible for all bacterial organisms, in particular if they are infective. Results We developed a machine learning technique to identify essential genes using the experimental data of genome-wide knock-out screens from one bacterial organism to infer essential genes of another related bacterial organism. We used a broad variety of topological features, sequence characteristics and co-expression properties potentially associated with essentiality, such as flux deviations, centrality, codon frequencies of the sequences, co-regulation and phyletic retention. An organism-wise cross-validation on bacterial species yielded reliable results with good accuracies (area under the receiver-operator-curve of 75% - 81%). Finally, it was applied to drug target predictions for Salmonella typhimurium. We compared our predictions to the viability of experimental knock-outs of S. typhimurium and identified 35 enzymes, which are highly relevant to be considered as potential drug targets. Specifically, we detected promising drug targets in the non-mevalonate pathway. Conclusions Using elaborated features characterizing network topology, sequence information and microarray data enables to predict essential genes from a bacterial reference organism to a related query organism without any knowledge about the essentiality of genes of the query organism. In general, such a method is beneficial for inferring drug targets when experimental data about genome-wide knockout screens is not available for the investigated organism. PMID:20438628

  3. A Survey of Essential Gene Function in the Yeast Cell Division Cycle

    PubMed Central

    Yu, Lisa; Castillo, Lourdes Peńa; Mnaimneh, Sanie

    2006-01-01

    Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO7 promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication. PMID:16943325

  4. Genome-wide characterization of essential, toxicity-modulating and no-phenotype genes in S. cerevisiae.

    PubMed

    Yang, Lei; Hao, Dapeng; Lv, Yingli; Zuo, Yongchun; Jiang, Wei

    2015-03-15

    Based on the requirements for an organism's viability, genes can be classified into essential genes and non-essential genes. Non-essential genes can be further classified into toxicity-modulating genes and no-phenotype genes based on the fitness phenotype of yeast cells when the gene is deleted under DNA-damaging conditions. In this study, graph theoretical approaches were used to characterize essential, toxicity-modulating and no-phenotype genes for S. cerevisiae in the physical interaction (PI) network and the perturbation sensitivity (PS) network. We also gained previously published biological datasets to gain a more complete understanding of the differences and relationships between essential, toxicity-modulating genes and no-phenotype genes. The analysis results indicate that toxicity-modulating genes have similar properties as essential genes, and toxicity-modulating genes might represent a middle ground between essential genes and no-phenotype genes, suggesting that cells initiate highly coordinated responses to damage that are similar to those needed for vital cellular functions. These findings may elucidate the mechanisms for understanding toxicity-modulating processes relevant to certain diseases. PMID:25576218

  5. 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

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

    PubMed Central

    Ianiri, Giuseppe

    2015-01-01

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

  7. Genes of the N-Methylglutamate Pathway Are Essential for Growth of Methylobacterium extorquens DM4 with Monomethylamine

    PubMed Central

    Gruffaz, Christelle; Muller, Emilie E. L.; Louhichi-Jelail, Yousra; Nelli, Yella R.; Guichard, Gilles

    2014-01-01

    Monomethylamine (MMA, CH3NH2) can be used as a carbon and nitrogen source by many methylotrophic bacteria. Methylobacterium extorquens DM4 lacks the MMA dehydrogenase encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by the N-methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genes gmaS, mgsABC, and mgdABCD for the three enzymes of the pathway, ?-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmaS and mgdABCD and their induction during growth with MMA. The genes mgdABCD and mgsABC were found to be essential for utilization of MMA as a carbon and nitrogen source. The gene gmaS was essential for MMA utilization as a carbon source, but residual growth of mutant DM4gmaS growing with succinate and MMA as a nitrogen source was observed. Plasmid copies of gmaS and the gmaS homolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4gmaS and DM4gmaS?metdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4gmaS and DM4gmaS?metdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source. PMID:24682302

  8. 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

  9. Predicting the Proportion of Essential Genes in Mouse Duplicates Based on Biased Mouse Knockout Genes

    E-print Network

    Gu, Xun

    (paralogous) gene has been thought to be an important factor in the genetic robustness (Conant and Wagner 2004 single-copy genes in both the yeast and the nematode (Gu et al. 2003; Conant and Wagner 2004; Kamath et

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

    Houël, Emeline; Gonzalez, German; Bessičre, 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

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

    PubMed

    Houël, Emeline; Gonzalez, German; Bessičre, 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

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

    PubMed Central

    Putiri, Emily; Pelegri, Francisco

    2011-01-01

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

  14. Functional Study of Genes Essential for Autogamy and Nuclear Reorganization in Paramecium?§

    PubMed Central

    Nowak, Jacek K.; Gromadka, Robert; Juszczuk, Marek; Jerka-Dziadosz, Maria; Maliszewska, Kamila; Mucchielli, Marie-Hélčne; Gout, Jean-François; Arnaiz, Olivier; Agier, Nicolas; Tang, Thomas; Aggerbeck, Lawrence P.; Cohen, Jean; Delacroix, Hervé; Sperling, Linda; Herbert, Christopher J.; Zagulski, Marek; Bétermier, Mireille

    2011-01-01

    Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis. PMID:21257794

  15. 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

  16. Predicting essential genes and synthetic lethality via influence propagation in signaling pathways of cancer cell fates.

    PubMed

    Zhang, Fan; Wu, Min; Li, Xue-Juan; Li, Xiao-Li; Kwoh, Chee Keong; Zheng, Jie

    2015-06-01

    A major goal of personalized anti-cancer therapy is to increase the drug effects while reducing the side effects as much as possible. A novel therapeutic strategy called synthetic lethality (SL) provides a great opportunity to achieve this goal. SL arises if mutations of both genes lead to cell death while mutation of either single gene does not. Hence, the SL partner of a gene mutated only in cancer cells could be a promising drug target, and the identification of SL pairs of genes is of great significance in pharmaceutical industry. In this paper, we propose a hybridized method to predict SL pairs of genes. We combine a data-driven model with knowledge of signalling pathways to simulate the influence of single gene knock-down and double genes knock-down to cell death. A pair of genes is considered as an SL candidate when double knock-down increases the probability of cell death significantly, but single knock-down does not. The single gene knock-down is confirmed according to the human essential genes database. Our validation against literatures shows that the predicted SL candidates agree well with wet-lab experiments. A few novel reliable SL candidates are also predicted by our model. PMID:25669329

  17. Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation{

    E-print Network

    Palczewski, Krzysztof

    in outer hair cells (OHCs) than inner hair cells. Clrn12/2 mice showed early onset hearing loss of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cellUsher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural

  18. 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

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

    PubMed

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

    2015-01-01

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

  20. 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. Evolutionary conservation of essential and highly expressed genes in Pseudomonas aeruginosa

    PubMed Central

    2010-01-01

    Background The constant increase in development and spread of bacterial resistance to antibiotics poses a serious threat to human health. New sequencing technologies are now on the horizon that will yield massive increases in our capacity for DNA sequencing and will revolutionize the drug discovery process. Since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus. Results In this study we demonstrate that pooled sequencing is applicable for the analysis of sequence variations of strain collections with more than 10 individual isolates. Pooled sequencing of 36 clinical Pseudomonas aeruginosa isolates revealed that essential and highly expressed proteins evolve at lower rates, whereas extracellular proteins evolve at higher rates. We furthermore refined the list of experimentally essential P. aeruginosa genes, and identified 980 genes that show no sequence variation at all. Among the conserved nonessential genes we found several that are involved in regulation, motility and virulence, indicating that they represent factors of evolutionary importance for the lifestyle of a successful environmental bacterium and opportunistic pathogen. Conclusion The detailed analysis of a comprehensive set of P. aeruginosa genomes in this study clearly disclosed detailed information of the genomic makeup and revealed a large set of highly conserved genes that play an important role for the lifestyle of this microorganism. Sequencing strain collections enables for a detailed and extensive identification of sequence variations as potential bacterial adaptation processes, e.g., during the development of antibiotic resistance in the clinical setting and thus may be the basis to uncover putative targets for novel treatment strategies. PMID:20380691

  2. 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

  3. 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.

  4. 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 conditions of higher bicarbonate demand. PMID:12937954

  5. Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain.

    PubMed

    Lopez, Gabriel; Anderson, J Christopher

    2015-12-18

    Synthetic auxotrophs are organisms engineered to require the presence of a particular molecule for viability. They have potential applications in biocontainment and enzyme engineering. We show that these organisms can be generated by engineering ligand-dependence into essential genes. We demonstrate a method for generating a Synthetic auxotroph based on a Ligand-Dependent Essential gene (SLiDE) using 5 essential genes as test cases: pheS, dnaN, tyrS, metG, and adk. We show that a single SLiDE strain can have a 1 × 10(8)-fold increase in viability when chemically complemented with the ligand benzothiazole. The optimized SLiDE strain engineering protocol required less than 1 week and $100 USD. We combined multiple SLiDE strain alleles into the industrial Escherichia coli strain BL21(DE3), yielding an organism that exceeds the biosafety criteria with an escape frequency below the limit of detection of 3 × 10(-11). PMID:26072987

  6. Dependence Relationships between Gene Ontology Terms based TIGR Gene Product Annotations

    E-print Network

    Borgelt, Christian

    , biological processes annotation of genes and gene products. These constitute three separate ontologiesDependence Relationships between Gene Ontology Terms based TIGR Gene Product Annotations Anand.de/~borgelt/ Abstract Gene Ontology important tool representation and processing information about gene products

  7. Membrane-bound alcohol dehydrogenase is essential for glyceric acid production in Acetobacter tropicalis.

    PubMed

    Habe, Hiroshi; Sato, Shun; Fukuoka, Tokuma; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2011-01-01

    Acetobacter tropicalis NBRC16470 can produce highly enantiomerically pure D-glyceric acid (D-GA; >99 % enantiomeric excess) from glycerol. To investigate whether membrane-bound alcohol dehydrogenase (mADH) is involved in GA production in A. tropicalis, we amplified part of the gene encoding mADH subunit I (adhA) using polymerase chain reaction and constructed an adhA-disrupted mutant of A. tropicalis (?adhA). Because ?adhA did not produce GA, we confirmed that mADH is essential for the conversion of glycerol to GA. We also cloned and sequenced the entire region corresponding to adhA and adhB, which encodes mADH subunit II. The sequences showed high identities (84-86 %) with the equivalent mADH subunits from other Acetobacter spp. PMID:21852749

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

    PubMed Central

    2014-01-01

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

  9. 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 ? 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

  10. Rapid evolution of RNA editing sites in a small non-essential plastid gene

    PubMed Central

    Fiebig, Andreas; Stegemann, Sandra; Bock, Ralph

    2004-01-01

    Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b6f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this ‘relative neutrality hypothesis for the evolution of plastid editing sites’ is discussed. PMID:15240834

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  13. Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa.

    PubMed

    Morita, Yuji; Narita, Shin-ichiro; Tomida, Junko; Tokuda, Hajime; Kawamura, Yoshiaki

    2010-09-01

    The Phi CTX-based integration vector pYM101 harboring a tightly controlled modified phage T7 early gene promoter/LacI(q) repressor (T7/LacI) system was constructed for the generation of unmarked conditional mutants in Pseudomonas aeruginosa. Promoter activity of the T7/LacI system was demonstrated to be dependent on the presence of the inducer isopropyl -beta-D-1-thiogalactopyranoside (IPTG), as evaluated by measuring beta-galactosidase activity. In the absence of the inducer, the promoter was silent as its activity was lower than those of a promoter-less lacZ control. Unmarked conditional mutants of four predicted essential genes (lolCDE (PA2988-86), lpxC (PA4406), rho (PA5239), and def (PA0019)) were successfully constructed using this recombination system. In the absence of IPTG, the growth of all mutants was repressed; however, the addition of either 0.1 or 1mM IPTG restored growth rates to levels nearly identical to wild-type cells. It was therefore demonstrated that the inducible integration vector pYM101 is suitable for the creation of unmarked conditional mutants of P. aeruginosa, and is particularly useful for examining the function of essential genes. PMID:20538017

  14. 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

  15. Haem uptake is essential for egg production in the haematophagous blood fluke of humans, Schistosoma mansoni.

    PubMed

    Toh, Shu Qin; Gobert, Geoffrey N; Malagón Martínez, David; Jones, Malcolm K

    2015-09-01

    Schistosomes ingest host erythrocytes, liberating large quantities of haem. Despite its toxicity, haem is an essential factor for numerous biological reactions, and may be an important iron source for these helminths. We used a fluorescence haem analogue, palladium mesoporphyrin, to investigate pathways of haem acquisition, and showed that palladium mesoporphyrin accumulates in the vitellaria (eggshell precursor glands) and ovary of female Schistosoma mansoni. Furthermore, incubation of adult females in 10-100 ?m cyclosporin A (IC50 = 2.3 ?m) inhibits the uptake of palladium mesoporphyrin to these tissues, with tenfold reductions in fluorescence intensity of the ovary. In vitro exposure to cyclosporin A resulted in significant perturbation of egg production, reducing egg output from 34 eggs per female to 5.7 eggs per female over the incubation period, and retardation of egg development. We characterized a S. mansoni homologue of the haem-responsive genes of Caenorhabditis elegans. The gene (Smhrg-1) encodes a protein with a molecular weight of approximately 17 kDa. SmHRG-1 was able to rescue growth in haem transport-deficient HEM1? yeast. Transcriptional suppression of Smhrg-1 in adult S. mansoni worms resulted in significant delay in egg maturation, with 47% of eggs from transcriptionally suppressed worms being identified as immature compared with only 27% of eggs laid by control worms treated with firefly luciferase. Our findings indicate the presence of transmembrane haem transporters in schistosomes, with a high abundance of these molecules being present in tissues involved in oogenesis. PMID:26153121

  16. 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

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

    PubMed Central

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

    2012-01-01

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

  18. The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans.

    PubMed

    Hao, Limin; Aspöck, Gudrun; Bürglin, Thomas R

    2006-02-15

    The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity. PMID:16413526

  19. Functional Domains of ZFP809 Essential for Nuclear Localization and Gene Silencing

    PubMed Central

    Ichida, Yu; Utsunomiya, Yuko; Yasuda, Toru; Nakabayashi, Kazuhiko; Sato, Toshinori; Onodera, Masafumi

    2015-01-01

    Zinc finger protein 809 (ZFP809) is a member of the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family, and is highly expressed in mouse immature cells. ZFP809 is known to inhibit the expression of transduced genes driven by Moloney murine leukemia virus (MoMLV)-typed retroviral vectors by binding to the primer binding site (PBS) located downstream of the MLV-long terminal repeat (LTR) of the vectors and recruiting protein complexes that introduce epigenetic silencing marks such as histone modifications and DNA methylation at the MLV-LTR. However, it remains undetermined what domains of ZFP809 among the KRAB domain at N-terminus and the seven zinc fingers are critical for gene silencing. In this study, we assessed subcellular localization, gene silencing ability, and binding ability to the PBS of a series of truncated and mutated ZFP809 proteins. We revealed the essential role of the KRAB A box for all functions assessed, together with the accessory roles of a subset of zinc fingers. Our data also suggest that interaction between KAP1 and the KRAB A box of ZFP809 is critical in KAP1-dependent control of gene silencing for ZFP809 targets. PMID:26417948

  20. 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.

  1. Identification of an essential gene responsible for D-Asp incorporation in the Lactococcus lactis peptidoglycan crossbridge.

    PubMed

    Veiga, Patrick; Piquet, Sandra; Maisons, Aurélie; Furlan, Sylviane; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2006-12-01

    Bacteria such as Lactococcus lactis have D-aspartate (D-Asp) or its amidated derivative D-asparagine (D-Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing D-amino acids in their PG crossbridge, but absent from those that instead insert L-amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate L-Ser-L-Ala or L-Ala-L-Ala in the PG crossbridge. Our results show that (i) yxbA encodes D-Asp ligase responsible for incorporation of D-Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of D-Asp may be complemented by L-Ser-L-Ala or L-Ala-L-Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having L-amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG sidechain composition. PMID:17083466

  2. 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

  3. Structure and Function of Fusion Gene Products in

    E-print Network

    Spang, Rainer

    Structure and Function of Fusion Gene Products in Childhood Acute Leukemia #12;Chromosomal;Transcriptional Activation RNA Polymerase II Gene Transcription Histon Acetylation Complex Chromatin Remodeling

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

    PubMed

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

    2015-02-10

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

  5. Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

    PubMed Central

    Tao, Xue Ying; Choi, Jae Young; Kim, Woo Jin; An, Saes Byeol; Liu, Qin; Kim, Song Eun; Lee, Seok Hee; Kim, Jong Hoon; Woo, Soo Dong; Jin, Byung Rae

    2014-01-01

    ABSTRACT ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5? rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. IMPORTANCE Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles. PMID:25320313

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

    PubMed Central

    2013-01-01

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

  7. Amplification-control element ACE-3 is important but not essential for autosomal chorion gene amplification.

    PubMed Central

    Swimmer, C; Delidakis, C; Kafatos, F C

    1989-01-01

    We have further characterized the cis-acting elements that control the amplification of the third chromosomal cluster of chorion genes in Drosophila melanogaster; these include the amplification-control element ACE-3 and four amplification-enhancing regions (AER-a to -d). We have used two types of deletions in the chorion cluster: the first was in vitro generated deletions of the ACE-3 region that were subsequently introduced into the germ line, and the second was deletions induced in vivo within a transposon at a preexisting chromosomal location, thus avoiding the complication of position effects. Some of the lines bearing deletions of either type showed amplification, albeit at drastically reduced levels. These unexpected results indicate that, despite its importance, ACE-3 is not essential for low-level amplification and that cis-acting amplification elements are functionally redundant within the autosomal chorion replicon. Images PMID:2554333

  8. Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression

    PubMed Central

    Levay, Konstantin; Slepak, Vladlen Z.

    2007-01-01

    We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation. PMID:17717601

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

    SciTech Connect

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

    1988-03-01

    Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36/sup 0/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 identify 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. cerevisia 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. cerevisia in vitro splicing extract, confirming that RNA8 plays an essential role in splicing.

  10. Transcriptional regulation of genes related to progesterone production [Review].

    PubMed

    Mizutani, Tetsuya; Ishikane, Shin; Kawabe, Shinya; Umezawa, Akihiro; Miyamoto, Kaoru

    2015-09-30

    Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3?-hydroxysteroid dehydrogenase/?(5)-?(4) isomerase (3?-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert ?(5)-?(4) isomerization activity to produce progesterone in a cooperative fashion with 3?-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-? coactivator-1? (PGC-1?), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production. PMID:26135521

  11. siRNAs with decreased off-target effect facilitate the identification of essential genes in cancer cells

    PubMed Central

    Liu, Wensheng; Wang, Di; Dong, Encheng; Wang, Yu; Wu, Chung-I; Lu, Xuemei

    2015-01-01

    Since the essential genes are crucial to the proliferation and survival of cancer cells, the interference of these genes is promising to be an option for cancer therapy to overcome heterogeneity. However, the essential genes are highly overestimated by RNA interference (RNAi) screenings, which is mainly caused by the pervasive off-target effect of small interference RNA (siRNA) and short hairpin RNA (shRNA). In the present study, we designed Match-Mismatch paired siRNAs to discriminate the on-target effect from off-target effect of siRNAs on cell viability. Only one of the 7 potential essential genes was validated as essential to cell viability, which demonstrates the high false positive rate in RNAi screenings. We modified the siRNA by introducing random nucleotides (N) into the guide strand to mitigate the off-target effect, without significantly compromising the on-target effect. The whole transcriptome profile analysis of cells transfected with siRNAs with or without Nindicates that siRNA-dN (with Ns on both the 2nd and the 18th bases of the guide strand) weakens the off-target effect by decreasing the unintended targets. The optimized siRNAs can be applied in the characterization of essential genes in cancer cells. PMID:26057633

  12. Essential Roles for Caenorhabditis elegans Lamin Gene in Nuclear Organization, Cell Cycle Progression, and Spatial Organization of Nuclear Pore Complexes

    PubMed Central

    Liu, Jun; Ben-Shahar, Tom Rolef; Riemer, Dieter; Treinin, Millet; Spann, Perah; Weber, Klaus; Fire, Andrew; Gruenbaum, Yosef

    2000-01-01

    Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs. PMID:11071918

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

    PubMed Central

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

    2014-01-01

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

  14. Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

    PubMed Central

    Mitchell, A P; Driscoll, S E; Smith, H E

    1990-01-01

    In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression. Images PMID:2183020

  15. 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

  16. 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

  17. 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

  18. 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

    2015-10-01

    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 ?-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

  19. Microarray Analysis of Differential Gene Expression Profile in Peripheral Blood Cells of Patients with Human Essential Hypertension

    PubMed Central

    Korkor, Melvin T.; Meng, Fan Bo; Xing, Shen Yang; Zhang, Mu Chun; Guo, Jin Rui; Zhu, Xiao Xue; Yang, Ping

    2011-01-01

    The polygenic nature of essential hypertension and its dependence on environmental factors pose a challenge for biomedical research. We hypothesized that the analysis of gene expression profiles from peripheral blood cells would distinguish patients with hypertension from normotensives. In order to test this, total RNA from peripheral blood cells was isolated. RNA was reversed-transcribed and labeled and gene expression analyzed using significance Analysis Microarrays (Stanford University, CA, USA). Briefly, Significance Analysis Microarrays (SAM) thresholding identified 31 up-regulated and 18 down-regulated genes with fold changes of ?2 or?0.5 and q-value ?5 % in expression. Statistically significantly gene ontology (GO) function and biological process differentially expressed in essential hypertension were MHC class II receptor activity and immune response respectively. Biological pathway analysis identified several related pathways which are associated with immune/inflammatory responses. Quantitative Real- Time RT-PCR results were consistent with the microarray results. The levels of C - reactive protein were higher in hypertensive patients than normotensives and inflammation-related genes were increased as well. In conclusion, genes enriched for “immune/inflammatory responses” may be associated with essential hypertension. In addition, there is a correlation between systemic inflammation and hypertension. It is anticipated that these findings may provide accurate and efficient strategies for prevention, diagnosis and control of this disorder. PMID:21369372

  20. 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; Rulličre, 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

  1. 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; Rulličre, 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

  2. 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

  3. Drosophila homologue of the Rothmund-Thomson syndrome gene: essential function in DNA replication during development.

    PubMed

    Wu, Jianhong; Capp, Christopher; Feng, Liping; Hsieh, Tao-shih

    2008-11-01

    Members of the RecQ family play critical roles in maintaining genome integrity. Mutations in human RecQL4 cause a rare genetic disorder, Rothmund-Thomson syndrome. Transgenic mice experiments showed that the RecQ4 null mutant causes embryonic lethality. Although biochemical evidence suggests that the Xenopus RecQ4 is required for the initiation of DNA replication in the oocyte extract, its biological functions during development remain to be elucidated. We present here our results in establishing the use of Drosophila as a model system to probe RecQ4 functions. Immunofluorescence experiments monitoring the cellular distribution of RecQ4 demonstrated that RecQ4 expression peaks during S phase, and RecQ4 is expressed only in tissues active in DNA replication, but not in quiescent cells. We have isolated Drosophila RecQ4 hypomorphic mutants, recq(EP) and recq4(23), which specifically reduce chorion gene amplification of follicle cells by 4-5 fold, resulting in thin and fragile eggshells, and female sterility. Quantitative analysis on amplification defects over a 14-kb domain in chorion gene cluster suggests that RecQ4 may have a specific function at or near the origin of replication. A null allele recq4(19) causes a failure in cell proliferation, decrease in DNA replication, chromosomal fragmentation, and lethality at the stage of first instar larvae. The mosaic analysis indicates that cell clones with homozygous recq4(19) fail to proliferate. These results indicate that RecQ4 is essential for viability and fertility, and is required for most aspects of DNA replication during development. PMID:18755177

  4. 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

  5. The phenotypic patterns of essential hypertension are the key to identifying "high blood pressure" genes.

    PubMed

    Korner, P I

    2010-01-01

    The genes that cause or increase susceptibility to essential hypertension (EH) and related animal models remain unknown. Their identification is unlikely to be realized with current genetic approaches, because of ambiguities in the genotype-phenotype relationships in these polygenic disorders. In turn, the phenotype is not just an aggregate of traits, but needs to be related to specific components of the circulatory control system at different stages of EH. Hence, clues about important genes must come through the phenotype, reversing the order of current approaches. A recent systems analysis has highlighted major differences in circulatory control in the two main syndromes of EH: (1) stress-and-salt-related EH (SSR-EH)--a constrictor hypertension with low blood volume; (2) hypertensive obesity--SSR-EH plus obesity. Each is initiated through sensitization of central synapses linking the cerebral cortex to the hypothalamic defense area. Several mechanisms are probably involved, including cerebellar effects on baroreflexes. The result is a sustained increase in sympathetic neural activity at stimulus levels that have no effect in normal subjects. Subsequent progression of EH is largely through interactions with non-neural mechanisms, including changes in concentration of vascular autacoids (e.g., nitric oxide) and the amplifying effect of structural changes in large resistance vessels. The rising vasoconstriction increases heterogeneity of blood flow, causing rarefaction (decreased microvascular density) and deterioration of vital organs. SSR-EH also increases food intake in response to stress, but only 40% of these individuals develop hypertensive obesity. Their brain ignores the adiposity signals that normally reduce eating. Hyperinsulinemia masks the sympathetic vasoconstriction through its dilator action, raises blood volume, whilst renal nephropathy and other diabetic complications are common. In each syndrome the neural and non-neural determinants of hypertension provide targets for identifying high BP genes. Reading the genome from the phenotype will require new approaches, such as those used in developmental genetics. In addition, transgenic technology may help verify hypotheses and examine whether an observed effect is through single or multiple mechanisms. To obtain answers will require substantial collaborative efforts between physiologists and geneticists. PMID:21208016

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

    PubMed

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

    2015-10-01

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

  7. TSHZ1-dependent gene regulation is essential for olfactory bulb development and olfaction

    PubMed Central

    Ragancokova, Daniela; Rocca, Elena; Oonk, Anne M.M.; Schulz, Herbert; Rohde, Elvira; Bednarsch, Jan; Feenstra, Ilse; Pennings, Ronald J.E.; Wende, Hagen; Garratt, Alistair N.

    2014-01-01

    The olfactory bulb (OB) receives odor information from the olfactory epithelium and relays this to the olfactory cortex. Using a mouse model, we found that development and maturation of OB interneurons depends on the zinc finger homeodomain factor teashirt zinc finger family member 1 (TSHZ1). In mice lacking TSHZ1, neuroblasts exhibited a normal tangential migration to the OB; however, upon arrival to the OB, the neuroblasts were distributed aberrantly within the radial dimension, and many immature neuroblasts failed to exit the rostral migratory stream. Conditional deletion of Tshz1 in mice resulted in OB hypoplasia and severe olfactory deficits. We therefore investigated olfaction in human subjects from families with congenital aural atresia that were heterozygous for TSHZ1 loss-of-function mutations. These individuals displayed hyposmia, which is characterized by impaired odor discrimination and reduced olfactory sensitivity. Microarray analysis, in situ hybridization, and ChIP revealed that TSHZ1 bound to and regulated expression of the gene encoding prokineticin receptor 2 (PROKR2), a G protein–coupled receptor essential for OB development. Mutations in PROKR2 lead to Kallmann syndrome, characterized by anosmia and hypogonadotrophic hypogonadism. Our data indicate that TSHZ1 is a key regulator of mammalian OB development and function and controls the expression of molecules involved in human Kallmann syndrome. PMID:24487590

  8. Vitamin D receptor gene polymorphisms and plasma renin activity in essential hypertensive individuals.

    PubMed

    Cottone, S; Guarino, L; Arsena, R; Scazzone, C; Tornese, F; Guarneri, M; Guglielmo, C; Bono, A; Mulč, G

    2015-08-01

    Several studies analyzed 25-hydroxyvitamin D (25[OH]D) and blood pressure (BP) relationship with mixed results. Moreover, a relationship between the risk of hypertension and vitamin D receptor (VDR) gene polymorphisms, FokI and BsmI, was reported. This study was aimed to analyze these relationships in essential hypertensive (EH) patients. Seventy-one EH patients, 18-75 years old, were enrolled. Patients underwent clinical BP, 24-h ambulatory BP monitoring, 25[OH]D and plasma renin activity (PRA) evaluations. FokI and BsmI VDR polymorphisms were analyzed and compared with those of 72 healthy controls. In EH patients, the median 25[OH]D levels were lower than 30 ng ml(-1). We found a significant negative correlation between 25[OH]D and 24-h systolic BP (r = -0.277, P = 0.043). This correlation persisted in backward stepwise multivariate analyses (? = -0.337; P = 0.022), after adjustment for age, gender, body mass index, glomerular filtration rate, and PRA. We did not observe statistically significant correlation between 25[OH]D and PRA. We compared the allelic frequencies and genotype distribution between patients and controls, and FokI and BsmI VDR polymorphisms were not associated either with hypertensive status or with PRA. Further wide studies are needed to clarify this relationship. PMID:25500899

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

    PubMed Central

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

    2015-01-01

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

  10. The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis.

    PubMed Central

    Komori, M; Rasmussen, S W; Kiel, J A; Baerends, R J; Cregg, J M; van der Klei, I J; Veenhuis, M

    1997-01-01

    We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14-1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome-inducing growth conditions. In cells of both pex14-1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild-type H. polymorpha cells resulted in a peroxisome-deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import. PMID:9009266

  11. Integrating the genetic and physical maps of Arabidopsis thaliana: identification of mapped alleles of cloned essential (EMB) genes.

    PubMed

    Meinke, David; Sweeney, Colleen; Muralla, Rosanna

    2009-01-01

    The classical genetic map of Arabidopsis includes more than 130 genes with an embryo-defective (emb) mutant phenotype. Many of these essential genes remain to be cloned. Hundreds of additional EMB genes have been cloned and catalogued (www.seedgenes.org) but not mapped. To facilitate EMB gene identification and assess the current level of saturation, we updated the classical map, compared the physical and genetic locations of mapped loci, and performed allelism tests between mapped (but not cloned) and cloned (but not mapped) emb mutants with similar chromosome locations. Two hundred pairwise combinations of genes located on chromosomes 1 and 5 were tested and more than 1100 total crosses were screened. Sixteen of 51 mapped emb mutants examined were found to be disrupted in a known EMB gene. Alleles of a wide range of published EMB genes (YDA, GLA1, TIL1, AtASP38, AtDEK1, EMB506, DG1, OEP80) were discovered. Two EMS mutants isolated 30 years ago, T-DNA mutants with complex insertion sites, and a mutant with an atypical, embryo-specific phenotype were resolved. The frequency of allelism encountered was consistent with past estimates of 500 to 1000 EMB loci. New EMB genes identified among mapped T-DNA insertion mutants included CHC1, which is required for chromatin remodeling, and SHS1/AtBT1, which encodes a plastidial nucleotide transporter similar to the maize Brittle1 protein required for normal endosperm development. Two classical genetic markers (PY, ALB1) were identified based on similar map locations of known genes required for thiamine (THIC) and chlorophyll (PDE166) biosynthesis. The alignment of genetic and physical maps presented here should facilitate the continued analysis of essential genes in Arabidopsis and further characterization of a broad spectrum of mutant phenotypes in a model plant. PMID:19812694

  12. Solvent-Free Production of Bioflavors by Enzymatic Esterification of Citronella (Cymbopogon winterianus) Essential Oil.

    PubMed

    Paroul, Natália; Grzegozeski, Luana Paula; Chiaradia, Viviane; Treichel, Helen; Cansian, Rogério L; Oliveira, J Vladimir; de Oliveira, Débora

    2011-10-01

    Enzymatic esterification of citronella essential oil towards the production of geranyl and citronellyl esters may present great scientific and technological interest due to the well-known drawbacks of the chemical-catalyzed route. In this context, this work reports the maximization of geranyl and citronellyl esters production by esterification of oleic and propionic acids in a solvent-free system using a commercial immobilized lipase as catalyst. Results of the reactions showed that the strategy adopted for the experimental design proved to be useful in evaluating the effects of the studied variables on the reaction conversion using Novozym 435 as catalyst. The operating conditions that maximized the production of each ester were determined, leading, in a general way, to conversions of about 90% for all systems. New experimental data on enzymatic esterification of crude citronella essential oil for geranyl and citronellyl esters production in solvent-free system are reported in this work. PMID:21976151

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

    PubMed

    Jeršek, Barbara; Poklar Ulrih, Nataša; Skrt, Mihaela; Gavari?, 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 ˝ MIC (0.0625 mg mL-1), but menthol at Ľ and ˝ 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 Ľ MIC (0.0313 mg mL-1). Essential oil of oregano (Ľ MIC, 0.2930 ?L mL-1) and carvacrol (˝ MIC, 0.1953 ?L 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

  14. 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...

  15. Affordable, clean and secure energy is essential for improving U.S. economic productivity, enhancing our

    E-print Network

    Affordable, clean and secure energy is essential for improving U.S. economic productivity in fossil, nuclear, renewable, and efficiency technologies, all with a view toward the future clean energy Laboratory System is leading transformational research, development, demonstration, and deployment for clean

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

    PubMed Central

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

    2015-01-01

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

  17. Identification of a novel vinyl reductase gene essential for the biosynthesis of monovinyl chlorophyll in Synechocystis sp. PCC6803.

    PubMed

    Ito, Hisashi; Yokono, Makio; Tanaka, Ryouichi; Tanaka, Ayumi

    2008-04-01

    The vast majority of oxygenic photosynthetic organisms use monovinyl chlorophyll for their photosynthetic reactions. For the biosynthesis of this type of chlorophyll, the reduction of the 8-vinyl group that is located on the B-ring of the macrocycle is essential. Previously, we identified the gene encoding 8-vinyl reductase responsible for this reaction in higher plants and termed it DVR. Among the sequenced genomes of cyanobacteria, only several Synechococcus species contain DVR homologues. Therefore, it has been hypothesized that many other cyanobacteria producing monovinyl chlorophyll should contain a vinyl reductase that is unrelated to the higher plant DVR. To identify the cyanobacterial gene that is responsible for monovinyl chlorophyll synthesis, we developed a bioinformatics tool, correlation coefficient calculation tool, which calculates the correlation coefficient between the distributions of a certain phenotype and genes among a group of organisms. The program indicated that the distribution of a gene encoding a putative dehydrogenase protein is best correlated with the distribution of the DVR-less cyanobacteria. We subsequently knocked out the corresponding gene (Slr1923) in Synechocystis sp. PCC6803 and characterized the mutant. The knock-out mutant lost its ability to synthesize monovinyl chlorophyll and accumulated 3,8-divinyl chlorophyll instead. We concluded that Slr1923 encodes the vinyl reductase or a subunit essential for monovinyl chlorophyll synthesis. The function and evolution of 8-vinyl reductase genes are discussed. PMID:18230620

  18. 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

  19. 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

  20. Dependence Relationships between Gene Ontology Terms based on TIGR Gene Product Annotations

    E-print Network

    Borgelt, Christian

    vocabularies for the designations of cellular components, molecular functions, and biological processes usedDependence Relationships between Gene Ontology Terms based on TIGR Gene Product Annotations Anand for the representation and processing of information about gene products and functions. It provides controlled

  1. Intronic and 5' flanking sequences of the Drosophila beta 3 tubulin gene are essential to confer ecdysone responsiveness.

    PubMed

    Bruhat, A; Dréau, D; Drake, M E; Tourmente, S; Chapel, S; Couderc, J L; Dastugue, B

    1993-07-01

    The expression of the beta 3 tubulin gene is regulated, at the transcriptional level, by the steroid hormone ecdysone, in Drosophila Kc cells. Using a transient expression assay, we show that 360 bp from the first intron of the beta 3 tubulin gene, associated with the 5' flanking sequences, are essential to confer ecdysone inducibility on a minimum promoter driving the chloramphenicol acetyl transferase (CAT) gene. The 5' flanking region contains ecdysone-independent cis-positive elements located in proximity to the promoter. Deletion analysis of the 360 bp intronic region reveals that a fragment of 57 bp is crucial for the ecdysone response of the beta 3 tubulin gene. This fragment contains 5'-TGA(A/C)C-3' motifs homologous to ecdysone responsive elements (EcRE) half sites. Band shift assays show that this 57-bp fragment is bound by three specific complexes. One of them appears to be involved in the level of the ecdysone response. PMID:8375576

  2. Novel genes induce uterine receptivity: the characterization of a specific gene product in the ewe uterus 

    E-print Network

    De Graauw, Jennifer Ann

    2013-02-22

    -1 NOVEL GENES INDUCE UTERINE RECEPTIVITY: THF. CHARACTERIZATION OF A SPECIFIC GENE PRODUCT IN THE EWE UTERUS A Senior Honors Thesis By JENNIFER ANN DE GRAAUW Submitted to the Office of Honors Programs 4 Academic Scholarships Texas A&M University... In partial fulfillment of the requirements of the UNIVERSITY UNDERGRADUATE RESEARCH FELLOWS APRIL 2000 Group: Molecular Genetics NOVEL GENES INDUCE UTERINE RECEPTIVITY: THE CHARACTERIZATION OF A SPECIFIC GENE PRODUCT IN THE EWE UTERUS A Senior Honors...

  3. 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

  4. 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.

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

    PubMed Central

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

    2015-01-01

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

  6. The Essential Amino Acid Content of Cottonseed, Peanut and Soybean Products

    E-print Network

    Hale, Fred; Kuiken, Kenneth A. (Kenneth Alfred); Lyman, Carl M. (Carl Morris)

    1947-01-01

    STATlON R. D. LEWIS, Director College Station, Texaa BULLETIN NO. 692 SEPTEMBER 1947 The Essential Amino Acid Content of Cottonseed, Peanut and Soybean Products CARL M. LYMAN, KENNETH KUIKEN and FRED HALE With the technical assistance of Shirley... Dieterich, Marjory Bradford and Mary Trant AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS GIBB GILCHRIST, President - [Blank Page in Original Bulletin] Preface . - - -- - . meals seed, Ili5tid hmino acids are the chemical structural units from which...

  7. Products of lipid, protein and RNA oxidation as signals and regulators of gene expression in plants

    PubMed Central

    Chmielowska-B?k, Jagna; Izbia?ska, Karolina; Deckert, Joanna

    2015-01-01

    Reactive oxygen species (ROS) are engaged in several processes essential for normal cell functioning, such as differentiation, anti-microbial defense, stimulus sensing and signaling. Interestingly, recent studies imply that cellular signal transduction and gene regulation are mediated not only directly by ROS but also by the molecules derived from ROS-mediated oxidation. Lipid peroxidation leads to non-enzymatic formation of oxylipins. These molecules were shown to modulate expression of signaling associated genes including genes encoding phosphatases, kinases and transcription factors. Oxidized peptides derived from protein oxidation might be engaged in organelle-specific ROS signaling. In turn, oxidation of particular mRNAs leads to decrease in the level of encoded proteins and thus, contributes to the post-transcriptional regulation of gene expression. Present mini review summarizes latest findings concerning involvement of products of lipid, protein and RNA oxidation in signal transduction and gene regulation. PMID:26082792

  8. glnD and mviN Are Genes of an Essential Operon in Sinorhizobium meliloti

    PubMed Central

    Rudnick, Paul A.; Arcondéguy, Tania; Kennedy, Christina K.; Kahn, Daniel

    2001-01-01

    To evaluate the role of uridylyl-transferase, the Sinorhizobium meliloti glnD gene was isolated by heterologous complementation in Azotobacter vinelandii. The glnD gene is cotranscribed with a gene homologous to Salmonella mviN. glnD1::? or mviN1::? mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that glnD and mviN are members of an indispensable operon in S. meliloti. PMID:11274131

  9. 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 ?g/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on F. graminearum were recorded as 1250 and 1800 ?g/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 ?g/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 ?g/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 ?g/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

  10. 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 ?g/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on F. graminearum were recorded as 1250 and 1800 ?g/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 ?g/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 ?g/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 ?g/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

  11. 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

  12. 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

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

    PubMed

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

    2014-01-01

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

  14. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  16. 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

  17. Effects of Lactobacillus reuteri-derived biosurfactant on the gene expression profile of essential adhesion genes (gtfB, gtfC and ftf) of Streptococcus mutans

    PubMed Central

    Salehi, Rasoul; Savabi, Omid; Kazemi, Mohammad; kamali, Sara; Salehi, Ahmad Reza; Eslami, Gilda; Tahmourespour, Arezoo

    2014-01-01

    Background: Streptococci are the main causative agents in plaque formation and mutans streptococci are the principle etiological agent of dental plaque and caries. The process of biofilm formation is a step-wise process, starting with adhesion of planktonic cells to the surfaces. It is now a well known fact that expression of glucosyltransferases (gtfs) and fructosyltransferase (ftf) genes play a critical role in the initial adhesion of Streptococcus mutans to the tooth surface, which results in the formation of dental plaques and consequently caries and other periodontal diseases. Materials and Methods: In the present study, we have determined the effect of biosurfactants purified from Lactobacillus reuteri (DSM20016) culture on gene expression profile of gftB/C and fft of S. mutans (ATCC35668) using quantitative real-time polymerase chain reaction. Results: The application of biosurfactant caused considerable down-regulation of the expression of all three genes under study. The reduction in gene expression was statistically very significant (P > 0.0001 for all three genes). Conclusions: Inhibition of these genes by the extracted L. reuteri biosurfactant shows the emergence of a powerful alternative to the presently practicing alternatives. In view of the importance of these gene products for S. mutans attachment to the tooth surface, which is the initial important step in biofilm production and dental caries, we believe that the biosurfactant prepared in this study could be considered as a step ahead in dental caries prevention. PMID:25221772

  18. Non-Hsp genes are essential for HSF1-mediated maintenance of whole body homeostasis.

    PubMed

    Hayashida, Naoki

    2015-10-23

    Mammalian tissues are always exposed to diverse threats from pathological conditions and aging. Therefore, the molecular systems that protect the cells from these threats are indispensable for cell survival. A variety of diseases, including neurodegenerative diseases, cause intracellular damage and disturb homeostasis. Heat shock transcription factor 1 (HSF1) positively regulates heat shock protein (Hsp) and maintains the precise folding of proteins. Moreover, HSF1 induces the non-Hsp genes expression, and degrades damaged/misfolded protein. Recently, my colleagues and I revealed non-Hsp genes have more protective roles than Hsps at the cellular level. However, whether these protective systems are similarly important to cellular defense in each tissue is still elusive. In this study, I compared polyglutamine (polyQ) protein aggregations/inclusion development in each tissue of WT- and HSF1KO-Huntington's disease (HD) mice, and examined the expression of the eight non-Hsp HSF1 target genes that have a strong suppressive effect on polyQ protein aggregation. Of these genes, Nfatc2, Pdzk3, Cryab, Csrp2, and Prame were detected in most tissues, but the other genes were not. Surprisingly, the obvious effect of HSF1 deficiency on the expression of these five genes was detected in only heart, spleen, and stomach. In addition, polyQ protein aggregations/inclusion was not detected in any tissues of WT-HD and HSF1KO-HD mice, but higher level of pre-aggregative polyQ protein was detected in HSF1KO-HD tissues. These results indicate non-Hsp genes are indispensable for the maintenance of intracellular homeostasis in mammalian tissues, resulting in whole body homeostasis. PMID:26251235

  19. Non-Hsp genes are essential for HSF1-mediated maintenance of whole body homeostasis

    PubMed Central

    Hayashida, Naoki

    2015-01-01

    Mammalian tissues are always exposed to diverse threats from pathological conditions and aging. Therefore, the molecular systems that protect the cells from these threats are indispensable for cell survival. A variety of diseases, including neurodegenerative diseases, cause intracellular damage and disturb homeostasis. Heat shock transcription factor 1 (HSF1) positively regulates heat shock protein (Hsp) and maintains the precise folding of proteins. Moreover, HSF1 induces the non-Hsp genes expression, and degrades damaged/misfolded protein. Recently, my colleagues and I revealed non-Hsp genes have more protective roles than Hsps at the cellular level. However, whether these protective systems are similarly important to cellular defense in each tissue is still elusive. In this study, I compared polyglutamine (polyQ) protein aggregations/inclusion development in each tissue of WT- and HSF1KO-Huntington’s disease (HD) mice, and examined the expression of the eight non-Hsp HSF1 target genes that have a strong suppressive effect on polyQ protein aggregation. Of these genes, Nfatc2, Pdzk3, Cryab, Csrp2, and Prame were detected in most tissues, but the other genes were not. Surprisingly, the obvious effect of HSF1 deficiency on the expression of these five genes was detected in only heart, spleen, and stomach. In addition, polyQ protein aggregations/inclusion was not detected in any tissues of WT-HD and HSF1KO-HD mice, but higher level of pre-aggregative polyQ protein was detected in HSF1KO-HD tissues. These results indicate non-Hsp genes are indispensable for the maintenance of intracellular homeostasis in mammalian tissues, resulting in whole body homeostasis. PMID:26251235

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

    PubMed

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

    2015-08-13

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

  1. 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

  2. The RNase III Enzyme DROSHA Is Essential for MicroRNA Production and Spermatogenesis*

    PubMed Central

    Wu, Qiuxia; Song, Rui; Ortogero, Nicole; Zheng, Huili; Evanoff, Ryan; Small, Chris L.; Griswold, Michael D.; Namekawa, Satoshi H.; Royo, Helene; Turner, James M.; Yan, Wei

    2012-01-01

    DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). To investigate the role of canonical miRNA and/or endogenous siRNA production in spermatogenesis, we generated Drosha or Dicer conditional knock-out (cKO) mouse lines by inactivating Drosha or Dicer exclusively in spermatogenic cells in postnatal testes using the Cre-loxp strategy. Both Drosha and Dicer cKO males were infertile due to disrupted spermatogenesis characterized by depletion of spermatocytes and spermatids leading to oligoteratozoospermia or azoospermia. The developmental course of spermatogenic disruptions was similar at morphological levels between Drosha and Dicer cKO males, but Drosha cKO testes appeared to be more severe in spermatogenic disruptions than Dicer cKO testes. Microarray analyses revealed transcriptomic differences between Drosha- and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally. Our data demonstrate that unlike DICER, which is required for the biogenesis of several small RNA species, DROSHA is essential mainly for the canonical miRNA production, and DROSHA-mediated miRNA production is essential for normal spermatogenesis and male fertility. PMID:22665486

  3. ideR, An essential gene in mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response.

    PubMed

    Rodriguez, G Marcela; Voskuil, Martin I; Gold, Benjamin; Schoolnik, Gary K; Smith, Issar

    2002-07-01

    The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that ideR is an essential gene in Mycobacterium tuberculosis. ideR cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare ideR mutant was obtained in which the lethal effects of ideR inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from ideR inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type, ideR mutant, and ideR-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism. PMID:12065475

  4. CO2?–?Intrinsic Product, Essential Substrate, and Regulatory Trigger of Microbial and Mammalian Production Processes

    PubMed Central

    Blombach, Bastian; Takors, Ralf

    2015-01-01

    Carbon dioxide formation mirrors the final carbon oxidation steps of aerobic metabolism in microbial and mammalian cells. As a consequence, CO2/HCO3? dissociation equilibria arise in fermenters by the growing culture. Anaplerotic reactions make use of the abundant CO2/HCO3? levels for refueling citric acid cycle demands and for enabling oxaloacetate-derived products. At the same time, CO2 is released manifold in metabolic reactions via decarboxylation activity. The levels of extracellular CO2/HCO3? depend on cellular activities and physical constraints such as hydrostatic pressures, aeration, and the efficiency of mixing in large-scale bioreactors. Besides, local CO2/HCO3? levels might also act as metabolic inhibitors or transcriptional effectors triggering regulatory events inside the cells. This review gives an overview about fundamental physicochemical properties of CO2/HCO3? in microbial and mammalian cultures effecting cellular physiology, production processes, metabolic activity, and transcriptional regulation. PMID:26284242

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

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Female Aging Alters Expression of Human Cumulus Cells Genes that Are Essential for Oocyte Quality

    PubMed Central

    Assou, Said; Ferričres, Alice; Bringer Deutsch, Sophie; Gala, Anna; Lecellier, Charles-Henri; Aďt-Ahmed, Ounissa; Hamamah, Samir

    2014-01-01

    Impact of female aging is an important issue in human reproduction. There was a need for an extensive analysis of age impact on transcriptome profile of cumulus cells (CCs) to link oocyte quality and developmental potential with patient's age. CCs from patients of three age groups were analyzed individually using microarrays. RT-qPCR validation was performed on independent CC cohorts. We focused here on pathways affected by aging in CCs that may explain the decline of oocyte quality with age. In CCs collected from patients >37 years, angiogenic genes including ANGPTL4, LEPR, TGFBR3, and FGF2 were significantly overexpressed compared to patients of the two younger groups. In contrast genes implicated in TGF-? signaling pathway such as AMH, TGFB1, inhibin, and activin receptor were underexpressed. CCs from patients whose ages are between 31 and 36 years showed an overexpression of genes related to insulin signaling pathway such as IGFBP3, PIK3R1, and IGFBP5. A bioinformatic analysis was performed to identify the microRNAs that are potential regulators of the differentially expressed genes of the study. It revealed that the pathways impacted by age were potential targets of specific miRNAs previously identified in our CCs small RNAs sequencing. PMID:25276836

  8. 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

  9. Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100? §

    PubMed Central

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

    2011-01-01

    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

  10. Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification

    PubMed Central

    Brown, Jamie L.; Snir, Mirit; Noushmehr, Houtan; Kirby, Martha; Hong, Sung-Kook; Elkahloun, Abdel G.; Feldman, Benjamin

    2008-01-01

    A major goal for developmental biologists is to define the behaviors and molecular contents of differentiating cells. We have devised a strategy for isolating cells from diverse embryonic regions and stages in the zebrafish, using computer-guided laser photoconversion of injected Kaede protein and flow cytometry. This strategy enabled us to perform a genome-wide transcriptome comparison of germ layer precursor cells. Mesendoderm and ectoderm precursors cells isolated by this method differentiated appropriately in transplantation assays. Microarray analysis of these cells reidentified known genes at least as efficiently as previously reported strategies that relied on artificial mesendoderm activation or inhibition. We also identified a large set of uncharacterized mesendoderm-enriched genes as well as ectoderm-enriched genes. Loss-of-function studies revealed that one of these genes, the MAP kinase inhibitor dusp4, is essential for early development. Embryos injected with antisense morpholino oligonucleotides that targeted Dusp4 displayed necrosis of head tissues. Marker analysis during late gastrulation revealed a specific loss of sox17, but not of other endoderm markers, and analysis at later stages revealed a loss of foregut and pancreatic endoderm. This specific loss of sox17 establishes a new class of endoderm specification defect. PMID:18719100

  11. Genome-Wide High-Throughput Screening to Investigate Essential Genes Involved in Methicillin-Resistant Staphylococcus aureus Sequence Type 398 Survival

    E-print Network

    Christiansen, Mette T.; Kaas, Rolf S.; Chaudhuri, Roy R.; Holmes, Mark A.; Hasman, Henrik; Aarestrup, Frank M.

    2014-02-12

    and function of all genes within the genome is essential to understand bacterial survival and adaptation, especially for bacteria that may change between ecological stages as colonizers and pathogens and for those that may infect multiple hosts. Homology... in Salmonella Typhi and Salmonella Typhimurium [9–11,13], but has not been applied previously to study S. aureus or other Gram positive bacteria. Importantly, this procedure not only identifies essential genes under different environmental conditions, but also...

  12. 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

  13. 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

  14. 76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-16

    ...Potency Tests for Cellular and Gene Therapy Products; Availability AGENCY...Potency Tests for Cellular and Gene Therapy Products'' dated January 2011...provides manufacturers of cellular and gene therapy (CGT) products with...

  15. 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

  16. 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

  17. 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

  18. 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)-?-ocimene, linalool, ?-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

  19. Essential genes for astroglial development and axon pathfinding during zebrafish embryogenesis

    PubMed Central

    Barresi, Michael J.F.; Burton, Sean; DiPietrantonio, Kristina; Amsterdam, Adam; Hopkins, Nancy; Karlstrom, Rolf O.

    2010-01-01

    The formation of the central nervous system depends on the coordinated development of neural and glial cell types that arise from a common precursor. Using an existing group of zebrafish mutants generated by viral insertion, we performed a “shelf-screen” to identify genes necessary for astroglial development and axon scaffold formation. We screened 274 of 315 viral insertion lines using antibodies that label axons (anti-Acetylated Tubulin) and astroglia (anti-Gfap) and identified 25 mutants with defects in gliogenesis, glial patterning, neurogenesis, and axon guidance. We also identified a novel class of mutants affecting radial glial cell numbers. Defects in astroglial patterning were always associated with axon defects, supporting an important role for axon-glial interactions during axon scaffold development. The genes disrupted in these viral lines have all been identified, providing a powerful new resource for the study of axon guidance, glio- and neurogenesis, and neuron-glial interactions during development of the vertebrate CNS. PMID:20806318

  20. Introns within Ribosomal Protein Genes Regulate the Production and Function

    E-print Network

    Zhang, Jianzhi

    -specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergeneIntrons within Ribosomal Protein Genes Regulate the Production and Function of Yeast Ribosomes In budding yeast, the most abundantly spliced pre- mRNAs encode ribosomal proteins (RPs). To investi- gate

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

    PubMed Central

    Kondratov, Roman V.; Jamasbi, Roudabeh J.

    2015-01-01

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

  2. Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168.

    PubMed

    Dorenbos, Ronald; Stein, Torsten; Kabel, Jorrit; Bruand, Claude; Bolhuis, Albert; Bron, Sierd; Quax, Wim J; Van Dijl, Jan Maarten

    2002-05-10

    Thiol-disulfide oxidoreductases are required for disulfide bond formation in proteins that are exported from the cytoplasm. Four enzymes of this type, termed BdbA, BdbB, BdbC, and BdbD, have been identified in the Gram-positive eubacterium Bacillus subtilis. BdbC and BdbD have been shown to be critical for the folding of a protein required for DNA uptake during natural competence. In contrast, no function has been assigned so far to the BdbA and BdbB proteins. The bdbA and bdbB genes are located in one operon that also contains the genes specifying the lantibiotic sublancin 168 and the ATP-binding cassette transporter SunT. Interestingly sublancin 168 contains two disulfide bonds. The present studies demonstrate that SunT and BdbB, but not BdbA, are required for the production of active sublancin 168. In addition, the BdbB paralogue BdbC is at least partly able to replace BdbB in sublancin 168 production. These observations show the unprecedented involvement of thiol-disulfide oxidoreductases in the synthesis of a peptide antibiotic. Notably BdbB cannot complement BdbC in competence development, showing that these two closely related thiol-disulfide oxidoreductases have different, but partly overlapping, substrate specificities. PMID:11872755

  3. Autophagy-related gene Atg5 is essential for astrocyte differentiation in the developing mouse cortex

    PubMed Central

    Wang, Shukun; Li, Baoguo; Qiao, Huimin; Lv, Xiaohui; Liang, Qingli; Shi, Zixiao; Xia, Wenlong; Ji, Fen; Jiao, Jianwei

    2014-01-01

    Astrocyte differentiation is essential for late embryonic brain development, and autophagy is active during this process. However, it is unknown whether and how autophagy regulates astrocyte differentiation. Here, we show that Atg5, which is necessary for autophagosome formation, regulates astrocyte differentiation. Atg5 deficiency represses the generation of astrocytes in vitro and in vivo. Conversely, Atg5 overexpression increases the number of astrocytes substantially. We show that Atg5 activates the JAK2-STAT3 pathway by degrading the inhibitory protein SOCS2. The astrocyte differentiation defect caused by Atg5 loss can be rescued by human Atg5 overexpression, STAT3 overexpression, and SOCS2 knockdown. Together, these data demonstrate that Atg5 regulates astrocyte differentiation, with potential implications for brain disorders with autophagy deficiency. PMID:25227738

  4. An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Kato, R; Ogawa, H

    1994-01-01

    A new mutant, which was sensitive to both methyl-methanesulfonate (MMS) and ultra-violet light (UV) and defective in meiotic recombination, was isolated from Saccharomyces cerevisiae. The gene, ESR1, was cloned by complementation of the MMS sensitivity of the mutant and found to be essential for cell growth, as the deleted haploid strain was lethal. The ESR1 gene was adjacent to the CKS1 gene on chromosome II and encoded a putative 2368-amino acid protein with a molecular weight of 273 k. The ESR1 transcript was 8.0 kb long and was induced during meiosis. The predicted Esr1 protein had a mosaic structure composed of homologous regions and showed amino acid sequence similarities to Schizosaccharomyces pombe rad3+ protein, which monitors completion of DNA repair synthesis, and cut1+ protein, which is required for spindle pole body (SPB) duplication. The Esr1 protein was also similar to phosphatidylinositol (PI) 3-kinases, including Saccharomyces cerevisiae TOR2 (and DRR1), which are involved in G1 progression. These results suggest that ESR1 is multi-functional throughout mitosis and meiosis. Images PMID:8065923

  5. Addition of Escherichia coli K-12 Growth Observation and Gene Essentiality Data to the EcoCyc Database

    PubMed Central

    Mackie, Amanda; Paley, Suzanne; Keseler, Ingrid M.; Shearer, Alexander; Paulsen, Ian T.

    2014-01-01

    The sets of compounds that can support growth of an organism are defined by the presence of transporters and metabolic pathways that convert nutrient sources into cellular components and energy for growth. A collection of known nutrient sources can therefore serve both as an impetus for investigating new metabolic pathways and transporters and as a reference for computational modeling of known metabolic pathways. To establish such a collection for Escherichia coli K-12, we have integrated data on the growth or nongrowth of E. coli K-12 obtained from published observations using a variety of individual media and from high-throughput phenotype microarrays into the EcoCyc database. The assembled collection revealed a substantial number of discrepancies between the high-throughput data sets, which we investigated where possible using low-throughput growth assays on soft agar and in liquid culture. We also integrated six data sets describing 16,119 observations of the growth of single-gene knockout mutants of E. coli K-12 into EcoCyc, which are relevant to antimicrobial drug design, provide clues regarding the roles of genes of unknown function, and are useful for validating metabolic models. To make this information easily accessible to EcoCyc users, we developed software for capturing, querying, and visualizing cellular growth assays and gene essentiality data. PMID:24363340

  6. Estrogen-related receptor {alpha} is essential for the expression of antioxidant protection genes and mitochondrial function

    SciTech Connect

    Rangwala, Shamina M. . E-mail: shamina.rangwala@novartis.com; Li, Xiaoyan; Lindsley, Loren; Wang, Xiaomei; Shaughnessy, Stacey; Daniels, Thomas G.; Szustakowski, Joseph; Nirmala, N.R.; Wu, Zhidan; Stevenson, Susan C.

    2007-05-25

    Estrogen-related receptor {alpha} (ERR{alpha}) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERR{alpha} null mice. Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) stimulated mitochondrial gene expression program in control cells, but not in the ERR{alpha} null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1{alpha} levels was dependent on ERR{alpha}. Furthermore, we found that the PGC-1{alpha}-mediated induction of estrogen-related receptor {gamma} and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERR{alpha}. Basal levels of NRF-2 were decreased in the absence of ERR{alpha}. The absence of ERR{alpha} resulted in a decrease in citrate synthase enzyme activity in response to PGC-1{alpha} overexpression. Our results indicate an essential role for ERR{alpha} as a key regulator of oxidative metabolism.

  7. A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

    PubMed

    Chettoor, Antony M; Yi, Gibum; Gomez, Elisa; Hueros, Gregorio; Meeley, Robert B; Becraft, Philip W

    2015-03-01

    Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function. PMID:24985738

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  9. Design and construction of a first-generation high-throughput integrated molecular biology platform for production of optimized synthetic genes and improved industrial strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular biological techniques for plasmid-based assembly and cloning of synthetic assembled gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. These techniques involve the production of full-length cDNA libraries as a source of plasmid-bas...

  10. Profiling Essential Genes in Human Mammary Cells by Multiplex RNAi Screening

    PubMed Central

    Silva, Jose M.; Marran, Krista; Parker, Joel S.; Silva, Javier; Golding, Michael; Schlabach, Michael R.; Elledge, Stephen J.; Hannon, Gregory J.; Chang, Kenneth

    2010-01-01

    By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line–specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities. PMID:18239125

  11. A FUSCA gene of Arabidopsis encodes a novel protein essential for plant development.

    PubMed Central

    Castle, L A; Meinke, D W

    1994-01-01

    Arabidopsis fusca mutants display striking purple coloration due to anthocyanin accumulation in their cotyledons. We describe six recessive fusca mutants isolated from Agrobacterium-transformed Arabidopsis families. These mutants first become defective during embryogenesis and exhibit limited seedling development. Double mutant constructs revealed that developmental defects were not simply a consequence of anthocyanin accumulation. fusca seedlings showed altered responses to several environmental and endogenous factors. Allelism tests established that three fusca loci are represented by mutants previously described as defective in light-regulated responses. To study the molecular basis of the fusca phenotype, we cloned the FUS6 gene. FUS6 encodes a novel protein that is hydrophilic, alpha-helical, and contains potential protein kinase C phosphorylation sites. The FUSCA proteins appear to act in a network of signal transduction pathways critical for plant development. PMID:8130643

  12. NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements

    PubMed Central

    Weischenfeldt, Joachim; Damgaard, Inge; Bryder, David; Theilgaard-Mönch, Kim; Thoren, Lina A.; Nielsen, Finn Cilius; Jacobsen, Sten Eirik W.; Nerlov, Claus; Porse, Bo Torben

    2008-01-01

    Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival. PMID:18483223

  13. Tomato tos1 mutation identifies a gene essential for osmotic tolerance and abscisic acid sensitivity

    E-print Network

    Málaga, Universidad de

    , in turn, affects water availability to the plant (Hasegawa et al., 2000). In addition to the hyper) is important for salt and/or osmotic plant tolerance, because tss2 is hypersensitive to growth inh stress severely limits plant growth and agricultural productivity. We have used mutagenesis to identify

  14. Kaposi's Sarcoma-Associated Herpesvirus ORF6 Gene Is Essential in Viral Lytic Replication

    PubMed Central

    Peng, Can; Chen, Jungang; Tang, Wei; Liu, Chunlan; Chen, Xulin

    2014-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC?6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BAC?6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BAC?6 cell supernatants. DNA synthesis was defective in 293T-BAC?6 cells. Expression of ORF6 in trans in BAC?6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BAC?6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV. PMID:24911362

  15. Novel Essential Role of Ethanol Oxidation Genes at Low Temperature Revealed by Transcriptome Analysis in the Antarctic Bacterium Pseudomonas extremaustralis

    PubMed Central

    Tribelli, Paula M.; Solar Venero, Esmeralda C.; Ricardi, Martiniano M.; Gómez-Lozano, Maria; Raiger Iustman, Laura J.; Molin, Sřren; López, Nancy I.

    2015-01-01

    Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures. PMID:26671564

  16. Trastuzumab Alters the Expression of Genes Essential for Cardiac Function and Induces Ultrastructural Changes of Cardiomyocytes in Mice

    PubMed Central

    ElZarrad, M. Khair; Mukhopadhyay, Partha; Mohan, Nishant; Hao, Enkui; Dokmanovic, Milos; Hirsch, Dianne S.; Shen, Yi; Pacher, Pal; Wu, Wen Jin

    2013-01-01

    Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity. PMID:24255707

  17. Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila.

    PubMed

    Yung, Philip Yuk Kwong; Stuetzer, Alexandra; Fischle, Wolfgang; Martinez, Anne-Marie; Cavalli, Giacomo

    2015-06-01

    Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis. PMID:26004180

  18. PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis

    PubMed Central

    1991-01-01

    Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau, 1989, Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3- mutants enabled us to clone the PAS3 gene by functional complementation. DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer. To verify these predictions antibodies were raised against a truncated portion of the PAS3 coding region overexpressed in E. coli. Pas3p was identified as a 48 kD peroxisomal integral membrane protein. It is shown that a lack of this protein causes the peroxisome- deficient phenotype and the cytosolic mislocalization of peroxisomal matrix enzymes. Based on protease digestion experiments Pas3p is discussed to be anchored in the peroxisomal membrane by its amino- terminus while the bulk of the molecule is exposed to the cytosol. These findings are consistent with the possibility that Pas3p is one component of the peroxisomal import machinery. PMID:1894692

  19. Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

    PubMed

    Lin, Yi-Chu; Wang, Po-Yu; Tsai, Shih-Chih; Lin, Chien-Liang; Tai, Hsuen-Yung; Lo, Chi-Fang; Wu, Shiow-Ing; Chiang, Yu-Mei; Liu, Li-Ling

    2015-01-01

    Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients. PMID:26374219

  20. 29 CFR 776.17 - Employment in a “closely related process or occupation directly essential to” production of goods.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... directness or indirectness of the activity's effect in relation to such production, or in terms of employment... 29 Labor 3 2010-07-01 2010-07-01 false Employment in a âclosely related process or occupation... § 776.17 Employment in a “closely related process or occupation directly essential to” production...

  1. Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes.

    PubMed Central

    Horiuchi, T; Maki, H; Maruyama, M; Sekiguchi, M

    1981-01-01

    By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map. Images PMID:6267604

  2. The protein disulfide isomerase AGR2 is essential for production of intestinal mucus

    PubMed Central

    Park, Sung-Woo; Zhen, Guohua; Verhaeghe, Catherine; Nakagami, Yasuhiro; Nguyenvu, Louis T.; Barczak, Andrea J.; Killeen, Nigel; Erle, David J.

    2009-01-01

    Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production. PMID:19359471

  3. The essential autophagy gene ATG7 modulates organ fibrosis via regulation of endothelial-to-mesenchymal transition.

    PubMed

    Singh, Krishna K; Lovren, Fina; Pan, Yi; Quan, Adrian; Ramadan, Azza; Matkar, Pratiek N; Ehsan, Mehroz; Sandhu, Paul; Mantella, Laura E; Gupta, Nandini; Teoh, Hwee; Parotto, Matteo; Tabuchi, Arata; Kuebler, Wolfgang M; Al-Omran, Mohammed; Finkel, Toren; Verma, Subodh

    2015-01-30

    Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGF? signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments. PMID:25527499

  4. liver-enriched gene 1a and 1b Encode Novel Secretory Proteins Essential for Normal Liver Development in Zebrafish

    PubMed Central

    Chang, Changqing; Hu, Minjie; Zhu, Zhihui; Lo, Li Jan; Chen, Jun; Peng, Jinrong

    2011-01-01

    liver-enriched gene 1 (leg1) is a liver-enriched gene in zebrafish and encodes a novel protein. Our preliminary data suggested that Leg1 is probably involved in early liver development. However, no detailed characterization of Leg1 has been reported thus far. We undertook both bioinformatic and experimental approaches to study leg1 gene structure and its role in early liver development. We found that Leg1 identifies a new conserved protein superfamily featured by the presence of domain of unknown function 781 (DUF781). There are two copies of leg1 in zebrafish, namely leg1a and leg1b. Both leg1a and leg1b are expressed in the larvae and adult liver with leg1a being the predominant form. Knockdown of Leg1a or Leg1b by their respective morpholinos specifically targeting their 5?-UTR each resulted in a small liver phenotype, demonstrating that both Leg1a and Leg1b are important for early liver development. Meanwhile, we found that injection of leg1-ATGMO, a morpholino which can simultaneously block the translation of Leg1a and Leg1b, caused not only a small liver phenotype but hypoplastic exocrine pancreas and intestinal tube as well. Further examination of leg1-ATGMO morphants with early endoderm markers and early hepatic markers revealed that although depletion of total Leg1 does not alter the hepatic and pancreatic fate of the endoderm cells, it leads to cell cycle arrest that results in growth retardation of liver, exocrine pancreas and intestine. Finally, we proved that Leg1 is a secretory protein. This intrigued us to propose that Leg1 might act as a novel secreted regulator that is essential for liver and other digestive organ development in zebrafish. PMID:21857963

  5. Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

    PubMed

    Apfel, C M; Takács, B; Fountoulakis, M; Stieger, M; Keck, W

    1999-01-01

    The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6. PMID:9882662

  6. PLASMA PROTEIN PRODUCTION INFLUENCED BY AMINO ACID MIXTURES AND LACK OF ESSENTIAL AMINO ACIDS

    PubMed Central

    Madden, S. C.; Anderson, F. W.; Donovan, J. C.; Whipple, G. H.

    1945-01-01

    When blood plasma proteins are depleted by bleeding with return of red cells suspended in saline (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a constant level of plasma protein production if the diet nitrogen intake is controlled and limited. Such dogs are outwardly normal but have a lowered resistance to infection and intoxication and probably to vitamin deficiency. When the diet nitrogen is provided by certain mixtures of the ten growth essential amino acids plus glycine, given intravenously at a rapid rate, plasma protein production is good. The same mixture absorbed subcutaneously at a slower rate may be slightly better utilized. Fed orally the same mixture is better utilized and associated with a lower urinary nitrogen excretion. An ample amino acid mixture for the daily intake of a 10 kilo dog may contain in grams dl-threonine 1.4, dl-valine 3, dl-leucine 3, dl-isoleucine 2, l(+)-lysine·HCl·H2O 2.2, dl-tryptophane 0.3, dl-phenylalanine 2, dl-methionine 1.2, l(+)-histidine·HCl·H2O 1, l(+)-arginine·HCl 1, and glycine 2. Half this quantity is inadequate and not improved by addition of a mixture of alanine, serine, norleucine, proline, hydroxyproline, and tyrosine totalling 1.4 gm. Aspartic acid appears to induce vomiting when added to a mixture of amino acids. The same response has been reported for glutamic acid (8). Omission from the intake of leucine or of leucine and isoleucine results in negative nitrogen balance and rapid weight loss but plasma protein production may be temporarily maintained. It is possible that leucine may be captured from red blood cell destruction. Tryptophane deficiency causes an abrupt decline in plasma protein production. No decline occurred during 2 weeks of histidine deficiency but the urinary nitrogen increased to negative balance. Plasma protein production may be impaired during conditions of dietary deficiency not related to the protein or amino acid intake. Skin lesions and liver function impairment are described. Unidentified factors present in liver and yeast appear to be involved. PMID:19871490

  7. Characterization of the Bacillus subtilis ywsC Gene, Involved in ?-Polyglutamic Acid Production

    PubMed Central

    Urushibata, Yuji; Tokuyama, Shinji; Tahara, Yasutaka

    2002-01-01

    The genes required for ?-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in ?ywsC and ?ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the ?ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. 14C-labeled PGA was synthesized by the purified proteins from l-[14C]-glutamate in the presence of ATP and MnCl2, through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis. PMID:11751809

  8. Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

    A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

  9. The Schizosaccharomyces Pombe Rec16 Gene Product Regulates Multiple Meiotic Events

    PubMed Central

    Li, Y. F.; Smith, G. R.

    1997-01-01

    Previously isolated meiotic recombination (rec) mutants of Schizosaccharomyces pombe define 16 complementation groups. The rec genes cloned and sequenced to date reveal little amino acid sequence identity to other reported proteins. We examined the rec mutants for alterations in meiotic events other than recombination to gain insight into the rec gene functions and to assess whether they affect recombination directly or indirectly. While mutations in the rec6-12, 14, 15 and 19 genes appeared to affect only meiotic recombination, a mutation in rec16 delayed meiotic DNA synthesis and, in some instances, reduced its amount; mitotic DNA synthesis was not detectably altered, indicating that the rec16 effect is limited to meiosis. In the rec16 mutant some meiotically induced transcripts (e.g., rec7 and 15) were significantly reduced in abundance, whereas others (e.g., rec10 and exo1) were induced and degraded with normal timing and extent during meiosis, indicating that the rec16 mutation leaves the basic meiotic program intact. These results indicate that the rec genes other than rec16 have their primary effect on meiotic recombination. In contrast, the rec16 gene product is essential for normal meiotic replication, recombination, and induction of some transcripts. These meiotic events may be coupled via a dependence of recombination and transcription on replication or via a cascade of gene expression. PMID:9136000

  10. Characterization of essential genes by parasexual genetics in the human fungal pathogen Aspergillus fumigatus: impact of genomic rearrangements associated with electroporation of DNA.

    PubMed Central

    Firon, Arnaud; Beauvais, Anne; Latgé, Jean-Paul; Couvé, Elisabeth; Grosjean-Cournoyer, Marie-Claire; D'Enfert, Christophe

    2002-01-01

    We have evaluated the usefulness of parasexual genetics in the identification of genes essential for the growth of the human fungal pathogen Aspergillus fumigatus. First, essentiality of the A. fumigatus AfFKS1 gene, encoding the catalytic subunit of the beta-(1,3)-glucan synthase complex, was assessed by inactivating one allele of AfFKS1 in a diploid strain of A. fumigatus obtained using adequate selectable markers in spore color and nitrate utilization pathways and by performing haploidization under conditions that select for the occurrence of the disrupted allele. Haploid progeny could not be obtained, demonstrating that AfFKS1 and, hence, beta-(1,3)-glucan synthesis are essential in A. fumigatus. Second, random heterozygous insertional mutants were generated by electroporation of diploid conidia with a heterologous plasmid. A total of 4.5% of the transformants failed to produce haploid progeny on selective medium. Genomic analysis of these heterozygous diploids led in particular to the identification of an essential A. fumigatus gene encoding an SMC-like protein resembling one in Schizosacccharomyces pombe involved in chromosome condensation and cohesion. However, significant plasmid and genomic DNA rearrangements were observed at many of the identified genomic loci where plasmid integration had occurred, thus suggesting that the use of electroporation to build libraries of A. fumigatus insertional mutants has relatively limited value and cannot be used in an exhaustive search of essential genes. PMID:12136012

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

    PubMed

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

    2015-01-01

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

  12. Essential oils in insect control: low-risk products in a high-stakes world.

    PubMed

    Regnault-Roger, Catherine; Vincent, Charles; Arnason, John Thor

    2012-01-01

    In recent years, the use of essential oils (EOs) derived from aromatic plants as low-risk insecticides has increased considerably owing to their popularity with organic growers and environmentally conscious consumers. EOs are easily produced by steam distillation of plant material and contain many volatile, low-molecular-weight terpenes and phenolics. The major plant families from which EOs are extracted include Myrtaceae, Lauraceae, Lamiaceae, and Asteraceae. EOs have repellent, insecticidal, and growth-reducing effects on a variety of insects. They have been used effectively to control preharvest and postharvest phytophagous insects and as insect repellents for biting flies and for home and garden insects. The compounds exert their activities on insects through neurotoxic effects involving several mechanisms, notably through GABA, octopamine synapses, and the inhibition of acetylcholinesterase. With a few exceptions, their mammalian toxicity is low and environmental persistence is short. Registration has been the main bottleneck in putting new products on the market, but more EOs have been approved for use in the United States than elsewhere owing to reduced-risk processes for these materials. PMID:21942843

  13. Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

    PubMed Central

    Vallone, D; Battista, S; Pierantoni, G M; Fedele, M; Casalino, L; Santoro, M; Viglietto, G; Fusco, A; Verde, P

    1997-01-01

    The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation. PMID:9311991

  14. Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, plants (14) and essential oils (EO; 88) from plants that are naturalized to, or can be successfully grown in North America were evaluated in a batch culture in vitro screening experiments with ruminal fluid as potential anti-methanogenic additives for ruminant diets. Essential oils we...

  15. FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation.

    PubMed

    Kyono, Ai; Avishai, Nanthawan; Ouyang, Zhufeng; Landreth, Gary E; Murakami, Shunichi

    2012-01-01

    To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK-dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1(-/-); ERK2(flox/flox); Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1(-/-); ERK2 (flox/flox); Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation. PMID:21678127

  16. FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation

    PubMed Central

    Kyono, Ai; Avishai, Nanthawan; Ouyang, Zhufeng; Landreth, Gary E.; Murakami, Shunichi

    2011-01-01

    To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1?/?; ERK2 flox/flox; Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1?/?; ERK2 flox/flox; Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation. PMID:21678127

  17. Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.

    PubMed Central

    Cheng, C; Kacherovsky, N; Dombek, K M; Camier, S; Thukral, S K; Rhim, E; Young, E T

    1994-01-01

    Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p. Images PMID:8196627

  18. Reovirus guanylyltransferase is L2 gene product lambda 2.

    PubMed Central

    Cleveland, D R; Zarbl, H; Millward, S

    1986-01-01

    Reovirus guanylyltransferase, studied as a covalent enzyme-GMP intermediate, was used to guanylate appropriate acceptor molecules in vitro to produce authentic cap structures. Guanylyltransferase activity was associated with lambda 2, the 140-kilodalton product of the L2 gene segment of reovirus serotypes 1 and 3. Images PMID:3018296

  19. Genome-Wide High-Throughput Screening to Investigate Essential Genes Involved in Methicillin-Resistant Staphylococcus aureus Sequence Type 398 Survival

    PubMed Central

    Christiansen, Mette T.; Kaas, Rolf S.; Chaudhuri, Roy R.; Holmes, Mark A.; Hasman, Henrik; Aarestrup, Frank M.

    2014-01-01

    Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 isolate to evaluate genes important for bacterial survival in laboratory and host-specific environments. The transposon mutant library consisted of approximately 1 million mutants with around 140,000 unique insertion sites and an average number of unique inserts per gene of 44.8. We identified LA-MRSA ST398 essential genes comparable to other high-throughput S. aureus essential gene studies. As ST398 is the most common MRSA isolated from pigs, the transposon mutant library was screened in whole porcine blood. Twenty-four genes were specifically identified as important for bacterial survival in porcine blood. Mutations in 23 of these genes resulted in attenuated bacterial fitness. Seven of the 23 genes were of unknown function, whereas 16 genes were annotated with functions predominantly related to carbon metabolism, pH shock and a variety of regulations and only indirectly to virulence factors. Mutations in one gene of unknown function resulted in a hypercompetitive mutant. Further evaluation of these genes is required to determine their specific relevance in blood survival. PMID:24563689

  20. Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

    DOE PAGESBeta

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alex S.

    2015-03-27

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as ‘topologically important.’ Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termedmore »as ‘functionally important’ genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.« less

  1. Integrated in silico analyses of regulatory and metabolic networks of Synechococcus sp. PCC 7002 reveal relationships between gene centrality and essentiality

    SciTech Connect

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alex S.

    2015-03-27

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as ‘topologically important.’ Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as ‘functionally important’ genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.

  2. Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships between Gene Centrality and Essentiality

    PubMed Central

    Song, Hyun-Seob; McClure, Ryan S.; Bernstein, Hans C.; Overall, Christopher C.; Hill, Eric A.; Beliaev, Alexander S.

    2015-01-01

    Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as “topologically important.” Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as “functionally important” genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles. PMID:25826650

  3. The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

    PubMed Central

    Funk, C R; Zimniak, L; Dowhan, W

    1992-01-01

    To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified. Images PMID:1309518

  4. The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

    PubMed Central

    Atanasova, Lea; Fekete, Erzsébet; Paholcsek, Melinda; Sándor, Erzsébet; Aquino, Benigno; Druzhinina, Irina S.; Karaffa, Levente; Kubicek, Christian P.

    2014-01-01

    Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex. PMID:25386652

  5. The Arabidopsis thaliana GRF-INTERACTING FACTOR gene family plays an essential role in control of male and female reproductive development

    E-print Network

    Franks, Robert

    The Arabidopsis thaliana GRF-INTERACTING FACTOR gene family plays an essential role in control 27 November 2013 Accepted 9 December 2013 Keywords: Arabidopsis thaliana GRF-INTERACTING FACTOR of Arabidopsis thaliana (Arabidopsis) consists of three members and acts as a positive regulator of cell

  6. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-01-01

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer. PMID:19680546

  7. Effect of essential oils on Aspergillus spore germination, growth and mycotoxin production: a potential source of botanical food preservative

    PubMed Central

    Gemeda, Negero; Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw

    2014-01-01

    Objective To investigate effect of essential oils on Aspergillus spore germination, growth and mycotoxin production. Method In vitro antifungal and antiaflatoxigenic activity of essential oils was carried out using poisoned food techniques, spore germination assay, agar dilution assay, and aflatoxin arresting assay on toxigenic strains of Aspergillus species. Results Cymbopogon martinii, Foeniculum vulgare and Trachyspermum ammi (T. ammi) essential oils were tested against toxicogenic isolates of Aspergillus species. T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 µl/mL by essential oils of T. ammi. The oil also showed, complete inhibition of spore germination at a concentration of 2 µl/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting aflatoxin production from Aspergillus niger and Aspergillus flavus at 0.5 and 0.75 µl/mL, respectively. Cymbopogon martinii, Foeniculum vulgare and T. ammi oils as antifungal were found superior over synthetic preservative. Moreover, a concentration of 5?336.297 µl/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity and strengthening its traditional reputations. Conclusions In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by storage fungi. PMID:25183114

  8. Fumigant Toxicity and Oviposition Deterrency of the Essential Oil from Cardamom, Elettaria cardamomum, Against Three Stored—product Insects

    PubMed Central

    Abbasipour, Habib; Mahmoudvand, Mohammad; Rastegar, Fahimeh; Hosseinpour, Mohammad Hossein

    2011-01-01

    Use of insecticides can have disruptive effects on the environment. Replacing the chemical compounds in these insecticides with plant materials, however, can be a safe method with low environmental risk. In the current study, chemical composition and insecticidal activities of the essential oil from cardamom, Elettaria cardamomum L. (Maton) (Zingiberales: Zingiberaceae) on the adults of three stored product pests was investigated. Results indicated that essential oil of E. cardamomum toxic to the bruchid beetle, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae), the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), and the flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Adults of E. kuehniella were more sensitive than the Coleoptera. Also, the highest mortality of these insects was seen after 12 hours. Results of the LT50 tests showed that the lethal time of mortality occurred between 10–20 hours in various test concentrations. Essential oil of E. cardamomum had a good efficacy on oviposition deterrence of C. maculatus females, too. The chemical constituents of the essential oils were analyzed by gas chromatography—mass spectrometry. The major constituents of cardamom were identified as 1,8-cineol, ?-terpinyl acetate, terpinene and fenchyl alcohol. These results suggest that essential oil of E. cardamomum is a good choice for control of stored product pests. PMID:22242564

  9. Fumigant toxicity and oviposition deterrency of the essential oil from cardamom, Elettaria cardamomum, against three stored–product insects.

    PubMed

    Abbasipour, Habib; Mahmoudvand, Mohammad; Rastegar, Fahimeh; Hosseinpour, Mohammad Hossein

    2011-01-01

    Use of insecticides can have disruptive effects on the environment. Replacing the chemical compounds in these insecticides with plant materials, however, can be a safe method with low environmental risk. In the current study, chemical composition and insecticidal activities of the essential oil from cardamom, Elettaria cardamomum L. (Maton) (Zingiberales: Zingiberaceae) on the adults of three stored product pests was investigated. Results indicated that essential oil of E. cardamomum toxic to the bruchid beetle, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae), the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), and the flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Adults of E. kuehniella were more sensitive than the Coleoptera. Also, the highest mortality of these insects was seen after 12 hours. Results of the LT?? tests showed that the lethal time of mortality occurred between 10-20 hours in various test concentrations. Essential oil of E. cardamomum had a good efficacy on oviposition deterrence of C. maculatus females, too. The chemical constituents of the essential oils were analyzed by gas chromatography-mass spectrometry. The major constituents of cardamom were identified as 1,8-cineol, ?-terpinyl acetate, terpinene and fenchyl alcohol. These results suggest that essential oil of E. cardamomum is a good choice for control of stored product pests. PMID:22242564

  10. The Flavin-Containing Monooxygenase 3 Gene and Essential Hypertension: The Joint Effect of Polymorphism E158K and Cigarette Smoking on Disease Susceptibility

    PubMed Central

    Bushueva, Olga; Solodilova, Maria; Churnosov, Mikhail; Ivanov, Vladimir; Polonikov, Alexey

    2014-01-01

    Gene encoding flavin-containing monooxygenase 3 (FMO3), a microsomal antioxidant defense enzyme, has been suggested to contribute to essential hypertension (EH). The present study was designed to investigate whether common functional polymorphism E158K (rs2266782) of the FMO3 gene is associated with EH susceptibility in a Russian population. A total of 2?995 unrelated subjects from Kursk (1?362 EH patients and 843 healthy controls) and Belgorod (357 EH patients and 422 population controls) regions of Central Russia were recruited for this study. DNA samples from all study participants were genotyped for the FMO3 gene polymorphism through PCR followed by RFLP analysis. We found that the polymorphism E158K is associated with increased risk of essential hypertension in both discovery population from Kursk region (OR 1.36?95% CI 1.09–1.69, P = 0.01) and replication population from Belgorod region (OR 1.54 95% CI 1.07–1.89, P = 0.02) after adjustment for gender and age using logistic regression analysis. Further analysis showed that the increased hypertension risk in carriers of genotype 158KK gene occurred in cigarette smokers, whereas nonsmoker carriers of this genotype did not show the disease risk. This is the first study reporting the association of the FMO3 gene polymorphism and the risk of essential hypertension. PMID:25243081

  11. The Flavin-Containing Monooxygenase 3 Gene and Essential Hypertension: The Joint Effect of Polymorphism E158K and Cigarette Smoking on Disease Susceptibility.

    PubMed

    Bushueva, Olga; Solodilova, Maria; Churnosov, Mikhail; Ivanov, Vladimir; Polonikov, Alexey

    2014-01-01

    Gene encoding flavin-containing monooxygenase 3 (FMO3), a microsomal antioxidant defense enzyme, has been suggested to contribute to essential hypertension (EH). The present study was designed to investigate whether common functional polymorphism E158K (rs2266782) of the FMO3 gene is associated with EH susceptibility in a Russian population. A total of 2?995 unrelated subjects from Kursk (1?362 EH patients and 843 healthy controls) and Belgorod (357 EH patients and 422 population controls) regions of Central Russia were recruited for this study. DNA samples from all study participants were genotyped for the FMO3 gene polymorphism through PCR followed by RFLP analysis. We found that the polymorphism E158K is associated with increased risk of essential hypertension in both discovery population from Kursk region (OR 1.36?95% CI 1.09-1.69, P = 0.01) and replication population from Belgorod region (OR 1.54 95% CI 1.07-1.89, P = 0.02) after adjustment for gender and age using logistic regression analysis. Further analysis showed that the increased hypertension risk in carriers of genotype 158KK gene occurred in cigarette smokers, whereas nonsmoker carriers of this genotype did not show the disease risk. This is the first study reporting the association of the FMO3 gene polymorphism and the risk of essential hypertension. PMID:25243081

  12. piRNAs and piRNA-Dependent siRNAs Protect Conserved and Essential C. elegans Genes from Misrouting into the RNAi Pathway.

    PubMed

    Phillips, Carolyn M; Brown, Kristen C; Montgomery, Brooke E; Ruvkun, Gary; Montgomery, Taiowa A

    2015-08-24

    piRNAs silence foreign genes, such as transposons, to preserve genome integrity, but they also target endogenous mRNAs by mechanisms that are poorly understood. Caenorhabditis elegans piRNAs interact with both transposon and nontransposon mRNAs to initiate sustained silencing via the RNAi pathway. To assess the dysregulation of gene silencing caused by lack of piRNAs, we restored RNA silencing in RNAi-defective animals in the presence or absence of piRNAs. In the absence of piRNAs and a cellular memory of piRNA activity, essential and conserved genes are misrouted into the RNAi pathway to produce siRNAs that bind the nuclear Argonaute HRDE-1, resulting in dramatic defects in germ cell proliferation and function such that the animals are sterile. Inactivation of RNAi suppresses sterility, indicating that aberrant siRNAs produced in the absence of piRNAs target essential genes for silencing. Thus, by reanimating RNAi, we uncovered a role for piRNAs in protecting essential genes from RNA silencing. PMID:26279487

  13. The FHIT gene product: tumor suppressor and genome ‘caretaker’

    PubMed Central

    Waters, Catherine E.; Saldivar, Joshua C.; Hosseini, Seyed Ali; Huebner, Kay

    2014-01-01

    The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial-mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome ‘caretaker.’ Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability. PMID:25283145

  14. Essential Oil of Amomum maximum Roxb. and Its Bioactivities against Two Stored-Product Insects.

    PubMed

    Guo, Shan-Shan; You, Chun-Xue; Liang, Jun-Yu; Zhang, Wen-Juan; Yang, Kai; Geng, Zhu-Feng; Wang, Cheng-Fang; Du, Shu-Shan; Lei, Ning

    2015-12-01

    Amomum maximum Roxb. is a perennial herb distributed in South China and Southeast Asia. The objective of this work was to analyze the chemical constituents and assess insecticidal and repellent activities of the essential oil from Amomum maximum fruits against Tribolium castaneum (Herbst) and Liposcelis bostrychophila (Badonnel). The essential oil was obtained by hydrodistillation and analyzed by gas chromatography-flame ionization detector and gas chromatography-mass spectrometry. The main components of the essential oil were identified to be ?-pinene (23.39%), ?-caryophyllene (16.43%), ?-pinene (7.55%), sylvestrene (6.61%) and ç-cadinene (4.19%). It was found that the essential oil of A. maximum fruits possessed contact and fumigant toxicities against T. castaneum adults (LD50 = 29.57 ?g/adult and LC50 = 23.09 mg/L air, respectively) and showed contact toxicity against L. bostrychophila (LD50 = 67.46 ?g/cm(2)). Repellency of the crude oil was also evaluated. After 2 h treatment, the essential oil possessed 100% repellency at 78.63 nL/cm(2) against T. castaneum and 84% repellency at 63.17 nL/cm(2) against L. bostrychophila. The results indicated that the essential oil of A. maximum fruits had the potential to be developed as a natural insecticide and repellent for control of T. castaneum and L. bostrychophila. PMID:26582152

  15. A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production.

    PubMed Central

    Honjo, M; Nakayama, A; Fukazawa, K; Kawamura, K; Ando, K; Hori, M; Furutani, Y

    1990-01-01

    We have cloned a 2.5-kilobase fragment of the Bacillus subtilis genomic DNA which caused the reduction of extracellular and cell-associated protease levels when present in high copy number. This fragment, in multicopy, was also responsible for reduced levels of alpha-amylase, levansucrase, alkaline phosphatase, and sporulation inhibition. The gene relevant to this pleiotropic phenotype is referred to as pai. By DNA sequencing, two open reading frames--ORF1 and ORF2, encoding polypeptides of 172 and 207 amino acid residues, respectively--were found. These open reading frames seemed to form an operon. Deletion analysis revealed that an entire region for ORF1 and ORF2 was necessary for the pai phenotype. In addition, it was observed that the presence of the pai gene, in multicopy, caused overproduction of two proteins (molecular masses, 21 and 24 kilodaltons [kDa]). Analyses of the N-terminal amino acid sequences of these two proteins suggested that they were products of ORF1 and ORF2. Disruption of the pai gene at ORF1 in the genomic DNA resulted in the release of repression on protease synthesis and sporulation in glucose-enriched (2%) medium. The mutant carrying insertional disruption at ORF2 could not be constructed, suggesting that the ORF2 product, the 24-kDa protein, is essential for growth. The 21-kDa protein contains a helix-turn-helix domain observed in other DNA-binding proteins. Chromosomal mapping of pai indicated that this gene is located close to thr-5. These results suggest that the pai gene is a novel transcriptional-regulation gene involved in glucose repression. Images FIG. 2 FIG. 3 FIG. 6 PMID:2108124

  16. Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products.

    PubMed Central

    Ronson, C W; Astwood, P M; Nixon, B T; Ausubel, F M

    1987-01-01

    We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum. The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm. The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM. The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E. coli. PMID:3671068

  17. Functional analyses and identification of two arginine residues essential to the ATP-utilizing activity of the triple gene block protein 1 of bamboo mosaic potexvirus.

    PubMed

    Liou, D Y; Hsu, Y H; Wung, C H; Wang, W H; Lin, N S; Chang, B Y

    2000-11-25

    The TGBp1 of bamboo mosaic potexvirus (BaMV) is encoded by the first overlapping gene of the triple-gene-block (TGB), whose products are thought to play roles in virus movement between plant cells. This protein forms cytoplasmic inclusions associated with virus particles in the BaMV-infected tissues. It has been proposed that the inclusion is one of the active forms of TGBp1. To prove this idea, we purified the TGBp1 inclusions from both the BaMV-infected Chenopodium quinoa and Escherichia coli cells overexpressing this protein to test some of their biochemical activities. We found that the TGBp1 inclusions isolated from the infected plant leaves, but not from E. coli, possess the NTP-binding and NTPase activities. However, they lack the RNA-binding activity possessed by the soluble TGBp1. These results indicate that the TGBp1 proteins in the BaMV-infected tissues assume two different functional forms. Mutational analyses and competition experiments show that the two arginine residues, Arg-16 and Arg-21, essential to RNA binding, are also required for the ATP-utilizing activity of the soluble TGBp1. This indicates that a same-structure motif is required for the two functions of the soluble TGBp1. The location of the two arginine residues outside the seven conserved motifs of the NTP-utilizing superfamily I RNA helicases, to which TGBp1 belongs, suggests that an extra-structure motif, besides the seven conserved ones, is required for the NTP-utilizing activity of the TGBp1 protein of BaMV. PMID:11080481

  18. BILIVERDIN REDUCTASE IS A TRANSPORTER OF HEME INTO THE NUCLEUS AND IS ESSENTIAL TO REGULATION OF HO-1 GENE EXPRESSION BY HEMATIN

    PubMed Central

    Tudor, Cicerone; Lerner-Marmarosh, Nicole; Engelborghs, Yves; Gibbs, Peter E. M; Maines, Mahin D.

    2009-01-01

    Synopsis Human biliverdin reductase (hBVR) is an enzyme that reduces biliverdin (the product of heme oxygenase HO-1 and HO-2 activity) to the antioxidant bilirubin. It also functions as a kinase and as a transcription factor in the MAPK signaling cascade. Fluorescence correlation spectroscopy was used to investigate the mobility of hBVR in living cells and its function in the nuclear transport of hematin for induction of HO-1. In transiently transfected HeLa cells only kinase-competent hBVR translocates to the nucleus. A reduced mobility in the nucleus of hematintreated cells suggests formation of an hBVR:hematin complex and its further association with large nuclear components. The binding of hematin is specific, with the formation of a 1:1 molar complex and the C-terminal 7-residue fragment (KYCCSRK296) of hBVR contributes to the binding. The following data suggest formation of dynamic complexes of hBVR:hematin with chromatin: a) the reduction of hBVR mobility in the presence of hematin is greater in heterochromatic regions than in euchromatic domains and, b) hBVR mobility is not retarded by hematin in nuclear lysates that contain only soluble factors. Moreover, hBVR kinase activity is stimulated in the presence of double-stranded DNA fragments corresponding to HO-1 antioxidant and hypoxia response elements and by hematin. Experiments with nuclear localization and export signal mutants, and si-hBVR indicate that nuclear localization of hBVR is required for induction of HO-1 by hematin. Because gene regulation is energy-dependent and hematin regulates gene expression, our data suggest that hBVR functions as an essential component of the regulatory mechanisms for heme-responsive transcriptional activation. PMID:18412543

  19. An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes.

    PubMed

    Shirasu, K; Koukolíková-Nicola, Z; Hohn, B; Kado, C I

    1994-02-01

    The 9.5 kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84 kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T-DNA transfer process were determined by generating non-polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T-DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection- and tumorigenicity-negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T-DNA and virulence. Furthermore, by using anti-VirB4 serum, the protein product of virB4 was localized to the inner-membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti-VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T-DNA or to assemble the T-DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, respectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T-DNA from A. tumefaciens to plant cells. PMID:8152380

  20. Gene D5 product of bacteriophage T5: DNA-binding protein affecting DNA replication and late gene expression.

    PubMed Central

    McCorquodale, D J; Gossling, J; Benzinger, R; Chesney, R; Lawhorne, L; Moyer, R W

    1979-01-01

    Gene D5 is not only necessary for replication of bacteriophage T5 DNA and for shutoff of expression of some early genes, but has been found to be necessary also for the expression of late T5 genes. The polypeptide product of gene D5 has been identified, an intragenic map of gene D5 has been constructed, and the direction of transcription of gene D5 has been established. The polypeptide coded by gene D5 has been shown to be a DNA-binding protein with affinity for both double- and single-stranded DNA. Images PMID:219226

  1. Chemical Composition and Bioactivities of the Essential Oil from Etlingera yunnanensis against Two Stored Product Insects.

    PubMed

    Guo, Shan-Shan; You, Chun-Xue; Liang, Jun-Yu; Zhang, Wen-Juan; Geng, Zhu-Feng; Wang, Cheng-Fang; Du, Shu-Shan; Lei, Ning

    2015-01-01

    The chemical composition of the essential oil of Etlingera yunnanensis rhizomes and its contact and repellent activities against Tribolium castaneum (Herbst) and Liposcelis bostrychophila (Badonnel) were investigated. The essential oil obtained from E. yunnanensis rhizomes with hydrodistillation was performed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. The main components of the essential oil were identified to be estragole (65.2%), ?-caryophyllene (6.4%), 1,8-cineole (6.4%), limonene (5.2%), and ?-pinene (2.4%). It was found that the essential oil of E. yunnanensis rhizomes possessed contact toxicity against T. castaneum and L. bostrychophila (LD50 = 23.33 ?g/adult and LD50 = 47.38 ?g/cm˛, respectively). Estragole, 1,8-cineole, and limonene exhibited stronger contact toxicity (LD50 values of 20.41, 18.86, and 13.40 ?g/adult, respectively) than ?-caryophyllene (LD50 = 41.72 ?g/adult) against T. castaneum adults. Estragole possessed stronger contact toxicity (LD50 = 30.22 µg/cm˛) than ?-caryophyllene, 1,8-cineole, and limonene (LD50 values of 74.11, 321.20, and 239.62 ?g/adult, respectively) against L. bostrychophila adults. Repellency of the crude oil was also evaluated. The essential oil and constituents possessed strong repellent activity against T. castaneum adults. The four individual constituents showed weaker repellent activity than the essential oil against L. bostrychophila adults. The results indicated that the essential oil of E. yunnanensis rhizomes and the individual constituents had the potential to be developed as a natural insecticide and repellent for the control of T. castaneum and L. bostrychophila. PMID:26343627

  2. Natural Products Version 2.0: Connecting Genes to Molecules

    PubMed Central

    Walsh, Christopher T.; Fischbach, Michael A.

    2009-01-01

    Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this perspective, we explore how connecting Nature’s small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and nonribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future? PMID:20121095

  3. Anode Biofilm Transcriptomics Reveals Outer Surface Components Essential for High Density Current Production in Geobacter sulfurreducens Fuel Cells

    PubMed Central

    Glaven, Richard H.; Johnson, Jessica P.; Woodard, Trevor L.; Methé, Barbara A.; DiDonato, Raymond J.; Covalla, Sean F.; Franks, Ashley E.; Liu, Anna; Lovley, Derek R.

    2009-01-01

    The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 µm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes. PMID:19461962

  4. Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis.

    PubMed

    Moriyama, Daisuke; Hosono, Kouji; Fujii, Makoto; Washida, Motohisa; Nanba, Hirokazu; Kaino, Tomohiro; Kawamukai, Makoto

    2015-01-01

    Coenzyme Q10 (CoQ10) is essential for energy production and has become a popular supplement in recent years. In this study, CoQ10 productivity was improved in the fission yeast Schizosaccharomyces pombe. Ten CoQ biosynthetic genes were cloned and overexpressed in S. pombe. Strains expressing individual CoQ biosynthetic genes did not produce higher than a 10% increase in CoQ10 production. In addition, simultaneous expression of all ten coq genes did not result in yield improvements. Genes responsible for the biosynthesis of p-hydroxybenzoate and decaprenyl diphosphate, both of which are CoQ biosynthesis precursors, were also overexpressed. CoQ10 production was increased by overexpression of Eco_ubiC (encoding chorismate lyase), Eco_aroF(FBR) (encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), or Sce_thmgr1 (encoding truncated HMG-CoA reductase). Furthermore, simultaneous expression of these precursor genes resulted in two fold increases in CoQ10 production. PMID:25647499

  5. Lactobacillus reuteri-Specific Immunoregulatory Gene rsiR Modulates Histamine Production and Immunomodulation by Lactobacillus reuteri

    PubMed Central

    Hemarajata, P.; Gao, C.; Pflughoeft, K. J.; Thomas, C. M.; Saulnier, D. M.; Spinler, J. K.

    2013-01-01

    Human microbiome-derived strains of Lactobacillus reuteri potently suppress proinflammatory cytokines like human tumor necrosis factor (TNF) by converting the amino acid l-histidine to the biogenic amine histamine. Histamine suppresses mitogen-activated protein (MAP) kinase activation and cytokine production by signaling via histamine receptor type 2 (H2) on myeloid cells. Investigations of the gene expression profiles of immunomodulatory L. reuteri ATCC PTA 6475 highlighted numerous genes that were highly expressed during the stationary phase of growth, when TNF suppression is most potent. One such gene was found to be a regulator of genes involved in histidine-histamine metabolism by this probiotic species. During the course of these studies, this gene was renamed the Lactobacillus reuteri-specific immunoregulatory (rsiR) gene. The rsiR gene is essential for human TNF suppression by L. reuteri and expression of the histidine decarboxylase (hdc) gene cluster on the L. reuteri chromosome. Inactivation of rsiR resulted in diminished TNF suppression in vitro and reduced anti-inflammatory effects in vivo in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of acute colitis. A L. reuteri strain lacking an intact rsiR gene was unable to suppress colitis and resulted in greater concentrations of serum amyloid A (SAA) in the bloodstream of affected animals. The PhdcAB promoter region targeted by rsiR was defined by reporter gene experiments. These studies support the presence of a regulatory gene, rsiR, which modulates the expression of a gene cluster known to mediate immunoregulation by probiotics at the transcriptional level. These findings may point the way toward new strategies for controlling gene expression in probiotics by dietary interventions or microbiome manipulation. PMID:24123819

  6. Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.

    PubMed Central

    Sandler, S J; Chackerian, B; Li, J T; Clark, A J

    1992-01-01

    We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92% and 38% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations. PMID:1542576

  7. CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

    PubMed Central

    Khairnar, Vishal; Duhan, Vikas; Maney, Sathish Kumar; Honke, Nadine; Shaabani, Namir; Pandyra, Aleksandra A.; Seifert, Marc; Pozdeev, Vitaly; Xu, Haifeng C.; Sharma, Piyush; Baldin, Fabian; Marquardsen, Florian; Merches, Katja; Lang, Elisabeth; Kirschning, Carsten; Westendorf, Astrid M.; Häussinger, Dieter; Lang, Florian; Dittmer, Ulf; Küppers, Ralf; Recher, Mike; Hardt, Cornelia; Scheffrahn, Inka; Beauchemin, Nicole; Göthert, Joachim R.; Singer, Bernhard B.; Lang, Philipp A.; Lang, Karl S.

    2015-01-01

    B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-?B-axis. The absence of this signalling cascade in naive Ceacam1?/? mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1?/? mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses. PMID:25692415

  8. Modular optimization of multi-gene pathways for fumarate production.

    PubMed

    Chen, Xiulai; Zhu, Pan; Liu, Liming

    2016-01-01

    Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate. PMID:26241189

  9. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    SciTech Connect

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  10. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    PubMed Central

    Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

  11. Single gene insertion drives bioalcohol production by a thermophilic archaeon.

    PubMed

    Basen, Mirko; Schut, Gerrit J; Nguyen, Diep M; Lipscomb, Gina L; Benn, Robert A; Prybol, Cameron J; Vaccaro, Brian J; Poole, Farris L; Kelly, Robert M; Adams, Michael W W

    2014-12-01

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

  12. The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7.

    PubMed

    Mith, Hasika; Clinquart, Antoine; Zhiri, Abdesselam; Daube, Georges; Delcenserie, Véronique

    2015-01-01

    The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics. PMID:25790499

  13. A weighted multipath measurement based on gene ontology for estimating gene products similarity.

    PubMed

    Liu, Lizhen; Dai, Xuemin; Wang, Hanshi; Song, Wei; Lu, Jingli

    2014-12-01

    Many different methods have been proposed for calculating the semantic similarity of term pairs based on gene ontology (GO). Most existing methods are based on information content (IC), and the methods based on IC are used more commonly than those based on the structure of GO. However, most IC-based methods not only fail to handle identical annotations but also show a strong bias toward well-annotated proteins. We propose a new method called weighted multipath measurement (WMM) for estimating the semantic similarity of gene products based on the structure of the GO. We not only considered the contribution of every path between two GO terms but also took the depth of the lowest common ancestors into account. We assigned different weights for different kinds of edges in GO graph. The similarity values calculated by WMM can be reused because they are only relative to the characteristics of GO terms. Experimental results showed that the similarity values obtained by WMM have a higher accuracy. We compared the performance of WMM with that of other methods using GO data and gene annotation datasets for yeast and humans downloaded from the GO database. We found that WMM is more suited for prediction of gene function than most existing IC-based methods and that it can distinguish proteins with identical annotations (two proteins are annotated with the same terms) from each other. PMID:25229994

  14. A Weighted Multipath Measurement Based on Gene Ontology for Estimating Gene Products Similarity

    PubMed Central

    Liu, Lizhen; Dai, Xuemin; Song, Wei; Lu, Jingli

    2014-01-01

    Abstract Many different methods have been proposed for calculating the semantic similarity of term pairs based on gene ontology (GO). Most existing methods are based on information content (IC), and the methods based on IC are used more commonly than those based on the structure of GO. However, most IC-based methods not only fail to handle identical annotations but also show a strong bias toward well-annotated proteins. We propose a new method called weighted multipath measurement (WMM) for estimating the semantic similarity of gene products based on the structure of the GO. We not only considered the contribution of every path between two GO terms but also took the depth of the lowest common ancestors into account. We assigned different weights for different kinds of edges in GO graph. The similarity values calculated by WMM can be reused because they are only relative to the characteristics of GO terms. Experimental results showed that the similarity values obtained by WMM have a higher accuracy. We compared the performance of WMM with that of other methods using GO data and gene annotation datasets for yeast and humans downloaded from the GO database. We found that WMM is more suited for prediction of gene function than most existing IC-based methods and that it can distinguish proteins with identical annotations (two proteins are annotated with the same terms) from each other. PMID:25229994

  15. 76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-16

    ... SERVICES Food and Drug Administration Guidance for Industry: Potency Tests for Cellular and Gene Therapy...: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance document provides manufacturers of cellular and gene therapy (CGT) products with recommendations for developing tests to...

  16. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells’ Transcription Factors

    PubMed Central

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barańao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

  17. Vitreoscilla hemoglobin gene ( vgb) improves lutein production in Chlorella vulgaris

    NASA Astrophysics Data System (ADS)

    Ma, Ruijuan; Lin, Xiangzhi

    2014-03-01

    Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene ( vgb) into Chlorella vulgaris by Agrobacterium tumefaciens -mediated transformation (ATMT). PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.

  18. Associations between human aldosterone synthase CYP11B2 (-344T/C) gene polymorphism and antihypertensive response to valsartan in Chinese patients with essential hypertension

    PubMed Central

    Ji, Xu; Qi, Hua; Li, Dong-Bao; Liu, Rong-Kun; Zheng, Yang; Chen, Hai-Ling; Guo, Jin-Cheng

    2015-01-01

    Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is associated with essential hypertension. But if the genetic variations in aldosterone synthesis could influence the antihypertensive response to Valsartan is not clear. A Chinese sample of 502 persons (217 women) was studied, which was divided into the hypertensive group (EH) of 345 persons and the normotensive group (NB) of 157 persons. Subjects were genotyped through the use of the polymerase chain reaction for the diallelic polymorphisms in CYP11B2. 98 persons of the essential hypertension group received 4 weeks therapy with valsartan. Blood pressure, 24-hour ambulatory blood pressure, biochemical index were also determined. The frequency of CC+CT genotypes in hypertensive group was significantly higher than that in normotensive group (P<0.05), the frequency of C allele of gene CYP11B2 (-344T/C) in hypertensive group was significantly higher than that in normotensive group (P<0.01). The descending values of SBP (systolic blood pressure), DBP (diastolic blood pressure), MAP (mean arterial pressure), 24 h SBP (mean SBP of 24 hours), 24 h DBP (mean DBP of 24 hours), 24 h MAP (mean arterial pressure of 24 hours) of CC+CT genotype group were significantly higher than those of the TT genotype group (P<0.05). The aldosterone synthase CYP11B2 (-344T/C) gene polymorphism is associated with essential hypertension in Chinese. And the aldosterone synthase CYP11B2 (-344T/C) gene polymorphism may be the predictor of the antihypertensive response to Valsartan. PMID:25785110

  19. Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

    PubMed

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M; Verstrepen, Kevin J

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that ?gpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts. PMID:25764309

  20. Glycerol Production by Fermenting Yeast Cells Is Essential for Optimal Bread Dough Fermentation

    PubMed Central

    Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M.; Verstrepen, Kevin J.

    2015-01-01

    Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that ?gpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts. PMID:25764309

  1. Variability in the Stability and Productivity of Transfected Genes in Chinese Hamster Ovary (CHO) cells

    E-print Network

    Ng, Say Kong

    In the field of biologics production, productivity and stability of the transfected gene of interest are two very important attributes that dictate if a production process is viable. To further understand and improve these ...

  2. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2015-09-29

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  3. Genes related to xylose fermentation and methods of using same for enhanced biofuel production

    SciTech Connect

    Wohlbach, Dana J.; Gasch, Audrey P.

    2014-08-05

    The present invention provides isolated gene sequences involved in xylose fermentation and related recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast useful for biofuel production are also provided.

  4. Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

    SciTech Connect

    Kondou, Youhei; Kitazawa, Daisuke; Takeda, Shigeki; Yamashita, Eiki; Mizuguchi, Mineyuki; Kawano, Keiichi; Tsukihara, Tomitake

    2005-01-01

    Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Ĺ. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Ĺ. The crystals diffract X-rays to at least 2.1 Ĺ resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Ĺ resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

  5. Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology.

    PubMed Central

    Kranz, R G; Gabbert, K K; Locke, T A; Madigan, M T

    1997-01-01

    Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available. The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus. In R. capsulatus, the phaAB genes are not linked to the phaC gene. Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome. Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level. Consistent with this conclusion, it was shown that the R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids. Grown on acetone, caproate, or heptanoate, wild-type R. capsulatus produced high levels of PHAs. Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present. The nutritional versatility and bioenergetic versatility of R. capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production. PMID:9251189

  6. fficient crop production requires an adequate supply of all essential plant nutrients. However,

    E-print Network

    cycle is a complex system involving the air, soil and plant. #12;pounds of nitrogen equivalent per acre, the use of commercial nitrogen (N) fertilizers to increase production, maintain profits and provide low- cost food and fiber is a necessity of modern agricul- ture. In general, crops need nitrogen

  7. mTORC1 Is Essential for Early Steps during Schwann Cell Differentiation of Amniotic Fluid Stem Cells and Regulates Lipogenic Gene Expression

    PubMed Central

    Schörghofer, David; Kinslechner, Katharina; Schütz, Birgit; Thi Thanh Pham, Ha; Rosner, Margit; Joo, Gabor Jozsef; Röhrl, Clemens; Weichhart, Thomas; Stangl, Herbert; Lubec, Gert; Hengstschläger, Markus; Mikula, Mario

    2014-01-01

    Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway. PMID:25221943

  8. Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system.

    PubMed

    Panina, Svetlana; Stephan, Alexander; la Cour, Jonas M; Jacobsen, Kivin; Kallerup, Line K; Bumbuleviciute, Rasita; Knudsen, Kristoffer V K; Sánchez-González, Pablo; Villalobo, Antonio; Olesen, Uffe H; Berchtold, Martin W

    2012-05-25

    Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr(99)/Tyr(138) or trimethylated at Lys(115) survived and grew well. CaM mutated at both Ca(2+) binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM. PMID:22493455

  9. Cell, Vol. 118, 3144, July 9, 2004, Copyright 2004 by Cell Press Exploration of Essential Gene Functions

    E-print Network

    Morris, Quaid

    et112 College Street al., 2000).Toronto, ON M5G 1L6 A major outcome of the deletions consortium) are essential for haploid viability under stan-1 Kings College Circle dard laboratory conditions (growth at 30 C biosynthesis, translation, cell wall3 Department of Biology and membrane biogenesis, DNA replication, nuclear

  10. Stock-outs of essential health products in Mozambique-longitudinal analyses from 2011 to 2013

    PubMed Central

    Wagenaar, Bradley H.; Gimbel, Sarah; Hoek, Roxanne; Pfeiffer, James; Michel, Cathy; Manuel, Joăo Luis; Cuembelo, Fatima; Quembo, Titos; Afonso, Pires; Gloyd, Stephen; Sherr, Kenneth

    2015-01-01

    objectives To assess the relationship between health system factors and facility-level EHP stock-outs in Mozambique. methods Service provisions were assessed in 26 health facilities and 13 district warehouses in Sofala Province, Mozambique, from July to August in 2011–2013. Generalised estimating equations were used to model factors associated with facility-level availability of essential drugs, supplies and equipment. results Stock-out rates for drugs ranged from 1.3% for oral rehydration solution to 20.5% for Depo-Provera and condoms, with a mean stock-out rate of 9.1%; mean stock-out rates were 15.4% for supplies and 4.1% for equipment. Stock-outs at the district level accounted for 27.1% (29/107) of facility-level drug stock-outs and 44.0% (37/84) of supply stock-outs. Each 10-km increase in the distance from district distribution warehouses was associated with a 31% (CI: 22–42%), 28% (CI: 17–40%) or 27% (CI: 7–50%) increase in rates of drug, supply or equipment stock-outs, respectively. The number of heath facility staff was consistently negatively associated with the occurrence of stock-outs. conclusions Facility-level stock-outs of EHPs in Mozambique are common and appear to disproportionately affect those living far from district capitals and near facilities with few health staff. The majority of facility-level EHP stock-outs in Mozambique occur when stock exists at the district distribution centre. Innovative methods are urgently needed to improve EHP supply chains, requesting and ordering of drugs, facility and district communication, and forecasting of future EHP needs in Mozambique. Increased investments in public-sector human resources for health could potentially decrease the occurrence of EHP stock-outs. PMID:24724617

  11. Association of Matrix Metalloproteinase-9 Gene ?1562C/T Polymorphism with Essential Hypertension: A Systematic Review and Meta-Analysis Article

    PubMed Central

    YANG, Wenchao; LU, Jiaojiao; YANG, Liu; ZHANG, Jinjin

    2015-01-01

    Background: Matrix metalloproteinase-9 (MMP-9) gene ?1562C/T polymorphism could regulate its expression level and thus affected people’s predisposition to essential hypertension. However, related studies yielded inconsistent or contradictory results. Methods: To evaluate the association of MMP-9 ?1562C/T polymorphism with essential hypertension, we performed a meta-analysis by combining all available independent case-control studies (n=6).A systematic literature search of PubMed, Web of Science, Scopus and CNKI (Chinese National Knowledge Infrastructure) databases was conducted by two researchers independently for all relevant articles published before March 2015. Results: MMP-9 ?1562C/T polymorphism was associated with essential hypertension under the allelic model (T vs.C, OR=1.36, 95% CI=1.17–1.59, P<0.0001). Subsequent sensitivity analysis confirmed the stability of the results. Such association was also observed in the dominant (TT+CT vs. CC, OR=1.30, 95% CI=1.10–1.54, P=0.002) and co-dominant (CT vs. TT+CC, OR=1.27, 95% CI=1.05–1.53, P=0.01) models but not in the recessive model (TT vs. CT+CC, OR=1.30, 95% CI=0.50–3.36, P=0.59). Conclusion: MMP-9 ?1562C/T polymorphism was associated with the risk of essential hypertension. PMID:26744701

  12. Spink13, an Epididymis-specific Gene of the Kazal-type Serine Protease Inhibitor (SPINK) Family, Is Essential for the Acrosomal Integrity and Male Fertility*

    PubMed Central

    Ma, Li; Yu, Heguo; Ni, Zimei; Hu, Shuanggang; Ma, Wubin; Chu, Chen; Liu, Qiang; Zhang, Yonglian

    2013-01-01

    Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives. PMID:23430248

  13. Effect of gibberellic acid and calliterpenone on plant growth attributes, trichomes, essential oil biosynthesis and pathway gene expression in differential manner in Mentha arvensis L.

    PubMed

    Bose, Subir K; Yadav, Ritesh Kumar; Mishra, Smrati; Sangwan, Rajender S; Singh, A K; Mishra, B; Srivastava, A K; Sangwan, Neelam S

    2013-05-01

    Extensive research is going on throughout the world to find out new molecules from natural sources to be used as plant growth promoter. Mentha arvensis L. is the main source of menthol rich essential oil used commercially in various food, pharmaceutical and other preparations. Experiments were conducted on field grown plants for understanding the effect of calliterpenone (CA), a stereo-isomer of abbeokutone, in comparison to gibberellic acid (GA3) on growth attributes, trichomes, essential oil biosynthesis and expression of some oil biosynthetic pathway genes. The exogenous application of CA (1 ?M, 10 ?M and 100 ?M) was found to be better in improving plant biomass and stolon yield, leaf area, branching and leaf stem ratio than with counterpart GA3 at the same concentrations. CA treated plants showed higher glandular trichome number, density and diameter and also correlated with enhanced oil biogenetic capacity as revealed by feeding labeled (14)C-sucrose for 72 h to excised shoots. Semi-quantitative PCR analysis of key pathway genes revealed differential up regulation under CA treatments. Transcript level of menthol dehydrogenase/menthone reductase was found highly up regulated in CA treated plants with increased content of menthone and menthol in oil. These findings demonstrate that CA positively regulated the yields by enhanced branching and higher density of trichomes resulting into higher accumulation of essential oil. The results suggest CA as a novel plant derived diterpenoid with growth promoting action and opens up new possibilities for improving the crop yields and essential oil biosynthesis in qualitative and quantitative manner. PMID:23514759

  14. Sesquiterpenes from the essential oil of Curcuma wenyujin and their inhibitory effects on nitric oxide production.

    PubMed

    Xia, Guiyang; Zhou, Li; Ma, Jianghao; Wang, Ying; Ding, Liqin; Zhao, Feng; Chen, Lixia; Qiu, Feng

    2015-06-01

    Three new sesquiterpenes including a new elemane-type sesquiterpene, 5?H-elem-1,3,7,8-tetraen-8,12-olide (1), and two new carabrane-type sesquiterpenes, 7?,11-epoxy-6?-methoxy-carabrane-4,8-dione (2) and 8,11-epidioxy-8-hydroxy-4-oxo-6-carabren (3), together with eight known sesquiterpenes (4-11) were isolated from Curcuma wenyujin Y. H. Chen et C. Ling. Their structures were elucidated based on extensive spectroscopic analyses. A possible biogenetic scheme for the related compounds was postulated. All of the isolated compounds were tested for inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages. Meanwhile, preliminary structure-activity relationships for these compounds are discussed. PMID:25819782

  15. G9a is essential for epigenetic silencing of K(+) channel genes in acute-to-chronic pain transition.

    PubMed

    Laumet, Geoffroy; Garriga, Judit; Chen, Shao-Rui; Zhang, Yuhao; Li, De-Pei; Smith, Trevor M; Dong, Yingchun; Jelinek, Jaroslav; Cesaroni, Matteo; Issa, Jean-Pierre; Pan, Hui-Lin

    2015-12-01

    Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K(+) channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. We found that nerve injury increased dimethylation of Lys9 on histone H3 (H3K9me2) at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect levels of DNA methylation on these genes in DRGs. Nerve injury increased activity of euchromatic histone-lysine N-methyltransferase-2 (G9a), histone deacetylases and enhancer of zeste homolog-2 (EZH2), but only G9a inhibition consistently restored K(+) channel expression. Selective knockout of the gene encoding G9a in DRG neurons completely blocked K(+) channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K(+) channels but also normalized 638 genes down- or upregulated by nerve injury. Thus G9a has a dominant function in transcriptional repression of K(+) channels and in acute-to-chronic pain transition after nerve injury. PMID:26551542

  16. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens

    E-print Network

    Li, Wei

    We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK demonstrates better ...

  17. Genetic variation in glutathione S-transferase genes and risk of nonfatal cerebral stroke in patients suffering from essential hypertension.

    PubMed

    Polonikov, Alexey; Vialykh, Ekaterina; Vasil'eva, Oksana; Bulgakova, Irina; Bushueva, Olga; Illig, Thomas; Solodilova, Maria

    2012-07-01

    Oxidative stress resulting from an increased amount of reactive oxygen species and an imbalance between oxidants and antioxidants has been implicated in pathogenesis of cerebral stroke. The purpose of this study was to investigate the relationship between common polymorphisms of glutathione S-transferase M1, T1, and P1 genes and risk of stroke in hypertensive individuals. A total of 667 unrelated Russian individuals with hypertension, including 306 hypertensives who suffered from cerebral stroke and 361 hypertensives who did not have cerebrovascular accidents, were recruited for the study. The deletion polymorphisms of GSTM1 and GSTT1 genes and polymorphism Ile105Val of the GSTP1 gene were genotyped by a multiplex polymerase chain reaction and restriction analyses, respectively. No differences in GSTM1 and GSTP1 genotype distributions between the cases and controls have been observed. The null GSTT1 genotype was found to be associated with increased risk of cerebral stroke after Bonferroni correction and adjusting for confounding variables such as gender, blood pressure, body mass index, and antihypertensive medication use (odds ratio 1.51 95 % CI 1.09-2.07, P?=?0.01). The present study was the first to show the association of null genotype of the GSTT1 gene with increased risk of cerebral stroke. PMID:22528457

  18. Genetic Dissection of the mamAB and mms6 Operons Reveals a Gene Set Essential for Magnetosome Biogenesis in Magnetospirillum gryphiswaldense

    PubMed Central

    Lohße, Anna; Borg, Sarah; Raschdorf, Oliver; Kolinko, Isabel; Tompa, Éva; Pósfai, Mihály; Faivre, Damien; Baumgartner, Jens

    2014-01-01

    Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlled by a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomic magnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that the five previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineralization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon. While all other deletions resulted in size reduction, elimination of either mms36 or mms48 caused the synthesis of magnetite crystals larger than those in the wild type (WT). Whereas the mms6 operon encodes accessory factors for crystal maturation, the large mamAB operon contains several essential and nonessential genes involved in various other steps of magnetosome biosynthesis, as shown by single deletions of all mamAB genes. While single deletions of mamL, -P, -Q, -R, -B, -S, -T, and -U showed phenotypes similar to those of their orthologs in a previous study in the related M. magneticum, we found mamI and mamN to be not required for at least rudimentary iron biomineralization in M. gryphiswaldense. Thus, only mamE, -L, -M, -O, -Q, and -B were essential for formation of magnetite, whereas a mamI mutant still biomineralized tiny particles which, however, consisted of the nonmagnetic iron oxide hematite, as shown by high-resolution transmission electron microscopy (HRTEM) and the X-ray absorption near-edge structure (XANES). Based on this and previous studies, we propose an extended model for magnetosome biosynthesis in M. gryphiswaldense. PMID:24816605

  19. Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense.

    PubMed

    Lohße, Anna; Borg, Sarah; Raschdorf, Oliver; Kolinko, Isabel; Tompa, Eva; Pósfai, Mihály; Faivre, Damien; Baumgartner, Jens; Schüler, Dirk

    2014-07-01

    Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlled by a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomic magnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that the five previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineralization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon. While all other deletions resulted in size reduction, elimination of either mms36 or mms48 caused the synthesis of magnetite crystals larger than those in the wild type (WT). Whereas the mms6 operon encodes accessory factors for crystal maturation, the large mamAB operon contains several essential and nonessential genes involved in various other steps of magnetosome biosynthesis, as shown by single deletions of all mamAB genes. While single deletions of mamL, -P, -Q, -R, -B, -S, -T, and -U showed phenotypes similar to those of their orthologs in a previous study in the related M. magneticum, we found mamI and mamN to be not required for at least rudimentary iron biomineralization in M. gryphiswaldense. Thus, only mamE, -L, -M, -O, -Q, and -B were essential for formation of magnetite, whereas a mamI mutant still biomineralized tiny particles which, however, consisted of the nonmagnetic iron oxide hematite, as shown by high-resolution transmission electron microscopy (HRTEM) and the X-ray absorption near-edge structure (XANES). Based on this and previous studies, we propose an extended model for magnetosome biosynthesis in M. gryphiswaldense. PMID:24816605

  20. Human mediator MED17 subunit plays essential roles in gene regulation by associating with the transcription and DNA repair machineries.

    PubMed

    Kikuchi, Yuko; Umemura, Hiroyasu; Nishitani, Saori; Iida, Satoshi; Fukasawa, Rikiya; Hayashi, Hiroto; Hirose, Yutaka; Tanaka, Aki; Sugasawa, Kaoru; Ohkuma, Yoshiaki

    2015-03-01

    In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER. PMID:25482373

  1. The Werner syndrome gene product (WRN): a repressor of hypoxia-inducible factor-1 activity.

    PubMed

    Labbé, Adam; Lafleur, Véronique N; Patten, David A; Robitaille, Genevičve A; Garand, Chantal; Lamalice, Laurent; Lebel, Michel; Richard, Darren E

    2012-08-15

    Werner syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of genes essential for adaptation to low oxygen conditions. HIF-1 is also implicated in the molecular mechanisms of ageing. Here, we show that the cellular depletion of WRN protein (by siRNA targeting) leads to increased HIF-1 complex stabilization and activation. HIF-1 activation in the absence of WRN involves the generation of mitochondrial reactive oxygen species (mtROS) since SkQ1, a mitochondrial-targeted antioxidant, and stigmatellin, an inhibitor of mitochondrial complex III, blocked increased HIF-1 levels. Ascorbate, an essential co-factor involved in HIF-1 stability, was decreased in WRN-depleted cells. Interestingly, expression levels of GLUT1, a known dehydroascorbic acid transporter, were also decreased in WRN-depleted cells. Ascorbate supplementation of WRN-depleted cells led to a dose-dependent inhibition of HIF-1 activation. These results indicate that WRN protein regulates HIF-1 activation by affecting mitochondrial ROS production and intracellular ascorbate levels. This work provides a novel mechanistic link between HIF-1 activity and different age-associated pathologies. PMID:22659133

  2. Gender-specific protective effect of the -463G>A polymorphism of myeloperoxidase gene against the risk of essential hypertension in Russians.

    PubMed

    Bushueva, Olga; Solodilova, Maria; Ivanov, Vladimir; Polonikov, Alexey

    2015-11-01

    The purpose of this study was to investigate whether a common polymorphism -463G>A (rs2333227) in the promoter of myeloperoxidase (MPO) gene, an oxidant enzyme producing hypohalogenic radicals, is associated with the risk of essential hypertension (EH) in Russian population. A total of 2044 unrelated subjects including 1256 EH patients and 788 normotensive controls were recruited for this study. Genotyping of the MPO gene polymorphism was done using TaqMan-based assay. A genotype -463GA was associated with decreased risk of EH (odds ratio = 0.82; 95% confidence interval: 0.68-1.00) at a borderline significance level (P = .05). The gender-stratified analysis showed that a carriage of the -463GA and -463AA genotypes is associated with decreased EH risk only in females (odds ratio = 0.74, 95% confidence interval: 0.56-0.96; P = .02). To the best of our knowledge, this is the first study reporting a negative association between the -463G>A polymorphism of the MPO gene and EH risk. Molecular mechanisms by which MPO gene is involved in the pathogenesis of EH are discussed. PMID:26431910

  3. The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

    PubMed

    Gazanion, E; Garcia, D; Silvestre, R; Gérard, C; Guichou, J F; Labesse, G; Seveno, M; Cordeiro-Da-Silva, A; Ouaissi, A; Sereno, D; Vergnes, B

    2011-10-01

    NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target. PMID:21819459

  4. Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

    PubMed Central

    Heath, D G; Cleary, P P

    1989-01-01

    The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins. PMID:2660147

  5. Functional genomics identifies novel genes essential for clear cell renal cell carcinoma tumor cell proliferation and migration

    PubMed Central

    von Roemeling, Christina A.; Marlow, Laura A.; Radisky, Derek C.; Rohl, Austin; Larsen, Hege E.; Wei, Johnny; Sasinowska, Heather; Zhu, Heng; Drake, Richard; Sasinowski, Maciek; Tun, Han W.; Copland, John A.

    2014-01-01

    Currently there is a lack of targeted therapies that lead to long-term attenuation or regression of disease in patients with advanced clear cell renal cell carcinoma (ccRCC). Our group has implemented a high-throughput genetic analysis coupled with a high-throughput proliferative screen in order to investigate the genetic contributions of a large cohort of overexpressed genes at the functional level in an effort to better understand factors involved in tumor initiation and progression. Patient gene array analysis identified transcripts that are consistently elevated in patient ccRCC as compared to matched normal renal tissues. This was followed by a high-throughput lentivirus screen, independently targeting 195 overexpressed transcripts identified in the gene array in four ccRCC cell lines. This revealed 31 ‘hits’ that contribute to ccRCC cell proliferation. Many of the hits identified are not only presented in the context of ccRCC for the first time, but several have not been previously linked to cancer. We further characterize the function of a group of hits in tumor cell invasion. Taken together these findings reveal pathways that may be critical in ccRCC tumorigenicity, and identifies novel candidate factors that could serve as targets for therapeutic intervention or diagnostic/prognostic biomarkers for patients with advanced ccRCC. PMID:24979721

  6. Comparative Genomics of Cultured and Uncultured Strains Suggests Genes Essential for Free-Living Growth of Liberibacter

    PubMed Central

    Fagen, Jennie R.; Leonard, Michael T.; McCullough, Connor M.; Edirisinghe, Janaka N.; Henry, Christopher S.; Davis, Michael J.; Triplett, Eric W.

    2014-01-01

    The full genomes of two uncultured plant pathogenic Liberibacter, Ca. Liberibacter asiaticus and Ca. Liberibacter solanacearum, are publicly available. Recently, the larger genome of a closely related cultured strain, Liberibacter crescens BT-1, was described. To gain insights into our current inability to culture most Liberibacter, a comparative genomics analysis was done based on the RAST, KEGG, and manual annotations of these three organisms. In addition, pathogenicity genes were examined in all three bacteria. Key deficiencies were identified in Ca. L. asiaticus and Ca. L. solanacearum that might suggest why these organisms have not yet been cultured. Over 100 genes involved in amino acid and vitamin synthesis were annotated exclusively in L. crescens BT-1. However, none of these deficiencies are limiting in the rich media used to date. Other genes exclusive to L. crescens BT-1 include those involved in cell division, the stringent response regulatory pathway, and multiple two component regulatory systems. These results indicate that L. crescens is capable of growth under a much wider range of conditions than the uncultured Liberibacter strains. No outstanding differences were noted in pathogenicity-associated systems, suggesting that L. crescens BT-1 may be a plant pathogen on an as yet unidentified host. PMID:24416233

  7. Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila

    PubMed Central

    Yuva-Aydemir, Yeliz; Xu, Xia-Lian; Aydemir, Ozkan; Gascon, Eduardo; Sayin, Serkan; Zhou, Wenke; Hong, Yang; Gao, Fen-Biao

    2015-01-01

    Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3’UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development. PMID:26000445

  8. A Polymorphism of the Renin Gene rs6682082 Is Associated with Essential Hypertension Risk and Blood Pressure Levels in Korean Women

    PubMed Central

    Park, Jongkeun; Song, Kijun; Jang, Yangsoo

    2015-01-01

    Purpose The aim of the present study was to investigate associations between the renin gene (REN) and the risk of essential hypertension and blood pressure (BP) levels in Koreans. Materials and Methods To outline the functional role of a single nucleotide polymorphism in the transcription of the REN gene, we conducted a case-control study of 1975 individuals: 646 hypertension (HT) patients and 1329 ethnically and age-matched normotensive subjects. Results Logistic regression analysis indicated that the genotypes AA/AG were strongly associated with risk of HT (odds ratio, 1.493; 95% confidence interval, 1.069-2.086, p=0.018) in female subjects. The genotypes AA/AG also showed significant association with higher blood pressure levels, both systolic and diastolic, in postmenopausal HT women (p=0.003 and p=0.017, respectively). Analysis of the promoter containing rs6682082 revealed a 2.4±0.01-fold higher activity in the A variant promoter than the G variant promoter, suggesting that rs6682082 is itself a functional variant. Conclusion We suggest that the A allele of rs6682082 is a positive genetic marker for predisposition to essential hypertension and high BP in Korean women and may be mediated through the transcriptional activation of REN. PMID:25510769

  9. Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila.

    PubMed

    Yuva-Aydemir, Yeliz; Xu, Xia-Lian; Aydemir, Ozkan; Gascon, Eduardo; Sayin, Serkan; Zhou, Wenke; Hong, Yang; Gao, Fen-Biao

    2015-05-01

    Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3'UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development. PMID:26000445

  10. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  11. Association of CT dinucleotide repeat polymorphism in the 5'-flanking region of the guanylyl cyclase (GC)-A gene with essential hypertension in the Japanese.

    PubMed

    Usami, Satoru; Kishimoto, Ichiro; Saito, Yoshihiko; Harada, Masaki; Kuwahara, Koichiro; Nakagawa, Yasuaki; Nakanishi, Michio; Yasuno, Shinji; Kangawa, Kenji; Nakao, Kazuwa

    2008-01-01

    Guanylyl cyclase (GC)-A (natriuretic peptide receptor [NPR]-1), the receptor for atrial and brain natriuretic peptide, is important in the regulation of blood pressure in animal models and, possibly, in humans. In this study, we examined the association between dinucleotide repeat polymorphism within the 5'-flanking region of the GC-A gene and essential hypertension in a group of Japanese subjects. By genotyping 177 hypertensive and 170 normotensive subjects, we identified 5 allele types with 6, 9, 10, 11 and 12 CT dinucleotide repeats, respectively, around position -293, upstream of the ATG codon in the human GC-A gene. The frequency of the (CT)n=6 allele was significantly higher among hypertensive than normotensive subjects, while the frequencies of the other allele types did not differ between the two groups. We also examined the linkage between G/A polymorphism at position -77 (rs13306004), downstream of the (CT)n polymorphism, and found that the (CT)n=6 allele was tightly linked to an A at position -77, while all other (CT)n alleles were linked to G. Promoter-reporter analyses carried out in cultured human aortic smooth muscle cells using a luciferase gene fused to the 5'-flanking region of the GC-A gene revealed that the promoter containing (CT)n=6 drove less transcriptional activity than that containing (CT)n=10. Finally, site-directed mutation showed that the (CT)n and G/A polymorphisms act synergistically to affect GC-A promoter activity. Our results thus define the (CT)n polymorphism in the 5'-flanking region of the GC-A gene as a potent and novel susceptibility marker for hypertension. PMID:18360023

  12. The dlx5a/dlx6a Genes Play Essential Roles in the Early Development of Zebrafish Median Fin and Pectoral Structures

    PubMed Central

    Heude, Églantine; Shaikho, Sarah; Ekker, Marc

    2014-01-01

    The Dlx5 and Dlx6 genes encode homeodomain transcription factors essential for the proper development of limbs in mammalian species. However, the role of their teleost counterparts in fin development has received little attention. Here, we show that dlx5a is an early marker of apical ectodermal cells of the pectoral fin buds and of the median fin fold, but also of cleithrum precursor cells during pectoral girdle development. We propose that early median fin fold establishment results from the medial convergence of dlx5a-expressing cells at the lateral edges of the neural keel. Expression analysis also shows involvement of dlx5a during appendage skeletogenesis. Using morpholino-mediated knock down, we demonstrate that disrupted dlx5a/6a function results in pectoral fin agenesis associated with misexpression of bmp4, fgf8a, and1 and msx genes. In contrast, the median fin fold presents defects in mesenchymal cell migration and actinotrichia formation, whereas the initial specification seems to occur normally. Our results demonstrate that the dlx5a/6a genes are essential for the induction of pectoral fin outgrowth, but are not required during median fin fold specification. The dlx5a/6a knock down also causes a failure of cleithrum formation associated with a drastic loss of runx2b and col10a1 expression. The data indicate distinct requirements for dlx5a/6a during median and pectoral fin development suggesting that initiation of unpaired and paired fin formation are not directed through the same molecular mechanisms. Our results refocus arguments on the mechanistic basis of paired appendage genesis during vertebrate evolution. PMID:24858471

  13. Oncogenic but non-essential role of N-myc downstream regulated gene 1 in the progression of esophageal squamous cell carcinoma

    PubMed Central

    Wei, Wei; Bracher-Manecke, Jacqueline C.; Zhao, Xiaohang; Davies, Neil H.; Zhou, Lanping; Ai, Runna; Oliver, Lisa; Vallette, Francois; Hendricks, Denver T.

    2013-01-01

    N-myc downstream regulated gene 1 (NDRG1/Cap43/Drg-1) has previously been shown to be dysregulated in esophageal squamous cell carcinoma (ESCC). In this study, we investigated the role of NDRG1 in the neoplastic progression of ESCC using ectopic gain-of-function and loss-of-function approaches. Stable transfectants of the KYSE30 ESCC cell line with altered NDRG1 levels were generated by lentiviral transduction. Although no measurable effects on in vitro cell proliferation were observed with altered NDRG1 expression, the ectopic overexpression of NDRG1 was positively linked to recognized markers of metastasis, angiogenesis and apoptotic evasion. Accordingly, in the nude mouse xenograft model system, NDRG1 overexpression promoted the in vivo growth of KYSE30 derived xenografts, which could be attributed to the reduced apoptotic and enhanced angiogenic activities associated with this gene. These processes were mediated in part by increased NF?B activity in NDRG1 overexpressing cells. Nevertheless, no significant phenotypic changes were observed in response to NDRG1 knock-down, suggesting that this gene might not be essential for the neoplastic progression of ESCC. Taken together, our results suggest that NDRG1 may play positive but dispensable roles in the progression of esophageal squamous cell carcinoma. PMID:23192272

  14. The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis.

    PubMed

    Lee, Chanhui; Zhong, Ruiqin; Richardson, Elizabeth A; Himmelsbach, David S; McPhail, Brooks T; Ye, Zheng-Hua

    2007-12-01

    Xylan, cellulose and lignin are the three major components of secondary walls in wood, and elucidation of the biosynthetic pathway of xylan is of importance for potential modification of secondary wall composition to produce wood with improved properties. So far, three Arabidopsis glycosyltransferases, FRAGILE FIBER8, IRREGULAR XYLEM8 and IRREGULAR XYLEM9, have been implicated in glucuronoxylan (GX) biosynthesis. In this study, we demonstrate that PARVUS, which is a member of family GT8, is required for the biosynthesis of the tetrasaccharide primer sequence, beta-D-Xyl-(1 --> 3)-alpha-l-Rha-(1 --> 2)-alpha-D-GalA-(1 --> 4)-D-Xyl, located at the reducing end of GX. The PARVUS gene is expressed during secondary wall biosynthesis in fibers and vessels, and its encoded protein is predominantly localized in the endoplasmic reticulum. Mutation of the PARVUS gene leads to a drastic reduction in secondary wall thickening and GX content. Structural analysis of GX using (1)H-nuclear magnetic resonance (NMR) spectroscopy revealed that the parvus mutation causes a loss of the tetrasaccharide primer sequence at the reducing end of GX and an absence of glucuronic acid side chains in GX. Activity assay showed that the xylan xylosyltransferase and glucuronyltransferase activities were not affected in the parvus mutant. Together, these findings implicate a possible role for PARVUS in the initiation of biosynthesis of the GX tetrasaccharide primer sequence and provide novel insights into the mechanisms of GX biosynthesis. PMID:17991630

  15. Extraction and refining of essential oil from Australian tea tree, Melaleuca alterfornia, and the antimicrobial activity in cosmetic products

    NASA Astrophysics Data System (ADS)

    Huynh, Q.; Phan, T. D.; Thieu, V. Q. Q.; Tran, S. T.; Do, S. H.

    2012-03-01

    Tea tree oil (TTO) comes from the leaves of Melaleuca alternifornia that belongs to the myrtle family (Myrtaceae). It is one of the most powerful immune system stimulants and sorts out most viral, bacterial and fungal infections in a snap, while it is great to heal wounds and acnes. In Vietnam, Melaleuca trees can grow on acid land that stretches in a large portion of lands in the Mekong Delta region. So, there are some Melaleuca plantations developed under the Vietnamese government plans of increasing plantation forests now. However, TTO contains various amounts of 1,8-cineole that causes skin irritant. So TTO purification is very necessary. In this study, the purification of TTO that meet International Standard ISO 4730 was carried out via two steps. The first step is steam distillation to obtain crude TTO (terpinen-4-ol 35% v/v) and the average productivity is among 2.37% (v/wet-wt) or 1.23% (v/dry-wt). In the second step, the cleaned TTO is collected by vacuum distillation column and extraction yield of the whole process is about 0.3% (w/w). Besides, high concentration essential oil was applied in the cosmetic products to increase its commercial value.

  16. Transition to farming more likely for small, conservative groups with property rights, but increased productivity is not essential.

    PubMed

    Gallagher, Elizabeth M; Shennan, Stephen J; Thomas, Mark G

    2015-11-17

    Theories for the origins of agriculture are still debated, with a range of different explanations offered. Computational models can be used to test these theories and explore new hypotheses; Bowles and Choi [Bowles S, Choi J-K (2013) Proc Natl Acad Sci USA 110(22):8830-8835] have developed one such model. Their model shows the coevolution of farming and farming-friendly property rights, and by including climate variability, replicates the timings for the emergence of these events seen in the archaeological record. Because the processes modeled occurred a long time ago, it can be difficult to justify exact parameter values; hence, we propose a fitting to idealized outcomes (FIO) method to explore the model's parameter space in more detail. We have replicated the model of Bowles and Choi, and used the FIO method to identify complexities and interactions of the model previously unidentified. Our results indicate that the key parameters for the emergence of farming are group structuring, group size, conservatism, and farming-friendly property rights (lending further support to Bowles and Choi's original proposal). We also find that although advantageous, it is not essential that farming productivity be greater than foraging productivity for farming to emerge. In addition, we highlight how model behaviors can be missed when gauging parameter sensitivity via a fix-all-but-one variation approach. PMID:26578766

  17. Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

    PubMed

    Chen, Y; Weeks, J; Mortin, M A; Greenleaf, A L

    1993-07-01

    We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype. PMID:8321225

  18. Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

    PubMed Central

    Chen, Y; Weeks, J; Mortin, M A; Greenleaf, A L

    1993-01-01

    We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype. Images PMID:8321225

  19. Fumigant Toxicity of Essential Oils from Basil and Spearmint Against Two Major Pyralid Pests of Stored Products.

    PubMed

    Eliopoulos, P A; Hassiotis, C N; Andreadis, S S; Porichi, A-E E

    2015-04-01

    The fumigant activity of essential oil vapors distilled from sweet basil Ocimum basilicum L. and spearmint Mentha spicata L. (Lamiaceae) were tested against two major stored products pests Ephestia kuehniella (Zeller) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). Various oil doses (0.5, 2.5, 5, 50, 250, 500, 1,000, and 1,500 µl/liter air), for an exposure period of 24?h, were tested. The essential oils were subjected to gas chromatography-mass spectrometry analysis and revealed that the major compounds were for spearmint oil carvone (67.1%) and limonene (+1,8 cineole; 14.3%) and for basil oil linalool (45.9%), 1,8 cineole (16.7%) and eugenol (10.3%). Apart from a few exceptions, no significant differences in insecticidal action were observed between basil and spearmint oil. Both oils were highly effective against adult moths, given that notable mortality (>80%) was recorded after exposure to low doses such as 2.5?µl/liter. Noteworthy, egg mortality was also recorded, reaching 73-79% for basil and 56-60% for spearmint. Toxicity data indicated that larvae and pupae were the most tolerant stages in all cases. Larval mortality never exceeded 21 and 18%, for basil and spearmint, respectively, irrespective of moth species. Basil and spearmint oils displayed mortalities as high as 38 and 28% in pupae. Lethal doses (LD50 and LD99) values were estimated via probit analysis. Developmental stage proved to be a significant factor, whereas the effect of oil species on insect mortality was insignificant. With the exception of adult individuals, basil and spearmint oils did not show satisfactory overall insecticidal activity against E. kuehniella and P. interpunctella. PMID:26470193

  20. Association of circadian genes with diurnal blood pressure changes and non-dipper essential hypertension: a genetic association with young-onset hypertension.

    PubMed

    Leu, Hsin-Bang; Chung, Chia-Min; Lin, Shing-Jong; Chiang, Kuang-Mao; Yang, Hsin-Chou; Ho, Hung-Yun; Ting, Chih-Tai; Lin, Tsung-Hsien; Sheu, Sheng-Hsiung; Tsai, Wei-Chuan; Chen, Jyh-Hong; Yin, Wei-Hsian; Chiu, Ting-Yu; Chen, Chin-Iuan; Fann, Cathy Sj; Chen, Yuan-Tsong; Pan, Wen-Harn; Chen, Jaw-Wen

    2015-02-01

    Recent studies have suggested that circadian genes have important roles in maintaining the circadian rhythm of the cardiovascular system. However, the associations between diurnal BP changes and circadian genes remain undetermined. We conducted a genetic association study of young-onset hypertension, in which 24-h ambulatory blood pressure (BP) monitoring was performed. A total of 23 tag single-nucleotide polymorphisms (SNPs) on 11 genes involved in circadian rhythms were genotyped for correlations with diurnal BP variation phenotypes. A permutation test was used to correct for multiple testing. Five tag SNPs within five loci, including rs3888170 in NPAS2, rs6431590 in PER2, rs1410225 in ROR??, rs3816358 in BMAL1 and rs10519096 in ROR?, were significantly associated with the non-dipper phenotype in 372 young hypertensive patients. A genetic risk score was generated by counting the risk alleles and effects for each individual. Genotyping was performed in an additional independent set of 619 young-onset hypertensive subjects. Altogether, non-dippers had a higher weighted genetic risk score than dippers (1.67±0.56 vs. 1.54±0.55, P<0.001), and the additive genetic risk score also indicated a graded association with decreased diurnal BP changes (P=0.006), as well as a non-dipper phenotype (P=0.031). After multivariable logistic analysis, only the circadian genetic risk score (odds ratio (OR), 1550; 95% confidence interval (CI), 1.225-1.961, P<0.001) and the use of ?-blockers (OR, 1.519; 95% CI, 1.164-1.982, P=0.003) were independently associated with the presence of non-dippers among subjects with young-onset hypertension. Genetic variants in circadian genes were associated with the diurnal phenotype of hypertension, suggesting a genetic association with diurnal BP changes in essential hypertension. PMID:25410879

  1. Identification of potentially hazardous human gene products in GMO risk assessment.

    PubMed

    Bergmans, Hans; Logie, Colin; Van Maanen, Kees; Hermsen, Harm; Meredyth, Michelle; Van Der Vlugt, Cécile

    2008-01-01

    Genetically modified organisms (GMOs), e.g. viral vectors, could threaten the environment if by their release they spread hazardous gene products. Even in contained use, to prevent adverse consequences, viral vectors carrying genes from mammals or humans should be especially scrutinized as to whether gene products that they synthesize could be hazardous in their new context. Examples of such potentially hazardous gene products (PHGPs) are: protein toxins, products of dominant alleles that have a role in hereditary diseases, gene products and sequences involved in genome rearrangements, gene products involved in immunomodulation or with an endocrine function, gene products involved in apoptosis, activated proto-oncogenes. For contained use of a GMO that carries a construct encoding a PHGP, the precautionary principle dictates that safety measures should be applied on a "worst case" basis, until the risks of the specific case have been assessed. The potential hazard of cloned genes can be estimated before empirical data on the actual GMO become available. Preliminary data may be used to focus hazard identification and risk assessment. Both predictive and empirical data may also help to identify what further information is needed to assess the risk of the GMO. A two-step approach, whereby a PHGP is evaluated for its conceptual dangers, then checked by data bank searches, is delineated here. PMID:18384725

  2. Requirements for Clinical Trials with Gene Therapy and Transplant Products in Switzerland.

    PubMed

    Marti, Andreas

    2015-01-01

    This chapter aims to describe and summarize the regulation of gene and cell therapy products in Switzerland and its legal basis. Product types are briefly described, as are Swiss-specific terminologies such as the term "transplant product," which means products manufactured from cells, tissues, or even whole organs. Although some parts of this chapter may show a guideline character, they are not legally binding, but represent the current thinking of Swissmedic, the Swiss Agency for Therapeutic Products. As so far the experience with marketing approval of gene therapy and cell therapy products in Switzerland is limited, this chapter focuses on the regulation of clinical trials conducted with these products. Quality, nonclinical, and clinical aspects are summarized separately for gene therapy products and transplant products. PMID:26374216

  3. Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation.

    PubMed

    van Workum, W A; Canter Cremers, H C; Wijfjes, A H; van der Kolk, C; Wijffelman, C A; Kijne, J W

    1997-03-01

    Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent. PMID:9057334

  4. The anti-apoptotic gene Anamorsin is essential for both autonomous and extrinsic regulation of murine fetal liver hematopoiesis.

    PubMed

    Tanimura, Akira; Shibayama, Hirohiko; Hamanaka, Yuri; Fujita, Natsuko; Ishibashi, Tomohiko; Sudo, Takao; Yokota, Takafumi; Ezoe, Sachiko; Tanaka, Hirokazu; Matsumura, Itaru; Oritani, Kenji; Kanakura, Yuzuru

    2014-05-01

    Anamorsin (AM) is an antiapoptotic molecule that confers factor-independent survival on hematopoietic cells. AM-deficient (AM(-/-)) mice are embryonic lethal because of a defect in definitive hematopoiesis; however, the significance of AM in embryonic hematopoiesis remains unknown. This study characterized the hematopoietic defects in AM(-/-) fetal livers. The AM(-/-) fetal liver displayed significantly reduced numbers of c-Kit(+)Sca-1(+)Lin(-) (KSL) cells. An in vitro colony-forming unit assay showed that fetal liver cells isolated from AM(-/-) embryos gave rise to fewer colonies in all cell types. The reconstitution activity in AM(-/-) hematopoietic stem cells (HSCs) was markedly reduced in all lineages. Furthermore, the limiting dilution assay revealed that the number of fetal liver HSCs was reduced because of AM deficiency. Retrovirus-mediated AM expression rescued the defective hematopoietic colony-forming activities of AM(-/-) KSL cells. We also investigated the effects of AM deficiency on fetal liver stromal cells, which support hematopoiesis. Interestingly, primary stromal cell cultures from wild type fetal liver supported the growth of AM(-/-) KSL cells, but stromal cultures from AM(-/-) fetal liver provided little support of wild type KSL cell growth. These results demonstrated that AM was essential for both autonomous and extrinsic regulation of fetal liver hematopoiesis. This study provided new insight into the molecular regulation of hematopoiesis. PMID:24440520

  5. The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.

    PubMed

    Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S; Lu, Yanling; Iyer, K Swaminathan; Trewhella, Jill; Fox, Archa H; Bond, Charles S

    2015-04-20

    SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Ĺ long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

  6. The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

    PubMed Central

    Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S.; Lu, Yanling; Iyer, K. Swaminathan; Trewhella, Jill; Fox, Archa H.; Bond, Charles S.

    2015-01-01

    SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Ĺ long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

  7. Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation.

    PubMed

    Tsuge, Yota; Yamamoto, Shogo; Kato, Naoto; Suda, Masako; Vertčs, Alain A; Yukawa, Hideaki; Inui, Masayuki

    2015-06-01

    We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale. PMID:25820644

  8. Mouse BAZ1A (ACF1) Is Dispensable for Double-Strand Break Repair but Is Essential for Averting Improper Gene Expression during Spermatogenesis

    PubMed Central

    Dowdle, James A.; Mehta, Monika; Kass, Elizabeth M.; Vuong, Bao Q.; Inagaki, Akiko; Egli, Dieter; Jasin, Maria; Keeney, Scott

    2013-01-01

    ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations. PMID:24244200

  9. Essential Tremor

    MedlinePLUS

    ... NINDS NINDS Essential Tremor Information Page Table of Contents (click to jump to sections) What is Essential ... 387-3667 Fax: 913-341-1296 Tremor Action Network P.O. Box 5013 Pleasanton, CA 94566-5013 ...

  10. Enhanced Hydrogen Production in Escherichia coli Through Chemical Mutagenesis, Gene Deletion, and Transposon Mutagenesis 

    E-print Network

    Garzon Sanabria, Andrea Juliana

    2011-08-08

    be increased by random mutagenesis using N-methyl-N?-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be further increased in the chemically-mutagenized strain by targeted gene deletion and overexpression of genes related to formate...), from chemical mutagen 3/86 increased hydrogen production 188 ? 0.50-fold (relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of formate hydrogen lyase (FHL), increased hydrogen production 232 ? 0.50-fold...

  11. Conservation of the coding regions of the glycine N-acyltransferase gene further suggests that glycine conjugation is an essential detoxification pathway.

    PubMed

    van der Sluis, Rencia; Badenhorst, Christoffel P S; Erasmus, Elardus; van Dyk, Etresia; van der Westhuizen, Francois H; van Dijk, Alberdina A

    2015-10-15

    Thorough investigation of the glycine conjugation pathway has been neglected. No defect of the glycine conjugation pathway has been reported and this could reflect the essential role of glycine conjugation in hepatic metabolism. Therefore, we hypothesised that genetic variation in the open reading frame (ORF) of the GLYAT gene should be low and that deleterious alleles would be found at low frequencies. This hypothesis was investigated by analysing the genetic variation of the human GLYAT ORF using data available in public databases. We also sequenced the GLYAT ORF of a small cohort of South African Afrikaner Caucasian individuals. In total, data from 1537 individuals was analysed. The two most prominent GLYAT haplotypes in all populations analysed, were S156 (70%) and T17S156 (20%). The S156C199 and S156H131 haplotypes, which have a negative effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. In the Afrikaner Caucasian cohort a novel Q61L SNP occurring at a high frequency (12%) was detected. The results of this study indicated that the GLYAT ORF is highly conserved and supported the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. These findings emphasise the importance of future investigations to determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. PMID:26149650

  12. Effects of Essential Oils on Methane Production and Fermentation by, and Abundance and Diversity of, Rumen Microbial Populations

    PubMed Central

    Patra, Amlan K.

    2012-01-01

    Five essential oils (EOs), namely, clove oil (CLO), eucalyptus oil (EUO), garlic oil (GAO), origanum oil (ORO), and peppermint oil (PEO), were tested in vitro at 3 different doses (0.25, 0.50, and 1.0 g/liter) for their effect on methane production, fermentation, and select groups of ruminal microbes, including total bacteria, cellulolytic bacteria, archaea, and protozoa. All the EOs significantly reduced methane production with increasing doses, with reductions by 34.4%, 17.6%, 42.3%, 87%, and 25.7% for CLO, EUO, GAO, ORO, and PEO, respectively, at 1.0 g/liter compared with the control. However, apparent degradability of dry matter and neutral detergent fiber also decreased linearly with increasing doses by all EOs except GAO. The concentrations of total volatile fatty acids were not affected by GAO, EUO, or PEO but altered linearly and quadratically by CLO and ORO, respectively. All the EOs also differed in altering the molar proportions of acetate, propionate, and butyrate. As determined by quantitative real-time PCR, all the EOs decreased the abundance of archaea, protozoa, and major cellulolytic bacteria (i.e., Fibrobacter succinogenes, Ruminococcus flavefaciens, and R. albus) linearly with increasing EO doses. On the basis of denaturing gradient gel electrophoresis analysis, different EOs changed the composition of both archaeal and bacterial communities to different extents. The Shannon-Wiener diversity index (H?) was reduced for archaea by all EOs in a dose-dependent manner but increased for bacteria at low and medium doses (0.25 and 0.50 g/liter) for all EOs except ORO. Due to the adverse effects on feed digestion and fermentation at high doses, a single EO may not effectively and practically mitigate methane emission from ruminants unless used at low doses in combinations with other antimethanogenic compounds. PMID:22492451

  13. Effects of essential oils on methane production and fermentation by, and abundance and diversity of, rumen microbial populations.

    PubMed

    Patra, Amlan K; Yu, Zhongtang

    2012-06-01

    Five essential oils (EOs), namely, clove oil (CLO), eucalyptus oil (EUO), garlic oil (GAO), origanum oil (ORO), and peppermint oil (PEO), were tested in vitro at 3 different doses (0.25, 0.50, and 1.0 g/liter) for their effect on methane production, fermentation, and select groups of ruminal microbes, including total bacteria, cellulolytic bacteria, archaea, and protozoa. All the EOs significantly reduced methane production with increasing doses, with reductions by 34.4%, 17.6%, 42.3%, 87%, and 25.7% for CLO, EUO, GAO, ORO, and PEO, respectively, at 1.0 g/liter compared with the control. However, apparent degradability of dry matter and neutral detergent fiber also decreased linearly with increasing doses by all EOs except GAO. The concentrations of total volatile fatty acids were not affected by GAO, EUO, or PEO but altered linearly and quadratically by CLO and ORO, respectively. All the EOs also differed in altering the molar proportions of acetate, propionate, and butyrate. As determined by quantitative real-time PCR, all the EOs decreased the abundance of archaea, protozoa, and major cellulolytic bacteria (i.e., Fibrobacter succinogenes, Ruminococcus flavefaciens, and R. albus) linearly with increasing EO doses. On the basis of denaturing gradient gel electrophoresis analysis, different EOs changed the composition of both archaeal and bacterial communities to different extents. The Shannon-Wiener diversity index (H') was reduced for archaea by all EOs in a dose-dependent manner but increased for bacteria at low and medium doses (0.25 and 0.50 g/liter) for all EOs except ORO. Due to the adverse effects on feed digestion and fermentation at high doses, a single EO may not effectively and practically mitigate methane emission from ruminants unless used at low doses in combinations with other antimethanogenic compounds. PMID:22492451

  14. Role of leptin receptor gene polymorphisms in susceptibility to the development of essential hypertension: a case-control association study in a Northern Han Chinese population.

    PubMed

    Liu, Y; Lou, Y Q; Liu, K; Liu, J L; Wang, Z G; Wen, J; Zhao, Q; Wen, S J; Xiao, L

    2014-09-01

    In order to explore the potential association between the leptin receptor (LEPR) gene polymorphisms and essential hypertension (EH) risk in the Northern Han Chinese population, we recruited 823 hypertensive subjects and 491 healthy control subjects from the Northern Han Chinese. Genotyping was performed to identify the Lys109Arg, Gln223Arg and Lys656Asn polymorphisms of the LEPR gene. Significant associations were found in a dominant genetic model ([GG+AG] vs. AA), P=0.007, odds ratio (OR)=3.697, 95% confidence interval (CI) 1.442-9.482), and in homozygote comparison (GG vs. AA, P=0.005, OR=3.890, 95% CI 1.501-10.077) for the Gln223Arg polymorphism. No significant association could be found between Lys109Arg or Lys656Asn polymorphism and EH risk. Linkage disequilibrium was detected between the Lys109Arg and Gln223Arg polymorphisms, and haplotype analyses identified that the G-A haplotype was a protective haplotype for EH. Our studies demonstrated that the LEPR Gln223Arg polymorphism had an important role in a patient's susceptibility to EH in the Northern Han Chinese population. PMID:24522342

  15. Role of leptin receptor gene polymorphisms in susceptibility to the development of essential hypertension: a case–control association study in a Northern Han Chinese population

    PubMed Central

    Liu, Y; Lou, Y-Q; Liu, K; Liu, J-L; Wang, Z-G; Wen, J; Zhao, Q; Wen, S-J; Xiao, L

    2014-01-01

    In order to explore the potential association between the leptin receptor (LEPR) gene polymorphisms and essential hypertension (EH) risk in the Northern Han Chinese population, we recruited 823 hypertensive subjects and 491 healthy control subjects from the Northern Han Chinese. Genotyping was performed to identify the Lys109Arg, Gln223Arg and Lys656Asn polymorphisms of the LEPR gene. Significant associations were found in a dominant genetic model ([GG+AG] vs AA), P=0.007, odds ratio (OR)=3.697, 95% confidence interval (CI) 1.442–9.482), and in homozygote comparison (GG vs AA, P=0.005, OR=3.890, 95% CI 1.501–10.077) for the Gln223Arg polymorphism. No significant association could be found between Lys109Arg or Lys656Asn polymorphism and EH risk. Linkage disequilibrium was detected between the Lys109Arg and Gln223Arg polymorphisms, and haplotype analyses identified that the G-A haplotype was a protective haplotype for EH. Our studies demonstrated that the LEPR Gln223Arg polymorphism had an important role in a patient's susceptibility to EH in the Northern Han Chinese population. PMID:24522342

  16. Extragenic bypass suppressors of mutations in the essential gene BLD2 promote assembly of basal bodies with abnormal microtubules in Chlamydomonas reinhardtii.

    PubMed Central

    Preble, A M; Giddings, T H; Dutcher, S K

    2001-01-01

    bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function. PMID:11139500

  17. The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

    PubMed Central

    Jamet, Stevie; Slama, Nawel; Domingues, Joana; Laval, Françoise; Texier, Pauline; Eynard, Nathalie; Quémard, Annaik; Peixoto, Antonio; Lemassu, Anne; Daffé, Mamadou; Cam, Kaymeuang

    2015-01-01

    Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs. PMID:26701652

  18. Lovastatin in Aspergillus terreus: Fermented Rice Straw Extracts Interferes with Methane Production and Gene Expression in Methanobrevibacter smithii

    PubMed Central

    Liang, Juan Boo; Ho, Yin Wan; Mohamad, Rosfarizan; Goh, Yong Meng; Shokryazdan, Parisa; Chin, James

    2013-01-01

    Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants. PMID:23710454

  19. Basal promoter of the rat connexin 32 gene: identification and characterization of an essential element and its DNA-binding protein.

    PubMed Central

    Bai, S; Schoenfeld, A; Pietrangelo, A; Burk, R D

    1995-01-01

    The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32-B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver. PMID:7862137

  20. Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations

    SciTech Connect

    Moore, R.C.; Redhead, N.J.; Selfridge, J.

    1995-09-01

    We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies. 43 refs., 6 figs., 1 tab.

  1. The dsbB gene product is required for protease production by Burkholderia cepacia.

    PubMed Central

    Abe, M; Nakazawa, T

    1996-01-01

    Burkholderia cepacia KF1, isolated from a pneumonia patient, produces a 37-kDa extracellular metalloprotease. A protease-deficient and lipase-proficient mutant, KFT1007, was complemented by a clone having an open reading frame coding for a 170-amino-acid polypeptide which showed significant homology to Escherichia coli DsbB. KFT1007, a presumed dsbB mutant, also failed to show motility, and both protease secretion and motility were restored by the introduction of the cloned dsbB gene of B. cepacia. The mutant KFT1007 excreted a 43-kDa polypeptide that is immunologically related to the 37-kDa mature protease. These results suggested that the dsbB mutant secretes a premature and catalytically inactive form of protease and that disulfide formation is required for the production of extracellular protease by B. cepacia. PMID:8926116

  2. Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product.

    PubMed Central

    Fukui, Y; Miyake, S; Satoh, M; Yamamoto, M

    1989-01-01

    Mutations in the Schizosaccharomyces pombe ral2 gene cause a phenotype indistinguishable from that of the ras1-defective mutant. Using cloned ral2 DNA, we disrupted the chromosomal gene. The disruptants showed the same phenotype as the original ral2 isolates, i.e., they had spherical cells, had no detectable mating activity, and exhibited no response to the mating pheromone, but their vegetative growth was apparently normal. Sequence analysis of the ral2 gene suggests that it encodes a polypeptide of 611 amino acid residues whose predicted amino acid sequence shows no strong homology to any known protein. Either multiple copies or even a single copy of the ras1Val-17 allele, which is an activated form of ras1, restored rodlike cell morphology and ability to respond to the mating factor to ral2 mutants. These results suggest that the ral2 and ras1 gene products interact intimately and that the ral2 gene product is involved in activation of the ras1 protein in S. pombe. Images PMID:2586528

  3. The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides.

    PubMed

    Zhang, Yueping; Choi, Yoon-E; Zou, Xuexiao; Xu, Jin-Rong

    2011-02-01

    Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity. PMID:20887797

  4. Comparative genomics of actinomycetes with a focus on natural product biosynthetic genes

    PubMed Central

    2013-01-01

    Background Actinomycetes are a diverse group of medically, industrially and ecologically important bacteria, studied as much for the diseases they cause as for the cures they hold. The genomes of actinomycetes revealed that these bacteria have a large number of natural product gene clusters, although many of these are difficult to tie to products in the laboratory. Large scale comparisons of these clusters are difficult to perform due to the presence of highly similar repeated domains in the most common biosynthetic machinery: polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). Results We have used comparative genomics to provide an overview of the genomic features of a set of 102 closed genomes from this important group of bacteria with a focus on natural product biosynthetic genes. We have focused on well-represented genera and determine the occurrence of gene cluster families therein. Conservation of natural product gene clusters within Mycobacterium, Streptomyces and Frankia suggest crucial roles for natural products in the biology of each genus. The abundance of natural product classes is also found to vary greatly between genera, revealing underlying patterns that are not yet understood. Conclusions A large-scale analysis of natural product gene clusters presents a useful foundation for hypothesis formulation that is currently underutilized in the field. Such studies will be increasingly necessary to study the diversity and ecology of natural products as the number of genome sequences available continues to grow. PMID:24020438

  5. Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics

    SciTech Connect

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. S.; Ahring, Birgitte K.; Linggi, Bryan E.

    2013-06-19

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. We found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.

  6. Id-1 and Id-2 genes and products as markers of epithelial cancer

    DOEpatents

    Desprez, Pierre-Yves (El Cerrito, CA); Campisi, Judith (Berkeley, CA)

    2011-10-04

    A method for detection and prognosis of breast cancer and other types of cancer. The method comprises detecting expression, if any, for both an Id-1 and an Id-2 genes, or the ratio thereof, of gene products in samples of breast tissue obtained from a patient. When expressed, Id-1 gene is a prognostic indicator that breast cancer cells are invasive and metastatic, whereas Id-2 gene is a prognostic indicator that breast cancer cells are localized and noninvasive in the breast tissue.

  7. Id-1 and Id-2 genes and products as markers of epithelial cancer

    DOEpatents

    Desprez, Pierre-Yves (El Cerrito, CA); Campisi, Judith (Berkeley, CA)

    2008-09-30

    A method for detection and prognosis of breast cancer and other types of cancer. The method comprises detecting expression, if any, for both an Id-1 and an Id-2 genes, or the ratio thereof, of gene products in samples of breast tissue obtained from a patient. When expressed, Id-1 gene is a prognostic indicator that breast cancer cells are invasive and metastatic, whereas Id-2 gene is a prognostic indicator that breast cancer cells are localized and noninvasive in the breast tissue.

  8. Major genes and QTL influencing wool production and quality: a review

    PubMed Central

    2005-01-01

    The opportunity exists to utilise our knowledge of major genes that influence the economically important traits in wool sheep. Genes with Mendelian inheritance have been identified for many important traits in wool sheep. Of particular importance are genes influencing pigmentation, wool quality and the keratin proteins, the latter of which are important for the morphology of the wool fibre. Gene mapping studies have identified some chromosomal regions associated with variation in wool quality and production traits. The challenge now is to build on this knowledge base in a cost-effective way to deliver molecular tools that facilitate enhanced genetic improvement programs for wool sheep. PMID:15601598

  9. Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I

    SciTech Connect

    Trucksis, M.; Depew, R.E.

    1981-04-01

    A gene that specifies production of Escherichia coli DNA topoisomerase I (..omega.. protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli ..omega.. protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX.

  10. The STM4195 Gene Product (PanS) Transports Coenzyme A Precursors in Salmonella enterica

    PubMed Central

    Ernst, Dustin C.

    2015-01-01

    ABSTRACT Coenzyme A (CoA) is a ubiquitous coenzyme involved in fundamental metabolic processes. CoA is synthesized from pantothenic acid by a pathway that is largely conserved among bacteria and eukaryotes and consists of five enzymatic steps. While higher organisms, including humans, must scavenge pantothenate from the environment, most bacteria and plants are capable of de novo pantothenate biosynthesis. In Salmonella enterica, precursors to pantothenate can be salvaged, but subsequent intermediates are not transported due to their phosphorylated state, and thus the pathway from pantothenate to CoA is considered essential. Genetic analyses identified the STM4195 gene product of Salmonella enterica serovar Typhimurium as a transporter of pantothenate precursors, ketopantoate and pantoate and, to a lesser extent, pantothenate. Further results indicated that STM4195 transports a product of CoA degradation that serves as a precursor to CoA and enters the biosynthetic pathway between PanC and CoaBC (dfp). The relevant CoA derivative is distinguishable from pantothenate, pantetheine, and pantethine and has spectral properties indicating the adenine moiety of CoA is intact. Taken together, the results presented here provide evidence of a transport mechanism for the uptake of ketopantoate, pantoate, and pantothenate and demonstrate a role for STM4195 in the salvage of a CoA derivative of unknown structure. The STM4195 gene is renamed panS to reflect participation in pantothenate salvage that was uncovered herein. IMPORTANCE This manuscript describes a transporter for two pantothenate precursors in addition to the existence and transport of a salvageable coenzyme A (CoA) derivative. Specifically, these studies defined a function for an STM protein in S. enterica that was distinct from the annotated role and led to its designation as PanS (pantothenate salvage). The presence of a salvageable CoA derivative and a transporter for it suggests the possibility that this compound is present in the environment and may serve a role in CoA synthesis for some organisms. As such, this work raises important question about CoA salvage that can be pursued with future studies in bacteria and other organisms. PMID:25645561

  11. Rapid effects of essential fatty acid deficiency on growth and development parameters and transcription of key fatty acid metabolism genes in juvenile barramundi (Lates calcarifer).

    PubMed

    Salini, Michael J; Turchini, Giovanni M; Wade, Nicholas M; Glencross, Brett D

    2015-12-01

    Barramundi (Lates calcarifer), a catadromous teleost of significant and growing commercial importance, are reported to have limited fatty acid bioconversion capability and therefore require preformed long-chain PUFA (LC-PUFA) as dietary essential fatty acid (EFA). In this study, the response of juvenile barramundi (47·0 g/fish initial weight) fed isolipidic and isoenergetic diets with 8·2 % added oil was tested. The experimental test diets were either devoid of fish oil (FO), and thus with no n-3 LC-PUFA (FO FREE diet), or with a low inclusion of FO (FO LOW diet). These were compared against a control diet containing only FO (FO CTRL diet) as the added lipid source, over an 8-week period. Interim samples and measurements were taken fortnightly during the trial in order to define the aetiology of the onset and progression of EFA deficiency. After 2 weeks, the fish fed the FO FREE and FO LOW diets had significantly lower live-weights, and after 8 weeks significant differences were detected for all performance parameters. The fish fed the FO FREE diet also had a significantly higher incidence of external abnormalities. The transcription of several genes involved in fatty acid metabolism was affected after 2 weeks of feeding, showing a rapid nutritional regulation. This experiment documents the aetiology of the onset and the progression of EFA deficiency in juvenile barramundi and demonstrates that such deficiencies can be detected within 2 weeks in juvenile fish. PMID:26411329

  12. Drosophila Rif1 is an essential gene and controls late developmental events by direct interaction with PP1-87B.

    PubMed

    Sreesankar, Easwaran; Bharathi, Vellaichamy; Mishra, Rakesh K; Mishra, Krishnaveni

    2015-01-01

    Rif1, identified as a regulator of telomerase in yeast, is an evolutionarily conserved protein and functions in diverse processes including telomere length regulation, epigenetic gene regulation, anti-checkpoint activity, DNA repair and establishing timing of firing at replication origins. Previously we had identified that all Rif1 homologues have PP1 interacting SILK-RVxF motif. In the present study, we show that Drosophila Rif1 is essential for normal fly development and loss of dRif1 impairs temporal regulation of initiation of DNA replication. In multiple tissues dRif1 colocalizes with HP1, a protein known to orchestrate timing of replication in fly. dRif1 associates with chromosomes in a mitotic stage-dependent manner coinciding with dephosphorylation of histones. Ectopic expression of dRif1 causes enlarged larval imaginal discs and early pupal lethality which is completely reversed by co-expression of PP1 87B, the major protein phosphatase in Drosophila, indicating genetic and functional interaction. These findings suggest that dRif1 is an adaptor for PP1 and functions by recruiting PP1 to multiple sites on the chromosome. PMID:26022086

  13. The effect of essential oils of dietary wormwood (Artemisia princeps), with and without added vitamin E, on oxidative stress and some genes involved in cholesterol metabolism.

    PubMed

    Chung, Mi Ja; Kang, Ah-Young; Park, Sung-Ok; Park, Kuen-Woo; Jun, Hee-Jin; Lee, Sung-Joon

    2007-08-01

    Wormwood (Artemisia princeps) due to the abundance of antioxidant in its essential oils (EO), has been used as a traditional drug and health food in Korea. Oxidative stress plays an important role in the etiology of atherosclerosis thus antioxidative chemicals improves hepatic lipid metabolism partly by reducing oxysterol formation. The antioxidant activity was assessed using two methods, human low-density lipoprotein (LDL) oxidation and the anti-DPPH free radical assays. It was found that the antioxidant activity of EO with vitamin E higher than EO alone. To study mechanisms accounting for the antiatherosclerotic properties of this wormwood EO, we examined the expression of key genes in cholesterol metabolism such as the LDL receptor, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and sterol regulatory element binding proteins. The induction was increased up to twofold at 0.05 mg/mL of EO treatment in HepG2 cells for 24h. When EO (0.2 mg/mL) was co-incubated with vitamin E, interestingly, the LDL receptor was dramatically induced by 5-6-folds. HMG-CoA reductase did not change. However, treatment with the higher concentration resulted in cytotoxicity. Our data suggest that wormwood EO with vitamin E may be anti-atherogenic due to their inhibition of LDL oxidation and upregulation of the LDL receptor. PMID:17368686

  14. Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition†

    PubMed Central

    Mohedano, M. Luz; Overweg, Karin; de la Fuente, Alicia; Reuter, Mark; Altabe, Silvia; Mulholland, Francis; de Mendoza, Diego; López, Paloma; Wells, Jerry M.

    2005-01-01

    The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism. PMID:15774879

  15. Drosophila Rif1 is an essential gene and controls late developmental events by direct interaction with PP1-87B

    PubMed Central

    Sreesankar, Easwaran; Bharathi, Vellaichamy; Mishra, Rakesh K.; Mishra, Krishnaveni

    2015-01-01

    Rif1, identified as a regulator of telomerase in yeast, is an evolutionarily conserved protein and functions in diverse processes including telomere length regulation, epigenetic gene regulation, anti-checkpoint activity, DNA repair and establishing timing of firing at replication origins. Previously we had identified that all Rif1 homologues have PP1 interacting SILK-RVxF motif. In the present study, we show that Drosophila Rif1 is essential for normal fly development and loss of dRif1 impairs temporal regulation of initiation of DNA replication. In multiple tissues dRif1 colocalizes with HP1, a protein known to orchestrate timing of replication in fly. dRif1 associates with chromosomes in a mitotic stage-dependent manner coinciding with dephosphorylation of histones. Ectopic expression of dRif1 causes enlarged larval imaginal discs and early pupal lethality which is completely reversed by co-expression of PP1 87B, the major protein phosphatase in Drosophila, indicating genetic and functional interaction. These findings suggest that dRif1 is an adaptor for PP1 and functions by recruiting PP1 to multiple sites on the chromosome. PMID:26022086

  16. Differential Gene Expression Profile of First- and Second-Generation T-Rapa Cell Clinical Products

    PubMed Central

    Castiello, Luciano; Mossoba, Miriam; Viterbo, Antonella; Sabatino, Marianna; Fellowes, Vicki; Foley, Jason E.; Winterton, Matthew; Halverson, David C.; Civini, Sara; Jin, Ping; Fowler, Daniel H.; Stroncek, David F.

    2013-01-01

    We completed a phase II clinical trial evaluating rapamycin-resistant allogeneic T cells (T-Rapa) and now are evaluating a T-Rapa product manufactured in 6-days (T-Rapa6) rather than 12-days (T-Rapa12). Using gene expression microarrays, we addressed our hypothesis that the two products would express a similar phenotype. The products had similar phenotypes using conventional comparison methods of cytokine secretion and surface markers. Unsupervised analysis of 34,340 genes revealed that T-Rapa6 and T-Rapa12 products clustered together, distinct from culture input CD4+ T cells (CD4). Statistical analysis of T-Rapa6 products revealed differential expression of 19.3% of genes (n=6641) compared to input CD4 cells; similarly, 17.8% of genes (n=6147) were differentially expressed between T-Rapa12 products and input CD4 cells. Compared to input CD4 cells, T-Rapa6 and T-Rapa12 products were similar in terms of major gene families up-regulated (cell cycle, stress response, glucose catabolism, DNA metabolism) and down-regulated (inflammatory response, immune response, apoptosis, transcriptional regulation). However, when directly compared, T-Rapa6 and T-Rapa12 products showed differential expression of 5.8% of genes (n=1994; T-Rapa6 vs. T-Rapa12). Second-generation T-Rapa6 cells therefore possess a similar yet distinct gene expression profile relative to first-generation T-Rapa12 cells, and thus may mediate differential effects after adoptive transfer. PMID:23352462

  17. Two Translation Products of Yersinia yscQ Assemble To Form a Complex Essential to Type III Secretion

    SciTech Connect

    Bzymek, Krzysztof P.; Hamaoka, Brent Y.; Ghosh, Partho

    2012-07-11

    The bacterial flagellar C-ring is composed of two essential proteins, FliM and FliN. The smaller protein, FliN, is similar to the C-terminus of the larger protein, FliM, both being composed of SpoA domains. While bacterial type III secretion (T3S) systems encode many proteins in common with the flagellum, they mostly have a single protein in place of FliM and FliN. This protein resembles FliM at its N-terminus and is as large as FliM but is more like FliN at its C-terminal SpoA domain. We have discovered that a FliN-sized cognate indeed exists in the Yersinia T3S system to accompany the FliM-sized cognate. The FliN-sized cognate, YscQ-C, is the product of an internal translation initiation site within the locus encoding the FliM-sized cognate YscQ. Both intact YscQ and YscQ-C were found to be required for T3S, indicating that the internal translation initiation site, which is conserved in some but not all YscQ orthologs, is crucial for function. The crystal structure of YscQ-C revealed a SpoA domain that forms a highly intertwined, domain-swapped homodimer, similar to those observed in FliN and the YscQ ortholog HrcQ{sub B}. A single YscQ-C homodimer associated reversibly with a single molecule of intact YscQ, indicating conformational differences between the SpoA domains of intact YscQ and YscQ-C. A 'snap-back' mechanism suggested by the structure can account for this. The 1:2 YscQ-YscQ-C complex is a close mimic of the 1:4 FliM-FliN complex and the likely building block of the putative Yersinia T3S system C-ring.

  18. Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics

    PubMed Central

    Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. Steven; Ahring, Birgitte K.; Linggi, Bryan

    2013-01-01

    The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions. PMID:23840410

  19. Genetics of essential tremor.

    PubMed

    Tio, Murni; Tan, Eng-King

    2016-01-01

    Essential tremor is one of the most common adult-onset movement disorders. While it is recognized that genes play a major role in ET with ?50% of the affected individuals having a positive family history, identifying underlying genes in both monogenic and complex forms of ET has been a challenging task. Recent discoveries linking LINGO1, FUS and TENM4 to essential tremor have been met with cautious optimism since reproducibility and pathogenicity have been contentious in previously implicated genes. The lack of gold standard diagnostic criteria together with clinical and genetic heterogeneity have presented considerable obstacles. Nevertheless, future genetic studies should adopt a multi-prong genomic approach with adequate sample size, supported by both functional in vitro and in vivo studies. Elucidation of the pathophysiologic mechanism will lead to better therapeutic strategies and management. PMID:26411503

  20. Bioactivity of Essential Oil of Zingiber purpureum Rhizomes and Its Main Compounds against Two Stored Product Insects.

    PubMed

    Wang, Y; You, C X; Yang, K; Wu, Y; Chen, R; Zhang, W J; Liu, Z L; Du, S S; Deng, Z W; Geng, Z F; Han, J

    2015-06-01

    The insecticidal and repellent activities of the essential oil extracted from Zingiber purpureum Roscoe rhizomes were evaluated against Tribolium castaneum (Herbst) and Lasioderma serricorne (L.) adults. During our screening program for agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of Z. purpureum rhizomes was found to possess strong contact toxicity against T. castaneum and L. serricorne adults, with LD50 values of 39.0 and 16.3?µg per adult, respectively, and also showed strong fumigant toxicity against the two grain storage insects with LC50 values of 13.6 and 9.3?mg/liter of air, respectively. The essential oil obtained by hydrodistillation was investigated by gas chromatography-mass spectrometry. The main components of the essential oil were identified to be sabinene (48.1%), terpinen-4-ol (25.1%), and ?-terpinene (6.7%), followed by ?-terpinene (4.3%), ?-thujene (3.4%), and ?-phellandrene (2.7%). Sabinene, terpinen-4-ol, and ?-terpinene were separated and purified by silica gel column chromatography and preparative thin-layer chromatography. Terpinen-4-ol showed the strongest contact toxicity against T. castaneum and L. serricorne (LD50=19.7 and 5.4?µg per adult, respectively) and also the strongest fumigant toxicity against T. castaneum and L. serricorne (LC50=3.7 and 1.3?mg/liter of air, respectively). Otherwise, sabinene and terpinen-4-ol were strongly repellent against T. castaneum as well as the essential oil, while ?-terpinene exhibited weaker repellency against T. castaneum compared with the positive control, DEET (N, N-diethyl-3-methylbenzamide). Moreover, only the essential oil exhibited strong repellency against L. serricorne, the three compounds exhibited weaker repellency against L. serricorne relative to DEET. PMID:26470212

  1. Whole transcriptome analysis reveals an 8-oxoguanine DNA glycosylase-1-driven DNA repair-dependent gene expression linked to essential biological processes.

    PubMed

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Wood, Thomas G; Widen, Steven G; Sur, Sanjiv; Ameredes, Bill T; Saavedra-Molina, Alfredo; Brasier, Allan R; Ba, Xueqing; Boldogh, Istvan

    2015-04-01

    Reactive oxygen species inflict oxidative modifications on various biological molecules, including DNA. One of the most abundant DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is repaired by 8-oxoguanine DNA glycosylase-1 (OGG1) during DNA base excision repair (OGG1-BER). 8-OxoG accumulation in DNA has been associated with various pathological and aging processes, although its role is unclear. The lack of OGG1-BER in Ogg1(-/-) mice resulted in decreased inflammatory responses and increased susceptibility to infections and metabolic disorders. Therefore, we proposed that OGG1 and/or 8-oxoG base may have a role in immune and homeostatic processes. To test our hypothesis, we challenged mouse lungs with OGG1-BER product 8-oxoG base and changes in gene expression were determined by RNA sequencing and data were analyzed by Gene Ontology and statistical tools. RNA-Seq analysis identified 1592 differentially expressed (? 3-fold change) transcripts. The upregulated mRNAs were related to biological processes, including homeostatic, immune-system, macrophage activation, regulation of liquid-surface tension, and response to stimulus. These processes were mediated by chemokines, cytokines, gonadotropin-releasing hormone receptor, integrin, and interleukin signaling pathways. Taken together, these findings point to a new paradigm showing that OGG1-BER plays a role in various biological processes that may benefit the host, but when in excess could be implicated in disease and/or aging processes. PMID:25614460

  2. Thaxtomin A production and virulence are controlled by several bld gene global regulators in Streptomyces scabies.

    PubMed

    Bignell, Dawn R D; Francis, Isolde M; Fyans, Joanna K; Loria, Rosemary

    2014-08-01

    Streptomyces scabies is the main causative agent of common scab disease, which leads to significant annual losses to potato growers worldwide. The main virulence factor produced by S. scabies is a phytotoxic secondary metabolite called thaxtomin A, which functions as a cellulose synthesis inhibitor. Thaxtomin A production is controlled by the cluster-situated regulator TxtR, which activates expression of the thaxtomin biosynthetic genes in response to cello-oligosaccharides. Here, we demonstrate that at least five additional regulatory genes are required for wild-type levels of thaxtomin A production and plant pathogenicity in S. scabies. These regulatory genes belong to the bld gene family of global regulators that control secondary metabolism or morphological differentiation in Streptomyces spp. Quantitative reverse-transcriptase polymerase chain reaction showed that expression of the thaxtomin biosynthetic genes was significantly downregulated in all five bld mutants and, in four of these mutants, this downregulation was attributed to the reduction in expression of txtR. Furthermore, all of the mutants displayed reduced expression of other known or predicted virulence genes, suggesting that the bld genes may function as global regulators of virulence gene expression in S. scabies. PMID:24678834

  3. Plant essential oils and allied volatile fractions as multifunctional additives in meat and fish-based food products: a review.

    PubMed

    Patel, Seema

    2015-01-01

    Essential oils are concentrated aromatic volatile compounds derived from botanicals by distillation or mechanical pressing. They play multiple, crucial roles as antioxidants, food pathogen inhibitors, shelf-life enhancers, texture promoters, organoleptic agents and toxicity-reducing agents. For their versatility, they appear promising as food preservatives. Several research findings in recent times have validated their potential as functional ingredients in meat and fish processing. Among the assortment of bioactive compounds in the essential oils, p-cymene, thymol, eugenol, carvacrol, isothiocyanate, cinnamaldehyde, cuminaldehyde, linalool, 1,8-cineol, ?-pinene, ?-terpineol, ?-terpinene, citral and methyl chavicol are most familiar. These terpenes (monoterpenes and sesquiterpenes) and phenolics (alcohols, esters, aldehydes and ketones) have been extracted from culinary herbs such as oregano, rosemary, basil, coriander, cumin, cinnamon, mint, sage and lavender as well as from trees such as myrtle, fir and eucalyptus. This review presents essential oils as alternatives to conventional chemical additives. Their synergistic actions with modified air packaging, irradiation, edible films, bacteriocins and plant byproducts are discussed. The decisive roles of metabolic engineering, microwave technology and metabolomics in quality and quantity augmentation of essential oil are briefly mooted. The limitations encountered and strategies to overcome them have been illuminated to pave way for their enhanced popularisation. The literature has been mined from scientific databases such as Pubmed, Pubchem, Scopus and SciFinder. PMID:25893282

  4. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  5. Reactivation of latent herpes simplex virus by adenovirus recombinants encoding mutant IE-0 gene products.

    PubMed Central

    Zhu, X X; Chen, J X; Young, C S; Silverstein, S

    1990-01-01

    We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system. Images PMID:2166826

  6. Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila.

    PubMed Central

    Cabrera, C V; Alonso, M C

    1991-01-01

    The achaete-scute complex (AS-C) and the daughterless (da) genes encode helix-loop-helix proteins which have been shown to interact in vivo and to be required for neurogenesis. We show in vitro that heterodimers of three AS-C products with DA bind DNA strongly, whereas DA homodimers bind weakly and homo or heterocombinations of AS-C products not at all. Proteins unable to dimerize did not bind DNA. Target sequences for the heterodimers were found in the promoters of the hunchback and the achaete genes. Using sequences of the former we show that the DNA binding results obtained in vitro fully correlate with the ability of different combinations to activate the expression of a reporter gene in yeast. Embryos deficient for the lethal of scute gene fail to activate hunchback in some neural lineages in a pattern consistent with the lack of a member of a multigene family. Images PMID:1915272

  7. Cc.snf5, a gene encoding a putative component of the SWI/SNF chromatin remodeling complex, is essential for sexual development in the agaricomycete Coprinopsis cinerea.

    PubMed

    Ando, Yuki; Nakazawa, Takehito; Oka, Kunihiko; Nakahori, Kiyoshi; Kamada, Takashi

    2013-01-01

    We characterized a Coprinopsis cinerea mutant strain, Spe20, defective in fruiting initiation, which was isolated after restriction enzyme-mediated integration (REMI) mutagenesis of a homokaryotic fruiting strain, 326. A plasmid rescue followed by complementation experiments, RACE, and cDNA analyses revealed that the gene, a mutation of which is responsible for the phenotype, is predicted to encode a protein that exhibits a high similarity to yeast Snf5p, a key component of the chromatin remodeling complex SWI/SNF, and named Cc.snf5. Cc.Snf5 is, however, different from Snf5p in that the former has, in addition to an Snf5 domain comprising N-terminal repeat1 (rp1) and C-terminal repeat2 (rp2) subdomains in a middle region, a GATA Zn-finger domain in a C-terminal region. In strain Spe20, plasmid pPHT1 used for REMI is inserted in the ORF encoding rp2. This raised the possibility that in strain Spe20, the disrupted Cc.Snf5 is functionally active albeit incompletely because it retains rp1. Thus, we disrupted the whole SNF5 domain and its downstream peptide and found that the disruption results in inhibition of not only fruiting initiation but also dikaryon development, a prerequisite for fruiting. We also found that specific disruption of the Zn-finger domain results in inhibition of fruiting initiation. These results indicate that Cc.Snf5 plays an essential role in sexual development of C. cinerea. PMID:23078835

  8. Protein Arginine Methyltransferase 6 (Prmt6) Is Essential for Early Zebrafish Development through the Direct Suppression of gadd45?a Stress Sensor Gene.

    PubMed

    Zhao, Xin-Xi; Zhang, Yun-Bin; Ni, Pei-Li; Wu, Zhi-Li; Yan, Yuan-Chang; Li, Yi-Ping

    2016-01-01

    Histone lysine methylation is important in early zebrafish development; however, the role of histone arginine methylation in this process remains unclear. H3R2me2a, generated by protein arginine methyltransferase 6 (Prmt6), is a repressive mark. To explore the role of Prmt6 and H3R2me2a during zebrafish embryogenesis, we identified the maternal characteristic of prmt6 and designed two prmt6-specific morpholino-oligos (MOs) to study its importance in early development, application of which led to early epiboly defects and significantly reduced the level of H3R2me2a marks. prmt6 mRNA could rescue the epiboly defects and the H3R2me2a reduction in the prmt6 morphants. Functionally, microarray data demonstrated that growth arrest and DNA damage-inducible, ?, a (gadd45?a) was a significantly up-regulated gene in MO-treated embryos, the activity of which was linked to the activation of the p38/JNK pathway and apoptosis. Importantly, gadd45?a MO and p38/JNK inhibitors could partially rescue the defect of prmt6 morphants, the downstream targets of Prmt6, and the apoptosis ratios of the prmt6 morphants. Moreover, the results of ChIP quantitative real time PCR and luciferase reporter assay indicated that gadd45?a is a repressive target of Prmt6. Taken together, these results suggest that maternal Prmt6 is essential to early zebrafish development by directly repressing gadd45?a. PMID:26487724

  9. Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

    DOEpatents

    Dai, Ziyu (Richland, WA); Lasure, Linda L. (Fall City, WA); Magnuson, Jon K. (Pasco, WA)

    2008-11-11

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  10. Isolated Fungal Promoters and Gene Transcription Terminators and Methods of Protein and Chemical Production in a Fungus

    DOEpatents

    Dai, Ziyu (Richland, WA); Lasure, Linda L. (Fall City, WA); Magnuson, Jon K. (Pasco, WA)

    2008-11-11

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  11. Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

    DOEpatents

    Dai, Ziyu; Lasure, Linda L; Magnuson, Jon K

    2014-05-27

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  12. Genetic resources for advanced biofuel production described with the Gene Ontology

    DOE PAGESBeta

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, Joao C.; Mukhopadhyay, Biswarup; Tyler, Brett M.

    2014-10-10

    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary.The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergymore »production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. We review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.« less

  13. Genetic resources for advanced biofuel production described with the Gene Ontology

    PubMed Central

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, Joăo C.; Mukhopadhyay, Biswarup; Tyler, Brett M.

    2014-01-01

    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology () project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way. PMID:25346727

  14. Genetic resources for advanced biofuel production described with the Gene Ontology

    SciTech Connect

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, Joao C.; Mukhopadhyay, Biswarup; Tyler, Brett M.

    2014-10-10

    Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary.The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. We review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.

  15. Antibacterial Discovery and Development: From Gene to Product and Back

    PubMed Central

    Fedorenko, Victor; Genilloud, Olga; Horbal, Liliya; Marcone, Giorgia Letizia; Marinelli, Flavia; Paitan, Yossi; Ron, Eliora Z.

    2015-01-01

    Concern over the reports of antibiotic-resistant bacterial infections in hospitals and in the community has been publicized in the media, accompanied by comments on the risk that we may soon run out of antibiotics as a way to control infectious disease. Infections caused by Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and other Enterobacteriaceae species represent a major public health burden. Despite the pharmaceutical sector's lack of interest in the topic in the last decade, microbial natural products continue to represent one of the most interesting sources for discovering and developing novel antibacterials. Research in microbial natural product screening and development is currently benefiting from progress that has been made in other related fields (microbial ecology, analytical chemistry, genomics, molecular biology, and synthetic biology). In this paper, we review how novel and classical approaches can be integrated in the current processes for microbial product screening, fermentation, and strain improvement. PMID:26339625

  16. The paf gene product modulates asexual development in Penicillium chrysogenum

    PubMed Central

    Hegedüs, Nikoletta; Sigl, Claudia; Zadra, Ivo; Pócsi, Istvan; Marx, Florentine

    2011-01-01

    Penicillium chrysogenum secretes a low molecular weight, cationic and cysteine-rich protein (PAF). It has growth inhibitory activity against the model organism Aspergillus nidulans and numerous zoo- and phytopathogenic fungi but shows only minimal conditional antifungal activity against the producing organism itself. In this study we provide evidence for an additional function of PAF which is distinct from the antifungal activity against putative ecologically concurrent microorganisms. Our data indicate that PAF enhances conidiation in P. chrysogenum by modulating the expression of brlA, the central regulatory gene for mitospore development. A paf deletion strain showed a significant impairment of mitospore formation which sustains our hypothesis that PAF plays an important role in balancing asexual differentiation in P. chrysogenum. PMID:21298690

  17. Engineering validamycin production by tandem deletion of ?-butyrolactone receptor genes in Streptomyces hygroscopicus 5008.

    PubMed

    Tan, Gao-Yi; Peng, Yao; Lu, Chenyang; Bai, Linquan; Zhong, Jian-Jiang

    2015-03-01

    Paired homologs of ?-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (?shbR1) and 20% (?shbR3). By double deletion, the ?shbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs. PMID:25527439

  18. Essential Questions

    ERIC Educational Resources Information Center

    Wilhelm, Jeffrey D.

    2012-01-01

    The secret to teaching may be as simple as asking students good questions--and then giving them the opportunity to find the answers. The author shares how he uses essential questions that set the class off on an inquiry. Rather than consuming information that he distributes and then repeating it on a test, students carry out their own…

  19. [Role of spot gene product in the degradation of pppGpp in bacteria].

    PubMed

    Belitski?, B R; Shakulov, R S

    1982-01-01

    In Baccillus subtilis cells, in contrast to Escherichia coli cells, chelating agent 1,10-phenanthroline induces the expansion of guanosine-5'-diphosphate,3'-diphosphate( (ppGpp) pool, as well as the pool of its precursor guanosine-5'-triphosphate,3'-diphosphate (pppGpp). Under these conditions the degradation rate of both nucleotides decreases greatly, which is the main cause of their accumulation in the cells. In E. coli phenanthroline inactivates the product of spoT gene, which is responsible for ppGpp degradation, as a result of combining Mn2+ ions necessary for the activity of this enzyme. The addition of Mn2+ ions to B. subtilis cells, treated with phenanthroline, leads to the decline in (p)ppGpp pools. Antibiotic tetracycline, which has the chelating properties at the concentration of 1 mg/ml, also inactivates spoT gene product in E. coli and slows down the decay of ppGpp, but not of pppGpp. The addition of high concentrations of tetracycline to B. subtilis cells leads to severe inhibition of the degradation of both nucleotides. Therefore in B. subtilis spoT gene product is involved in the degradation of pppGpp, as well as ppGpp. In E. coli cells with defective gpp gene product, taking part in the conversion of pppGpp to ppGpp, phenanthroline and tetracycline also inhibit the breakdown of both nucleotides. The similarity of B. subtilis and E. coli gpp cells in respect of spoT gene product functions and of enhanced pppGpp fraction in the total amount of guanosine polyphosphates during aminoacyl-tRNA limitation makes it plausible that in B. subtilis cells the product of gpp gene is missing or has low activity. PMID:6811860

  20. 1,3-Propanediol production by Escherichia coli using genes from Citrobacter freundii atcc 8090.

    PubMed

    Przysta?owska, Hanna; Zeyland, Joanna; Ko?mider, Alicja; Szalata, Marlena; S?omski, Ryszard; Lipi?ski, Daniel

    2015-01-01

    Compared with chemical synthesis, fermentation has the advantage of mass production at low cost, and has been used in the production of various industrial chemicals. As a valuable organic compound, 1,3-propanediol (1,3-PDO) has numerous applications in the production of polymers, lubricants, cosmetics and medicines. Here, conversion of glycerol (a renewable substrate and waste from biodiesel production) to 1,3-PDO by E. coli bacterial strain carrying altered glycerol metabolic pathway was investigated. Two gene constructs containing the 1,3-PDO operon from Citrobacter freundii (pCF1 and pCF2) were used to transform the bacteria. The pCF1 gene expression construct contained dhaBCE genes encoding the three subunits of glycerol dehydratase, dhaF encoding the large subunit of the glycerol dehydratase reactivation factor and dhaG encoding the small subunit of the glycerol dehydratase reactivating factor. The pCF2 gene expression construct contained the dhaT gene encoding the 1,3-propanediol dehydrogenase. Expression of the genes cloned in the above constructs was under regulation of the T7lac promoter. RT-PCR, SDS-PAGE analyses and functional tests confirmed that 1,3-PDO synthesis pathway genes were expressed at the RNA and protein levels, and worked flawlessly in the heterologous host. In a batch flask culture, in a short time applied just to identify the 1,3-PDO in a preliminary study, the recombinant E. coli bacteria produced 1.53 g/L of 1,3-PDO, using 21.2 g/L of glycerol in 72 h. In the Sartorius Biostat B Plus reactor, they produced 11.7 g/L of 1,3-PDO using 24.2 g/L of glycerol, attaining an efficiency of 0.58 [mol1,3-PDO/molglycerol]. PMID:26345096

  1. The ERCC1 and ERCC4 (XPF) genes and gene products.

    PubMed

    Manandhar, Mandira; Boulware, Karen S; Wood, Richard D

    2015-09-15

    The ERCC1 and ERCC4 genes encode the two subunits of the ERCC1-XPF nuclease. This enzyme plays an important role in repair of DNA damage and in maintaining genomic stability. ERCC1-XPF nuclease nicks DNA specifically at junctions between double-stranded and single-stranded DNA, when the single-strand is oriented 5' to 3' away from a junction. ERCC1-XPF is a core component of nucleotide excision repair and also plays a role in interstrand crosslink repair, some pathways of double-strand break repair by homologous recombination and end-joining, as a backup enzyme in base excision repair, and in telomere length regulation. In many of these activities, ERCC1-XPF complex cleaves the 3' tails of DNA intermediates in preparation for further processing. ERCC1-XPF interacts with other proteins including XPA, RPA, SLX4 and TRF2 to perform its functions. Disruption of these interactions or direct targeting of ERCC1-XPF to decrease its DNA repair function might be a useful strategy to increase the sensitivity of cancer cells to some DNA damaging agents. Complete deletion of either ERCC1 or ERCC4 is not compatible with viability in mice or humans. However, mutations in the ERCC1 or ERCC4 genes cause a remarkable array of rare inherited human disorders. These include specific forms of xeroderma pigmentosum, Cockayne syndrome, Fanconi anemia, XFE progeria and cerebro-oculo-facio-skeletal syndrome. PMID:26074087

  2. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

    PubMed Central

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence. PMID:25566225

  3. Overproduction and purification of the luxR gene product: transcriptional activator of the Vibrio fischeri luminescence system

    SciTech Connect

    Kaplan, H.B.; Greenberg, E.P.

    1987-10-01

    Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing luxR under control of a tac promoter was engineered to overproduce this gene product. The overproduced luxR gene product was active in vivo, and its apparent monomeric molecular weight was indistinguishable from that of the protein encoded by luxR under control of its own promoter (M/sub r/ 27,000). The new tac-luxR construct directed the synthesis of large quantities of the luxR gene product in induced Escherichia coli cells lacking other lux genes. In the presence of other lux genes, overexpression of the tac-luxR construct was not detected. The overproduced luxR gene product, which formed cytoplasmic inclusion bodies, was purified and used in subsequent studies. Nonequilibrium pH gradient electrophoresis indicated that the protein was basic, and the amino-terminal 15 amino acids were sequenced. DNA-binding activity was detected by membrane filter binding assays; under the conditions used, the binding was not lux DNA-specific. Binding of tritium-labeled autoinducer to the luxR gene product was not detected, and autoinducer enhancement of the binding of the luxR gene product to DNA could not be detected reproducibly.

  4. Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product.

    PubMed

    Reidt, W; Wohlfarth, T; Ellerström, M; Czihal, A; Tewes, A; Ezcurra, I; Rask, L; Bäumlein, H

    2000-03-01

    The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis-motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis-element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis-motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development. PMID:10758492

  5. Cinnamomum cassia essential oil inhibits ?-MSH-induced melanin production and oxidative stress in murine B16 melanoma cells.

    PubMed

    Chou, Su-Tze; Chang, Wen-Lun; Chang, Chen-Tien; Hsu, Shih-Lan; Lin, Yu-Che; Shih, Ying

    2013-01-01

    Essential oils extracted from aromatic plants exhibit important biological activities and have become increasingly important for the development of aromatherapy for complementary and alternative medicine. The essential oil extracted from Cinnamomum cassia Presl (CC-EO) has various functional properties; however, little information is available regarding its anti-tyrosinase and anti-melanogenic activities. In this study, 16 compounds in the CC-EO have been identified; the major components of this oil are cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%). CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities; however, cis-2-methoxycinnamic acid did not demonstrate tyrosinase inhibitory activity. In murine B16 melanoma cells stimulated with ?-melanocyte-stimulating hormone (?-MSH), CC-EO and cinnamaldehyde not only reduced the melanin content and tyrosinase activity of the cells but also down-regulated tyrosinase expression without exhibiting cytotoxicity. Moreover, CC-EO and cinnamaldehyde decreased thiobarbituric acid-reactive substance (TBARS) levels and restored glutathione (GSH) and catalase activity in the ?-MSH-stimulated B16 cells. These results demonstrate that CC-EO and its major component, cinnamaldehyde, possess potent anti-tyrosinase and anti-melanogenic activities that are coupled with antioxidant properties. Therefore, CC-EO may be a good source of skin-whitening agents and may have potential as an antioxidant in the future development of complementary and alternative medicine-based aromatherapy. PMID:24051402

  6. Cinnamomum cassia Essential Oil Inhibits ?-MSH-Induced Melanin Production and Oxidative Stress in Murine B16 Melanoma Cells

    PubMed Central

    Chou, Su-Tze; Chang, Wen-Lun; Chang, Chen-Tien; Hsu, Shih-Lan; Lin, Yu-Che; Shih, Ying

    2013-01-01

    Essential oils extracted from aromatic plants exhibit important biological activities and have become increasingly important for the development of aromatherapy for complementary and alternative medicine. The essential oil extracted from Cinnamomum cassia Presl (CC-EO) has various functional properties; however, little information is available regarding its anti-tyrosinase and anti-melanogenic activities. In this study, 16 compounds in the CC-EO have been identified; the major components of this oil are cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%). CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities; however, cis-2-methoxycinnamic acid did not demonstrate tyrosinase inhibitory activity. In murine B16 melanoma cells stimulated with ?-melanocyte-stimulating hormone (?-MSH), CC-EO and cinnamaldehyde not only reduced the melanin content and tyrosinase activity of the cells but also down-regulated tyrosinase expression without exhibiting cytotoxicity. Moreover, CC-EO and cinnamaldehyde decreased thiobarbituric acid-reactive substance (TBARS) levels and restored glutathione (GSH) and catalase activity in the ?-MSH-stimulated B16 cells. These results demonstrate that CC-EO and its major component, cinnamaldehyde, possess potent anti-tyrosinase and anti-melanogenic activities that are coupled with antioxidant properties. Therefore, CC-EO may be a good source of skin-whitening agents and may have potential as an antioxidant in the future development of complementary and alternative medicine-based aromatherapy. PMID:24051402

  7. A mutant gene that increases gibberellin production in Brassica

    SciTech Connect

    Rood, S.B. ); Williams, P.H. ); Pearce, D.; Pharis, R.P. ); Murofushi, Noboru ); Mander, L.N. )

    1990-07-01

    A single gene mutant (elongated internode (ein/ein)) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A{sub 3} (GA{sub 3}) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA{sub 1} and GA{sub 3} were estimated by gas chromatography-selected ion monitoring using ({sup 2}H)GA{sub 1} as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA{sub 20} and GA{sub 1}, and the rate of GA{sub 19} metabolism were simultaneously analyzed. Levels of GA{sub 1} and GA{sub 20} were 4.6- and 12.9-fold higher, respectively, and conversions to GA{sub 20} and GA{sub 1} were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA{sub 1} biosynthesis in ein, the conversion of ({sup 3}H)GA{sub 20} to ({sup 3}H) GA{sub 1} was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA{sub 1} biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A{sub 1} and A{sub 3}.

  8. O-linked glycosylation of retroviral envelope gene products

    SciTech Connect

    Pinter, A.; Honnen, W.J. )

    1988-03-01

    Treatment of ({sup 3}H)glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-M{sub r} (49K) product. For ({sup 3}H)mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an addition size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90{sup env}, the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80{sup env}, and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicting that O-linked glycosylation is a conserved feature of retroviral env proteins.

  9. Improving the safety of cell therapy products by suicide gene transfer

    PubMed Central

    Jones, Benjamin S.; Lamb, Lawrence S.; Goldman, Frederick; Di Stasi, Antonio

    2014-01-01

    Adoptive T-cell therapy can involve donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation, the administration of tumor infiltrating lymphocyte expanded ex-vivo, or more recently the use of T cell receptor or chimeric antigen receptor redirected T cells. However, cellular therapies can pose significant risks, including graft-vs.-host-disease and other on and off-target effects, and therefore strategies need to be implemented to permanently reverse any sign of toxicity. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The “ideal” suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of “all” and “only” the cells responsible for the unwanted toxicity. This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the most extensively validated suicide genes, including the clinical setting: herpes-simplex-thymidine-kinase and inducible-caspase-9. PMID:25505885

  10. Improving the safety of cell therapy products by suicide gene transfer.

    PubMed

    Jones, Benjamin S; Lamb, Lawrence S; Goldman, Frederick; Di Stasi, Antonio

    2014-01-01

    Adoptive T-cell therapy can involve donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation, the administration of tumor infiltrating lymphocyte expanded ex-vivo, or more recently the use of T cell receptor or chimeric antigen receptor redirected T cells. However, cellular therapies can pose significant risks, including graft-vs.-host-disease and other on and off-target effects, and therefore strategies need to be implemented to permanently reverse any sign of toxicity. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The "ideal" suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of "all" and "only" the cells responsible for the unwanted toxicity. This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the most extensively validated suicide genes, including the clinical setting: herpes-simplex-thymidine-kinase and inducible-caspase-9. PMID:25505885

  11. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled. PMID:26323274

  12. Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

  13. Mining novel genes resistant to blast disease for US rice production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice blast disease is one of the most damaging and prevalent diseases in the southern U.S. The Pi-ta resistance gene that conveys resistance to numerous races has been used in rice production for about 20 years, but is overcome by several new races including IB-33 and IE-1k. Banks is a high yielding...

  14. A MUTATION IN AN EXBD GENE REDUCES TAGETITOXIN PRODUCTION BY PSEUDOMONAS SYRINGAE PV TAGETIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to reduce the risks to the environment, water, and food supply from the use of synthetic chemicals, more efficacious biological control agents for weeds are needed. This purpose of this study was to identify genes required for the production of a phytotoxin, tagetitoxin, produced by a bact...

  15. Role of nitric oxide and flavohemoglobin homolo genes in Aspergillus nidulans sexual development and mycotoxin production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavohemoglobins are widely distributed proteins in both prokaryotic and eukaryotic organisms, conferring resistance against nitrosative stress. In the present study we investigated the role of two flavohemoglobin homologous genes, fhbA and fhbB, in morphogenesis and in the production of the mycotox...

  16. Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division

    E-print Network

    Erickson, Harold P.

    Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division Ruth for review July 30, 2013) Bacteriophages take over host resources primarily via the activity of proteins that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division in

  17. Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes

    DOEpatents

    Ingram, Lonnie O. (Gainesville, FL); Conway, Tyrrell (Lincoln, NE); Alterthum, Flavio (Gainesville, FL)

    1991-01-01

    A novel operon and plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase activities of Zymomonas mobilis are described. Also disclosed are methods for increasing the growth of microorganisms or eukaryotic cells and methods for reducing the accumulation of undesirable metabolic products in the growth medium of microorganisms or cells.

  18. Regulation of E. coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

    E-print Network

    Simon, Sharotka M. (Sharotka Maria)

    2007-01-01

    Products of the umuD gene in E. coli are involved in regulating the timing of error-free DNA repair processes and mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD2 is ...

  19. ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g ...

  20. PHYA GENE PRODUCT OF ASPERGILLUS FICUUM AND PENIOPHORA LYCII PRODUCES TWO DISSIMILAR PHYTASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PhyA gene product of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid resi...

  1. Respiratory activity is essential for post-exponential-phase production of type 5 capsular polysaccharide by Staphylococcus aureus.

    PubMed Central

    Dassy, B; Fournier, J M

    1996-01-01

    Capsule formation is believed to have a significant role in bacterial virulence. To examine the possible involvement of capsular polysaccharide (CP) from Staphylococcus aureus in the pathological mechanisms associated with staphylococcal infections, we investigated the influence of respiratory activity on type 5 CP production by S. aureus grown in the presence of various concentrations of dissolved oxygen or nitrate. The effects of several metabolic inhibitors (arsenite, cyanide, azide, trimethylamine N-oxide, 2-heptyl-4-hydroxyquinoline N-oxide, and 2,4-dinitrophenol) were also tested. The metabolism of the bacteria was estimated by measuring their reductive capacity and by monitoring the pH and concentrations of fermentation products. Type 5 CP was always produced by S. aureus during the exponential phase of growth under all culture conditions tested. In contrast, post-exponential-phase CP production appeared to be strictly dependent on the respiratory activity. Since post-exponential-phase CP production contributes at least two-thirds of the total CP obtained, the influence of S. aureus respiration on CP production might be of some importance in the process of infection. PMID:8698459

  2. Essential tremor.

    PubMed Central

    Murray, T. J.

    1981-01-01

    Essential tremor, including the juvenile and senile variations, may be a result of a disorder of the servomechanism that controls physiologic tremor. Hands and arms are affected most commonly, and the tremor can vary in amplitude as well as frequency. Long-term treatment with propranolol has been helpful for some patients, although older patients are less likely to benefit. Other drugs and behaviour modification therapy have been less successful. Surgical treatment is effective but should probably be reserved for severe cases. An effective instrument for measuring the subjective and objective aspects of the tremor is still needed, as is an effective long-term method of treatment. PMID:7018658

  3. Essential veterinary education in the cultural, political and biological complexities of international trade in animals and animal products.

    PubMed

    Brown, C C

    2009-08-01

    Globalisation has changed the veterinary profession in many ways and academic institutes may need to re-tool to help future professionals deal with the changes in a successful and productive way. The remarkably expanded and expanding volume of trade and traffic in animals and animal products means that to be effective veterinarians must grasp some of the complexities inherent in this trade. Being able to engage productively in cross-cultural dialogue will be important in negotiations over livestock shipments and also within the context of the delivery of medical services to companion animals in societies that are becoming increasingly diverse. Understanding the political landscapes that influence trade decisions will help to expedite agreements and facilitate the transfer of goods and materials that involve animal health. Disease emergence will continue to occur, and an awareness of the factors responsible and the response measures to undertake will help to contain any damage. PMID:20128459

  4. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  5. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

  6. An integrated approach utilizing chemometrics and GC/MS for classification of chamomile flowers, essential oils and commerical products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of an ongoing research program on authentication, safety and biological evaluation of phytochemicals and dietary supplements, an in-depth chemical investigation of different types of chamomile was performed. A collection of chamomile samples including authenticated plants, commercial product...

  7. Rhamnolipids in perspective: gene regulatory pathways, metabolic engineering, production and technological forecasting.

    PubMed

    Dobler, Leticia; Vilela, Leonardo F; Almeida, Rodrigo V; Neves, Bianca C

    2016-01-25

    Rhamnolipids have emerged as a very promising class of biosurfactants in the last decades, exhibiting properties of great interest in several industrial applications, and have represented a suitable alternative to chemically-synthesized surfactants. This class of biosurfactants has been extensively studied in recent years, aiming at their large-scale production based on renewable resources, which still require high financial costs. Development of non-pathogenic, high-producing strains has been the focus of a number of studies involving heterologous microbial hosts as platforms. However, the intricate gene regulation network controlling rhamnolipid biosynthesis represents a challenge to metabolic engineering and remains to be further understood and explored. This article provides an overview of the biosynthetic pathways and the main gene regulatory factors involved in rhamnolipid production within Pseudomonas aeruginosa, the prototypal producing species. In addition, we provide a perspective view into the main strategies applied to metabolic engineering and biotechnological production. PMID:26409933

  8. Modern plant metabolomics: Advanced natural product gene discoveries, improved technologies, and future prospects

    SciTech Connect

    Sumner, Lloyd W.; Lei, Zhentian; Nikolau, Basil J.; Saito, Kazuki

    2014-10-24

    Plant metabolomics has matured and modern plant metabolomics has accelerated gene discoveries and the elucidation of a variety of plant natural product biosynthetic pathways. This study highlights specific examples of the discovery and characterization of novel genes and enzymes associated with the biosynthesis of natural products such as flavonoids, glucosinolates, terpenoids, and alkaloids. Additional examples of the integration of metabolomics with genome-based functional characterizations of plant natural products that are important to modern pharmaceutical technology are also reviewed. This article also provides a substantial review of recent technical advances in mass spectrometry imaging, nuclear magnetic resonance imaging, integrated LC-MS-SPE-NMR for metabolite identifications, and x-ray crystallography of microgram quantities for structural determinations. The review closes with a discussion on the future prospects of metabolomics related to crop species and herbal medicine.

  9. Modern plant metabolomics: Advanced natural product gene discoveries, improved technologies, and future prospects

    DOE PAGESBeta

    Sumner, Lloyd W.; Lei, Zhentian; Nikolau, Basil J.; Saito, Kazuki

    2014-10-24

    Plant metabolomics has matured and modern plant metabolomics has accelerated gene discoveries and the elucidation of a variety of plant natural product biosynthetic pathways. This study highlights specific examples of the discovery and characterization of novel genes and enzymes associated with the biosynthesis of natural products such as flavonoids, glucosinolates, terpenoids, and alkaloids. Additional examples of the integration of metabolomics with genome-based functional characterizations of plant natural products that are important to modern pharmaceutical technology are also reviewed. This article also provides a substantial review of recent technical advances in mass spectrometry imaging, nuclear magnetic resonance imaging, integrated LC-MS-SPE-NMR formore »metabolite identifications, and x-ray crystallography of microgram quantities for structural determinations. The review closes with a discussion on the future prospects of metabolomics related to crop species and herbal medicine.« less

  10. Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

    PubMed

    Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

    2015-03-27

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome. PMID:25382643

  11. Direct Capture and Heterologous Expression of Salinispora Natural Product Genes for the Biosynthesis of Enterocin

    PubMed Central

    2015-01-01

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora’s promising secondary metabolome. PMID:25382643

  12. Preliminary validation of Albedo, FAPAR and LAI Essential Climate Variables products derived from PROBA-V observations in the Copernicus Global Land Service

    NASA Astrophysics Data System (ADS)

    Camacho, Fernando; Sanchez, Jorge; Lacaze, Roselyne; Smets, Bruno

    2015-04-01

    From 1st January 2013, the Copernicus Global Land Service is operational, providing in near real time a set of biophysical variables over the globe, including Surface Albedo (SA), Leaf Area Index (LAI) and Fraction of Absorbed Photosynthetically Active Radiation (FAPAR) Essential Climate Variables among other variables such as the Fraction of Vegetation Cover (FCover) are delivered at 1 km resolution and 10-days frequency. These ECVs are also key inputs for land surface applications such as agriculture monitoring, yield estimate, food security, environmental monitoring (e.g. desertification, drought). The first version of these Copernicus Global Land products were based on SPOT/VGT observations (1999-2004). The continuity of the production is currently based on PROBA-V 1 km observations, and the evolution of the services will provide enhanced spatial resolution (333m). This study presents the preliminary validation results of PROBA-V Albedo, FAPAR, LAI and FCover 1 km products, focused on the consistency with SPOT/VGT GEOV1 products during the overlap period (November 2013 - May 2014) and including intercomparison with MODIS C5 equivalent products. The procedure follows as much as possible guidelines and metrics defined by the Land Product Validation (LPV) group of the Committee on Earth Observation Satellite (CEOS) for the validation of satellite-derived land products and propose additional metrics to quantify spatial and temporal consistency among the several products. Several criteria of performance were evaluated including product completeness, spatial consistency, temporal consistency, inter-annual precision and accuracy. Inter-comparison with reference satellite products (SPOT/VGT GEOV1 and MODIS C5) are presented over a network of sites (BELMANIP-2). The accuracy of PROBA-V LAI and FAPAR products was evaluated against a number of agricultural sites using the ImagineS database, whereas for Albedo few homogeneous sites with available ground data were considered coming from SURFRAD and EFDC networks. The ground data was collected, compiled or processed in the context of the FP7 ImagineS project in support of the evolution of Copernicus Land Service. Our results demonstrate that PROBA-V GEOV1 LAI and FAPAR products were found spatially and temporally consistent with similar products (SPOT/VGT, MODISC5), and good agreement with limited ground truth data. Albedo products are currently under evaluation, and preliminary results will be shown at the conference.

  13. imaging essentials images defined,

    E-print Network

    Zhang, Yi

    imaging essentials images defined, editing strategies explained Please sign-in & take a handout (on corner of table...) imaging made easy with Beth Beighlie The Harvard Medical School is accredited, or distributing health care related goods, products or services. intro to digital images · digital images defined

  14. Leptin and leptin receptor gene polymorphisms are correlated with production performance in the Arctic fox.

    PubMed

    Zhang, M; Bai, X J

    2015-01-01

    The polymerase chain reaction-single-strand conformation polymorphism technique was employed to measure mononucleotide diversity in the coding region of the leptin and leptin receptor genes in the Arctic fox. The relationships between specific genetic mutations and reproductive performance in Arctic foxes were determined to im-prove breeding strategies. We found that a leptin gene polymorphism was significantly associated with body weight (P < 0.01), abdominal circumference (P < 0.01), and fur length (P < 0.01). Furthermore, a polymorphism in the leptin receptor gene was associated with carcass weight and guard hair length (P < 0.01). Leptin and leptin receptor gene combinatorial genotypes were significantly associated with abdominal circumference, fur length (P < 0.01), and body weight (P < 0.05). The leptin gene is thus a key gene affecting body weight, abdominal circumference, and fur length in Arctic foxes, whereas variations in the leptin receptor mainly affect carcass weight and guard hair. The marker loci identified in this study can be used to assist in the selection of Arctic foxes for breeding to raise the production performance of this species. PMID:26125753

  15. Effect of HCV infection on expression of several cancer-associated gene products in HCC

    PubMed Central

    Yang, Jian-Min; Wang, Rong-Quan; Bu, Bao-Guo; Zhou, Zi-Cheng; Fang, Dian-Chun; Luo, Yuan-Hui

    1999-01-01

    AIM To study hepatocarcinogenesis of hepatitis C virus (HCV). METHODS Expression of HCV antigens (CP10, NS3 and NS5) and several cancer-associated gene products (ras p21, c-myc,c-erbB-2, mutated p53 and p16 protein) in the tissues of hepato-cellular carcinoma (HCC, n = 46) and its surrounding liver tissue were studied by the ABC (avidin-biotin complex ) immu nohistochemical method. The effect of HCV infection on expres-sion of those gene products in HCC was analyzed by comparing HCV antigen-positive group with HCV antigen negative group. RESULTS Positive immunostaining with one, two or three HCV antigens was found in 20 (43.5%) cases, with either of two or three HCV antigens in 16 (34.8%) cases, and with three HCV antigens in 9 (19.6%) cases. Deletion rate of p16 protein expression in HCC with positive HCV antigen (80%, 16/20) was significantly higher than that in HCC with negative HCV anti-gen. Where as no significant difference of the other gene product expression was observed between the two groups. CONCLUSION HCV appears related to about one-third of cases of HCC in Chongqing, the south-west of China, and it may be involved in hepato-carcinogen esis by inhibiting the function of p16 gene, which acts as a negative regulator of cell cycle. PMID:11819378

  16. Use of Staby(®) technology for development and production of DNA vaccines free of antibiotic resistance gene.

    PubMed

    Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

    2013-10-01

    The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby(®) technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby(®) technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1). PMID:24051431

  17. Use of Staby® technology for development and production of DNA vaccines free of antibiotic resistance gene

    PubMed Central

    Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

    2013-01-01

    The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby® technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby® technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1). PMID:24051431

  18. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

    PubMed

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1?mM l-isoleucine, MV-KICF1 accumulated 47?mM KIC (6.1?g?l(-1)) with a yield of 0.20?mol/mol glucose and a volumetric productivity of 1.41?mmol?KIC?l(-1) ?h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. PMID:25488800

  19. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    PubMed

    Przysta?owska, Hanna; Zeyland, Joanna; Szymanowska-Powa?owska, Daria; Szalata, Marlena; S?omski, Ryszard; Lipi?ski, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol?,?-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming there is an increasing industrial need to exchange the chemical way of chemicals synthesis for biotechnological - more ecological manner. The 1,3-PDO production from glycerol is an desirable alternative to the traditional production from non-renewable resources. This work is a part of project, which opens a way to development of innovative "green chemistry" and new perspectives to chemical industry. PMID:25644946

  20. BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Enhanced biosynthetic gene expressions and production

    E-print Network

    Gu, Tingyue

    , a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum . Phenobarbital induction . Gene expression . Mushroom fermentation technology Introduction Some secondary

  1. Anthemis wiedemanniana essential oil prevents LPS-induced production of NO in RAW 264.7 macrophages and exerts antiproliferative and antibacterial activities in vitro.

    PubMed

    Conforti, Filomena; Menichini, Federica; Formisano, Carmen; Rigano, Daniela; Senatore, Felice; Bruno, Maurizio; Rosselli, Sergio; Celik, Sezgin

    2012-01-01

    Anthemis wiedemanniana is known in folk medicine for the treatment of microbial infections, cancer and also urinary and pulmonary problems. In this study, the chemical composition of the essential oil from A. wiedemanniana was evaluated and its antibacterial activity was tested against 10 bacterial strains. The oil was also tested for its potentiality to inhibit nitric oxide production in RAW 264.7 macrophages and for its cytotoxicity against four human cancer cell lines. A. wiedemanniana oil, rich of oxygenated monoterpenes (25.4%), showed a good antibacterial activity against Gram-positive bacteria and a good activity against the two Gram-negative bacteria, Escherichia coli and Proteus vulgaris. Besides that, it exhibited a high inhibitory effect on the LPS-induced nitrite production and a strong cytotoxic activity, especially against amelanotic melanoma (C32) and large lung cell carcinoma (COR-L23) cell lines. PMID:22124231

  2. The heterochronic genes lin-28a and lin-28b play an essential and evolutionarily conserved role in early zebrafish development.

    PubMed

    Ouchi, Yasuo; Yamamoto, Jyunya; Iwamoto, Takashi

    2014-01-01

    The Caenorhabditis elegans heterochronic gene pathway, which consists of a set of regulatory genes, plays an important regulatory role in the timing of stage-specific cell lineage development in nematodes. Research into the heterochronic gene pathway gave rise to landmark microRNA (miRNA) studies and showed that these genes are important in stem cell and cancer biology; however, their functions in vertebrate development are largely unknown. To elucidate the function of the heterochronic gene pathway during vertebrate development, we cloned the zebrafish homologs of the C. elegans let-7 miRNA-binding protein, Lin-28, and analyzed their function in zebrafish development. The zebrafish genome contains two Lin28-related genes, lin-28a and lin-28b. Similar to mammalian Lin28 proteins, both zebrafish Lin-28a and Lin-28b have a conserved cold-shock domain and a pair of CCHC zinc finger domains, and are ubiquitously expressed during early embryonic development. In a reciprocal fashion, the expression of downstream heterochronic genes, let-7 and lin-4/miR-125 miRNA, occurred subsequent to lin-28 expression. The knockdown of Lin-28a or Lin-28b function by morpholino microinjection into embryos resulted in severe cell proliferation defects during early morphogenesis. We found that the expression of let-7 miRNA was upregulated and its downstream target gene, lin-41, was downregulated in these embryos. Interestingly, the expression of miR-430, a key regulator of maternal mRNA decay, was downregulated in lin-28a and lin-28b morphant embryos, suggesting a role for Lin-28 in the maternal-to-zygotic transition in zebrafish. Taken together, our results suggest an evolutionarily conserved and pivotal role of the heterochronic gene pathway in early vertebrate embryogenesis. PMID:24516585

  3. Essential Metabolites of Mycobacterium tuberculosis and Their Mimics

    PubMed Central

    Lamichhane, Gyanu; Freundlich, Joel S.; Ekins, Sean; Wickramaratne, Niluka; Nolan, Scott T.; Bishai, William R.

    2011-01-01

    An organism requires a range of biomolecules for its growth. By definition, these are essential molecules which constitute the basic metabolic requirements of an organism. A small organic molecule with chemical similarity to that of an essential metabolite may bind to the enzyme that catalyzes its production and inhibit it, likely resulting in the stasis or death of the organism. Here, we report a high-throughput approach for identifying essential metabolites of an organism using genetic and biochemical approaches and then implement computational approaches to identify metabolite mimics. We generated and genotyped 5,126 Mycobacterium tuberculosis mutants and performed a statistical analysis to determine putative essential genes. The essential molecules of M. tuberculosis were classified as products of enzymes that are encoded by genes in this list. Although incomplete, as many enzymes of M. tuberculosis have yet to be identified and characterized, this is the first report of a large number of essential molecules of the organism. We identified essential metabolites of three distinct metabolic pathways in M. tuberculosis and selected molecules with chemical similarity using cheminformatics strategies that illustrate a variety of different pharmacophores. Our approach is aimed at systematic identification of essential molecules and their mimics as a blueprint for development of effective chemical probes of M. tuberculosis metabolism, with the ultimate goal of seeking drugs that can kill this pathogen. As an illustration of this approach, we report that compounds JFD01307SC and l-methionine-S-sulfoximine, which share chemical similarity with an essential molecule of M. tuberculosis, inhibited the growth of this organism at micromolar concentrations. PMID:21285434

  4. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    PubMed Central

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G. L.; Howlett, Allyn C.

    2015-01-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1? was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNF?, VCAM1, and NF?B1 were suppressed after 5 h with STE, whereas only TNF? and NF?B1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNF? were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. PMID:24927667

  5. Expression of POX2 gene and disruption of POX3 genes in the industrial Yarrowia lipolytica on the ?-decalactone production.

    PubMed

    Guo, Yanqiong; Song, Huanlu; Wang, Zhaoyue; Ding, Yongzhi

    2012-04-20

    The yeast Yarrowia lipolytica growing on methyl ricinoleate can produce ?-decalactone, the worthy aroma compound, which can exhibit fruity and creamy sensorial notes, and recognized internationally as a safe food additive. Unfortunately, the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded), which was responsible for continuation of oxidation after C(10) level and lactone reconsumption. In this paper, we chose the industrial Y. lipolytica (CGMCC accession number 2.1405), which is the diploid strain as the starting strain and constructed the recombinant strain Tp-12 by targeting the POX3 locus of the wild type, one copy of POX3 was deleted by CRF1+POX2 insertion. The other recombinant strain Tpp-11, which was a null mutant possessing multiple copies of POX2 and disrupted POX3 genes on two chromosomes, was constructed by inserting XPR2+hpt into the other copy of POX3 of Tp-12. The growth ability of the recombinants was changed after genetic modification in the fermentation medium. The production of ?-decalactone was increased, resulting from blocking ?-oxidation at the C(10) Aox level and POX2 overexpression. The recombinant strain Tpp-11 was stable. Because there was no reconsumption of ?-decalactone, the mutant strain could be grown in continuous fermentation of methyl ricinoleate to produce ?-decalactone. PMID:22115771

  6. ESSENTIAL CHROMIUM?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During the production of chrome-tanned leather, chromium is incorporated into the leather with at least three types of interactions. Most important is the chromium that is complexed with collagen to give leather its characteristic properties. Other interactions include nonproductive binding of chr...

  7. Discovery of a Linear Peptide for Improving Tumor Targeting of Gene Products and Treatment of Distal Tumors by IL-12 Gene Therapy

    PubMed Central

    Cutrera, Jeffry; Dibra, Denada; Xia, Xueqing; Hasan, Azeem; Reed, Scott; Li, Shulin

    2011-01-01

    Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments. PMID:21386825

  8. TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

    PubMed Central

    Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

    2014-01-01

    Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30?l inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

  9. Genome Wide Association Analysis Reveals New Production Trait Genes in a Male Duroc Population

    PubMed Central

    Wang, Kejun; Liu, Dewu; Hernandez-Sanchez, Jules; Chen, Jie; Liu, Chengkun; Wu, Zhenfang; Fang, Meiying; Li, Ning

    2015-01-01

    In this study, 796 male Duroc pigs were used to identify genomic regions controlling growth traits. Three production traits were studied: food conversion ratio, days to 100 KG, and average daily gain, using a panel of 39,436 single nucleotide polymorphisms. In total, we detected 11 genome-wide and 162 chromosome-wide single nucleotide polymorphism trait associations. The Gene ontology analysis identified 14 candidate genes close to significant single nucleotide polymorphisms, with growth-related functions: six for days to 100 KG (WT1, FBXO3, DOCK7, PPP3CA, AGPAT9, and NKX6-1), seven for food conversion ratio (MAP2, TBX15, IVL, ARL15, CPS1, VWC2L, and VAV3), and one for average daily gain (COL27A1). Gene ontology analysis indicated that most of the candidate genes are involved in muscle, fat, bone or nervous system development, nutrient absorption, and metabolism, which are all either directly or indirectly related to growth traits in pigs. Additionally, we found four haplotype blocks composed of suggestive single nucleotide polymorphisms located in the growth trait-related quantitative trait loci and further narrowed down the ranges, the largest of which decreased by ~60 Mb. Hence, our results could be used to improve pig production traits by increasing the frequency of favorable alleles via artificial selection. PMID:26418247

  10. Identification of the Pr1 Gene Product Completes the Anthocyanin Biosynthesis Pathway of Maize

    PubMed Central

    Sharma, Mandeep; Cortes-Cruz, Moises; Ahern, Kevin R.; McMullen, Michael; Brutnell, Thomas P.; Chopra, Surinder

    2011-01-01

    In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3?H encoding gene (Zmf3?h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3?H1 promoter–gene construct established that the encoded protein product was sufficient to perform a 3?-hydroxylation reaction. The Zmf3?h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5?-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize. PMID:21385724

  11. The transport of antibiotic resistance genes and residues in groundwater near swine production facilities

    NASA Astrophysics Data System (ADS)

    Lin, Y. F.; Yannarell, A. C.; Mackie, R. I.; Krapac, I. G.; Chee-Sanford, J. S.; Koike, S.

    2008-12-01

    The use of antibiotics at concentrated animal feeding operations (CAFOs) for disease prevention, disease treatment, and growth promotion can contribute to the spread of antibiotic compounds, their breakdown products, and antibiotic resistant bacteria and/or the genes that confer resistance. In addition, constitutive use of antibiotics at sub-therapeutic levels can select for antibiotic resistance among the bacteria that inhabit animal intestinal tracts, onsite manure treatment facilities, and any environments receiving significant inputs of manure (e.g. through waste lagoon leakage or fertilizer amendments to farm soils). If the antibiotic resistant organisms persist in these new environments, or if they participate in genetic exchanges with the native microflora, then CAFOs may constitute a significant reservoir for the spread of antibiotic resistance to the environment at large. Our results have demonstrated that leakage from waste treatment lagoons can influence the presence and persistence of tetracycline resistance genes in the shallow aquifer adjacent to swine CAFOs, and molecular phylogeny allowed us to distinguish "native" tetracycline resistance genes in control groundwater wells from manure-associated genes introduced from the lagoon. We have also been able to detect the presence of erythromycin resistance genes in CAFO surface and groundwater even though erythromycin is strictly reserved for use in humans and thus is not utilized at any of these sites. Ongoing research, including modeling of particle transport in groundwater, will help to determine the potential spatial and temporal extent of CAFO-derived antibiotic resistance.

  12. Discovering Genes Essential to the Hypothalamic Regulation of Human Reproduction Using a Human Disease Model: Adjusting to Life in the "-Omics" Era.

    PubMed

    Stamou, M I; Cox, K H; Crowley, William F

    2015-12-01

    The neuroendocrine regulation of reproduction is an intricate process requiring the exquisite coordination of an assortment of cellular networks, all converging on the GnRH neurons. These neurons have a complex life history, migrating mainly from the olfactory placode into the hypothalamus, where GnRH is secreted and acts as the master regulator of the hypothalamic-pituitary-gonadal axis. Much of what we know about the biology of the GnRH neurons has been aided by discoveries made using the human disease model of isolated GnRH deficiency (IGD), a family of rare Mendelian disorders that share a common failure of secretion and/or action of GnRH causing hypogonadotropic hypogonadism. Over the last 30 years, research groups around the world have been investigating the genetic basis of IGD using different strategies based on complex cases that harbor structural abnormalities or single pleiotropic genes, endogamous pedigrees, candidate gene approaches as well as pathway gene analyses. Although such traditional approaches, based on well-validated tools, have been critical to establish the field, new strategies, such as next-generation sequencing, are now providing speed and robustness, but also revealing a surprising number of variants in known IGD genes in both patients and healthy controls. Thus, before the field moves forward with new genetic tools and continues discovery efforts, we must reassess what we know about IGD genetics and prepare to hold our work to a different standard. The purpose of this review is to: 1) look back at the strategies used to discover the "known" genes implicated in the rare forms of IGD; 2) examine the strengths and weaknesses of the methodologies used to validate genetic variation; 3) substantiate the role of known genes in the pathophysiology of the disease; and 4) project forward as we embark upon a widening use of these new and powerful technologies for gene discovery. PMID:26394276

  13. Transcriptional Network Analysis Reveals that AT1 and AT2 Angiotensin II Receptors Are Both Involved in the Regulation of Genes Essential for Glioma Progression

    PubMed Central

    Azevedo, Hátylas; Fujita, André; Bando, Silvia Yumi; Iamashita, Priscila; Moreira-Filho, Carlos Alberto

    2014-01-01

    Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of Angiotensin II (Ang II) receptors has been associated with poor prognosis in human astrocytomas, the most common type of glioma. In this study, we investigated the role of Angiotensin II in glioma malignancy through transcriptional profiling and network analysis of cultured C6 rat glioma cells exposed to Ang II and to inhibitors of its membrane receptor subtypes. C6 cells were treated with Ang II and specific antagonists of AT1 and AT2 receptors. Total RNA was isolated after three and six hours of Ang II treatment and analyzed by oligonucleotide microarray technology. Gene expression data was evaluated through transcriptional network modeling to identify how differentially expressed (DE) genes are connected to each other. Moreover, other genes co-expressing with the DE genes were considered in these analyses in order to support the identification of enriched functions and pathways. A hub-based network analysis showed that the most connected nodes in Ang II-related networks exert functions associated with cell proliferation, migration and invasion, key aspects for glioma progression. The subsequent functional enrichment analysis of these central genes highlighted their participation in signaling pathways that are frequently deregulated in gliomas such as ErbB, MAPK and p53. Noteworthy, either AT1 or AT2 inhibitions were able to down-regulate different sets of hub genes involved in protumoral functions, suggesting that both Ang II receptors could be therapeutic targets for intervention in glioma. Taken together, our results point out multiple actions of Ang II in glioma pathogenesis and reveal the participation of both Ang II receptors in the regulation of genes relevant for glioma progression. This study is the first one to provide systems-level molecular data for better understanding the protumoral effects of Ang II in the proliferative and infiltrative behavior of gliomas. PMID:25365520

  14. Genetic resources for methane production from biomass described with the Gene Ontology.

    PubMed

    Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, Joăo C; Tyler, Brett M; Mukhopadhyay, Biswarup

    2014-01-01

    Methane (CH4) is a valuable fuel, constituting 70-95% of natural gas, and a potent greenhouse gas. Release of CH4 into the atmosphere contributes to climate change. Biological CH4 production or methanogenesis is mostly performed by methanogens, a group of strictly anaerobic archaea. The direct substrates for methanogenesis are H2 plus CO2, acetate, formate, methylamines, methanol, methyl sulfides, and ethanol or a secondary alcohol plus CO2. In numerous anaerobic niches in nature, methanogenesis facilitates mineralization of complex biopolymers such as carbohydrates, lipids and proteins generated by primary producers. Thus, methanogens are critical players in the global carbon cycle. The same process is used in anaerobic treatment of municipal, industrial and agricultural wastes, reducing the biological pollutants in the wastes and generating methane. It also holds potential for commercial production of natural gas from renewable resources. This process operates in digestive systems of many animals, including cattle, and humans. In contrast, in deep-sea hydrothermal vents methanogenesis is a primary production process, allowing chemosynthesis of biomaterials from H2 plus CO2. In this report we present Gene Ontology (GO) terms that can be used to describe processes, functions and cellular components involved in methanogenic biodegradation and biosynthesis of specialized coenzymes that methanogens use. Some of these GO terms were previously available and the rest were generated in our Microbial Energy Gene Ontology (MENGO) project. A recently discovered non-canonical CH4 production process is also described. We have performed manual GO annotation of selected methanogenesis genes, based on experimental evidence, providing "gold standards" for machine annotation and automated discovery of methanogenesis genes or systems in diverse genomes. Most of the GO-related information presented in this report is available at the MENGO website (http://www.mengo.biochem.vt.edu/). PMID:25520705

  15. Genetic resources for methane production from biomass described with the Gene Ontology

    PubMed Central

    Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, Joăo C.; Tyler, Brett M.; Mukhopadhyay, Biswarup

    2014-01-01

    Methane (CH4) is a valuable fuel, constituting 70–95% of natural gas, and a potent greenhouse gas. Release of CH4 into the atmosphere contributes to climate change. Biological CH4 production or methanogenesis is mostly performed by methanogens, a group of strictly anaerobic archaea. The direct substrates for methanogenesis are H2 plus CO2, acetate, formate, methylamines, methanol, methyl sulfides, and ethanol or a secondary alcohol plus CO2. In numerous anaerobic niches in nature, methanogenesis facilitates mineralization of complex biopolymers such as carbohydrates, lipids and proteins generated by primary producers. Thus, methanogens are critical players in the global carbon cycle. The same process is used in anaerobic treatment of municipal, industrial and agricultural wastes, reducing the biological pollutants in the wastes and generating methane. It also holds potential for commercial production of natural gas from renewable resources. This process operates in digestive systems of many animals, including cattle, and humans. In contrast, in deep-sea hydrothermal vents methanogenesis is a primary production process, allowing chemosynthesis of biomaterials from H2 plus CO2. In this report we present Gene Ontology (GO) terms that can be used to describe processes, functions and cellular components involved in methanogenic biodegradation and biosynthesis of specialized coenzymes that methanogens use. Some of these GO terms were previously available and the rest were generated in our Microbial Energy Gene Ontology (MENGO) project. A recently discovered non-canonical CH4 production process is also described. We have performed manual GO annotation of selected methanogenesis genes, based on experimental evidence, providing “gold standards” for machine annotation and automated discovery of methanogenesis genes or systems in diverse genomes. Most of the GO-related information presented in this report is available at the MENGO website (http://www.mengo.biochem.vt.edu/). PMID:25520705

  16. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex.

    PubMed

    Spear, Allyn; Ogram, Sushma A; Morasco, B Joan; Smerage, Lucia Eisner; Flanegan, James B

    2015-11-01

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showed that 3D entered the replication complex in the form of its precursor, P3 (or 3CD), and was cleaved to release active 3D polymerase. Furthermore, our results showed that P3 is the preferred precursor that binds to the 5'CL. Using reciprocal complementation assays, we showed that one molecule of P3 binds the 5'CL and that a second molecule of P3 provides 3D. In addition, we showed that a second molecule of P3 served as the VPg provider. These results support a model in which P3 binds to the 5'CL and recruits additional molecules of P3, which are cleaved to release either 3D or VPg to initiate RNA replication. PMID:26303005

  17. Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in Sphingomonas Strain S88 and Rhizobium leguminosarum

    PubMed Central

    Pollock, Thomas J.; van Workum, Wilbert A. T.; Thorne, Linda; Mikolajczak, Marcia J.; Yamazaki, Motohide; Kijne, Jan W.; Armentrout, Richard W.

    1998-01-01

    Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(?1?4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(?1?4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(?1?4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate. PMID:9457861

  18. Molecular and Functional Analyses of the Gene (eshA) Encoding the 52-Kilodalton Protein of Streptomyces coelicolor A3(2) Required for Antibiotic Production

    PubMed Central

    Kawamoto, Shinichi; Watanabe, Masakatsu; Saito, Natsumi; Hesketh, Andrew; Vachalova, Katerina; Matsubara, Keiko; Ochi, Kozo

    2001-01-01

    Analysis of proteins recovered in the S100 precipitate fraction of Streptomyces griseus after ultracentrifugation led to the identification of a 52-kDa protein which is produced during the late growth phase. The gene (eshA) which codes for this protein was cloned from S. griseus, and then its homologue was cloned from Streptomyces coelicolor A3(2). The protein was deduced to be 471 amino acids in length. The protein EshA is characterized by a central region that shows homology to the eukaryotic-type cyclic nucleotide-binding domains. Significant homology was also found to MMPI in Mycobacterium leprae, a major antigenic protein to humans. The eshA gene mapped near the chromosome end and was not essential for viability, as demonstrated by gene disruption experiments, but its disruption resulted in the abolishment of an antibiotic (actinorhodin but not undecylprodigiosin) production. Aerial mycelium was produced as abundantly as by the parent strain. Expression analysis of the EshA protein by Western blotting revealed that EshA is present only in late-growth-phase cells. The eshA gene was transcribed just preceding intracellular accumulation of the EshA protein, as determined by S1 nuclease protection, indicating that EshA expression is regulated at the transcription level. The expression of EshA was unaffected by introduction of the relA mutation, which blocks ppGpp synthesis. PMID:11567001

  19. New silk protein: modification of silk protein by gene engineering for production of biomaterials.

    PubMed

    Mori, H; Tsukada, M

    2000-08-01

    The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein. PMID:11763506

  20. Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

    USGS Publications Warehouse

    Mackie, R.I.; Koike, S.; Krapac, I.; Chee-Sanford, J.; Maxwell, Susan; Aminov, R.I.

    2006-01-01

    Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators-including inorganic ions, antibiotics, and antibiotic resistance genes-were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 ??g/L.Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes-tet(M), tet(O), tet(Q), and tet(W)-were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.

  1. Increased IL-6 gene expression and production in patients with common variable immunodeficiency.

    PubMed Central

    Pandolfi, F; Paganelli, R; Oliva, A; Quinti, I; Polidori, V; Fanales-Belasio, E; Guerra, E; Aiuti, F

    1993-01-01

    We have studied IL-6 gene expression and production by in vitro stimulated peripheral blood mononuclear cells (PBMC) isolated from common variable immunodeficiency (CVI) patients. A strong hybridization signal for the IL-6 probe was observed in mRNA extracted from phytohaemagglutinin (PHA)- and PHA/phorbol myristate acetate (PMA)-stimulated PBMC from most of 12 CVI patients analysed. IL-6 production by PHA-stimulated PBMC from 28 CVI patients was evaluated in ELISA and found to be significantly (P < 0.0001) higher than in normal controls. IL-6 production, however, did not correlate with the lymphocyte populations examined, nor with the absolute number of monocytes. We have also showed that IL-6 was able to increase IgM secretion by several Epstein-Barr virus (EBV)-transformed cell lines derived from both normal donors and CVI patients, but it failed to modify substantially the amounts of IgM and IgG produced in vitro by PBMC derived from CVI patients and activated with pokeweed mitogen (PWM) or anti-IgM. Our data indicate that IL-6 gene expression and production is increased in CVI, but CVI cells do not respond to IL-6 with increased production of immunoglobulin. Images Fig. 1 PMID:8485909

  2. Targeted Analysis Reveals an Important Role of JAK-STAT-SOCS Genes for Milk Production Traits in Australian Dairy Cattle

    PubMed Central

    Arun, Sondur J.; Thomson, Peter C.; Sheehy, Paul A.; Khatkar, Mehar S.; Raadsma, Herman W.; Williamson, Peter

    2015-01-01

    The Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway genes along with suppressors of cytokine signalling (SOCS) family genes play a crucial role in controlling cytokine signals in the mammary gland and thus mammary gland development. Mammary gene expression studies showed differential expression patterns for all the JAK-STAT pathway genes. Gene expression studies using qRT-PCR revealed differential expression of SOCS2, SOCS4, and SOCS5 genes across the lactation cycle in dairy cows. Using genotypes from 1,546 Australian Holstein-Friesian bulls, a statistical model for an association analysis based on SNPs within 500 kb of JAK-STAT pathway genes, and SOCS genes alone was constructed. The analysis suggested that these genes and pathways make a significant contribution to the Australian milk production traits. There were 24 SNPs close to SOCS1, SOCS3, SOCS5, SOCS7, and CISH genes that were significantly associated with Australian Profit Ranking (APR), Australian Selection Index (ASI), and protein yield (PY). This study supports the view that there may be some merit in choosing SNPs around functionally relevant genes for the selection and genetic improvement schemes for dairy production traits. PMID:26697059

  3. Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

    PubMed

    Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

    2012-05-01

    The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli. PMID:22460717

  4. Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells

    PubMed Central

    Hara, Toshifumi; Jones, Matthew F.; Subramanian, Murugan; Li, Xiao Ling; Ou, Oliver; Zhu, Yuelin; Yang, Yuan; Wakefield, Lalage M.; Hussain, S. Perwez; Gaedcke, Jochen; Ried, Thomas; Luo, Ji; Caplen, Natasha J.; Lal, Ashish

    2014-01-01

    MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors. PMID:25245095

  5. The production, purification and crystallization of a soluble form of the nonclassical MHC HLA-G: the essential role of cobalt

    SciTech Connect

    Clements, Craig S.; Kjer-Nielsen, Lars; Kostenko, Lyudmila; McCluskey, James; Rossjohn, Jamie

    2006-01-01

    X-ray diffraction data were collected to 1.9 Ĺ from crystals of HLA-G. Cobalt ions were found to be essential for the production of diffracting crystals. HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal–maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 Ĺ resolution, belonged to space group P3{sub 2}2{sub 1}, with unit-cell parameters a = b = 77.15, c = 151.72 Ĺ.

  6. Production of Chlorella vulgaris as a source of essential fatty acids in a tubular photobioreactor continuously fed with air enriched with CO2 at different concentrations.

    PubMed

    Ortiz Montoya, Erika Y; Casazza, Alessandro A; Aliakbarian, Bahar; Perego, Patrizia; Converti, Attilio; de Carvalho, Joăo C Monteiro

    2014-01-01

    To reduce CO2 emissions and simultaneously produce biomass rich in essential fatty acids, Chlorella vulgaris CCAP 211 was continuously grown in a tubular photobioreactor using air alone or air enriched with CO2 as the sole carbon source. While on one hand, nitrogen-limited conditions strongly affected biomass growth, conversely, they almost doubled its lipid fraction. Under these conditions using air enriched with 0, 2, 4, 8, and 16% (v/v) CO2 , the maximum biomass concentration was 1.4, 5.8, 6.6, 6.8, and 6.4 gDB L(-1) on a dry basis, the CO2 consumption rate 62, 380, 391, 433, and 430 mgCO2 L(-1) day(-1) , and the lipid productivity 3.7, 23.7, 24.8, 29.5, and 24.4 mg L(-1) day(-1) , respectively. C. vulgaris was able to grow effectively using CO2 -enriched air, but its chlorophyll a (3.0-3.5 g 100gDB (-1) ), chlorophyll b (2.6-3.0 g 100gDB (-1) ), and lipid contents (10.7-12.0 g 100gDB (-1) ) were not significantly influenced by the presence of CO2 in the air. Most of the fatty acids in C. vulgaris biomass were of the saturated series, mainly myristic, palmitic, and stearic acids, but a portion of no less than 45% consisted of unsaturated fatty acids, and about 80% of these were high added-value essential fatty acids belonging to the ?3 and ?6 series. These results highlight that C. vulgaris biomass could be of great importance for human health when used as food additive or for functional food production. PMID:24532479

  7. Recognition of adenovirus E1A gene products on immortalized cell surfaces by cytotoxic T lymphocytes.

    PubMed

    Bellgrau, D; Walker, T A; Cook, J L

    1988-05-01

    The experiments described in this report were designed to examine whether target cells transfected with the adenovirus E1A gene and exhibiting increased susceptibility to lysis by natural killer cells and activated macrophages (J. L. Cook, T. A. Walker, A. M. Lewis, Jr., H. E. Ruley, F. L. Graham, and S. H. Pilder, Proc. Natl. Acad. Sci. USA 83:6965-6969, 1986) also express E1A proteins on their surfaces. MT1A, 12S, and 13S are strain Fischer baby rat kidney (BRK) cell lines immortalized by transfection with plasmids containing only the E1A gene of nononcogenic adenovirus. All of these cell lines were effective in stimulating the generation of cytotoxic T lymphocytes (CTL) in vitro, provided that the cultures were supplemented with an exogenous source of lymphokine and that the responding lymphocytes were from syngeneic Fischer rats previously immunized with a cell line containing the intact E1A gene. HrA2, a Fischer BRK cell line immortalized by transfection with a plasmid containing only exon 1 of the E1A gene, did not generate, nor was it lysed by, E1A-specific CTL. The cytolytic activity of E1A-specific CTL was blocked by antiserum from Fischer rats immunized with purified E1A proteins synthesized in Escherichia coli, supporting the conclusion that an epitope on E1A proteins encoded by the intact E1A gene constitutes part of the CTL target structure on adenovirus-transformed cells. These data suggest that in addition to their functions within host cells, E1A gene products are important immunogenic determinants on the surfaces of adenovirus-transformed cells. PMID:2451753

  8. Silver Resistance Genes Are Overrepresented among Escherichia coli Isolates with CTX-M Production

    PubMed Central

    Edquist, Petra; Sandegren, Linus; Adler, Marlen; Tängdén, Thomas; Drobni, Mirva; Olsen, Björn; Melhus, Ĺsa

    2014-01-01

    Members of the Enterobacteriaceae with extended-spectrum beta-lactamases (ESBLs) of the CTX-M type have disseminated rapidly in recent years and have become a threat to public health. In parallel with the CTX-M type expansion, the consumption and widespread use of silver-containing products has increased. To determine the carriage rates of silver resistance genes in different Escherichia coli populations, the presence of three silver resistance genes (silE, silP, and silS) and genes encoding CTX-M-, TEM-, and SHV-type enzymes were explored in E. coli isolates of human (n = 105) and avian (n = 111) origin. The antibiotic profiles were also determined. Isolates harboring CTX-M genes were further characterized, and phenotypic silver resistance was examined. The silE gene was present in 13 of the isolates. All of them were of human origin. Eleven of these isolates harbored ESBLs of the CTX-M type (P = 0.007), and eight of them were typed as CTX-M-15 and three as CTX-M-14. None of the silE-positive isolates was related to the O25b-ST131 clone, but 10 out of 13 belonged to the ST10 or ST58 complexes. Phenotypic silver resistance (silver nitrate MIC > 512 mg/liter) was observed after silver exposure in 12 of them, and a concomitant reduced susceptibility to piperacillin-tazobactam developed in three. In conclusion, 12% of the human E. coli isolates but none of the avian isolates harbored silver resistance genes. This indicates another route for or level of silver exposure for humans than that caused by common environmental contamination. Since silE-positive isolates were significantly more often found in CTX-M-positive isolates, it is possible that silver may exert a selective pressure on CTX-M-producing E. coli isolates. PMID:25128339

  9. Prevalence of ten putative virulence genes in the emerging foodborne pathogen Arcobacter isolated from food products.

    PubMed

    Girbau, Cecilia; Guerra, Cristian; Martínez-Malaxetxebarria, Irati; Alonso, Rodrigo; Fernández-Astorga, Aurora

    2015-12-01

    Arcobacter spp. are considered to be emerging food- and waterborne pathogens for both humans and animals. However, their virulence mechanisms are still poorly understood. In this study the presence of ten virulence genes (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, tlyA and iroE) was assessed in a set of 47 strains of Arcobacter butzleri, 10 of Arcobacter cryaerophilus and 1 Arcobacter skirrowii strain recovered from different food products (pork, chicken, beef, milk, clams and mussels). Overall, the genes cadF, ciaB, cj1349, mviN, pldA and tlyA were detected in all A. butzleri and A. skirrowii strains. Lower detection rates were observed for irgA, iroE, hecA and hecB. The genes hecB and iroE were detected neither in A. cryaerophilus nor in A. skirrowii. The genes hecA and irgA were not detected in A. skirrowii. It was noteworthy that the genes hecA and hecB were significantly (P < 0.05) highly detected in A. butzleri strains isolated from clams compared with strains isolated from milk and chicken. Therefore, our findings underline clams as a source of A. butzleri strains with high prevalence of putative virulence genes. This could be hazardous to human health, especially because these bivalves are usually consumed raw or undercooked. PMID:26338128

  10. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    SciTech Connect

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  11. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu (Muko, JP); Hiraoka, Nobutsugu (Muko, JP); Obayashi, Akira (Uji, JP)

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  12. Pharmacotherapy of Essential Tremor

    PubMed Central

    Hedera, Peter; Cibul?ík, František; Davis, Thomas L.

    2013-01-01

    Essential tremor (ET) is a common movement disorder but its pathogenesis remains poorly understood. This has limited the development of effective pharmacotherapy. The current therapeutic armamentaria for ET represent the product of careful clinical observation rather than targeted molecular modeling. Here we review their pharmacokinetics, metabolism, dosing, and adverse effect profiles and propose a treatment algorithm. We also discuss the concept of medically refractory tremor, as therapeutic trials should be limited unless invasive therapy is contraindicated or not desired by patients. PMID:24385718

  13. Nonsteroidal anti-inflammatory drug activated gene-1 (NAG-1) modulators from natural products as anti-cancer agents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural products are rich source of gene modulators for prevention and treatment of cancer. In recent days, nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) has been focused as a new target of diverse cancers like colorectal, pancreatic, prostate, and breast. A variety of natural...

  14. Dissection of Maize Kernel Composition and Starch Production by Candidate Gene Association

    PubMed Central

    Wilson, Larissa M.; Whitt, Sherry R.; Ibáńez, Ana M.; Rocheford, Torbert R.; Goodman, Major M.; Buckler, Edward S.

    2004-01-01

    Cereal starch production forms the basis of subsistence for much of the world's human and domesticated animal populations. Starch concentration and composition in the maize (Zea mays ssp mays) kernel are complex traits controlled by many genes. In this study, an association approach was used to evaluate six maize candidate genes involved in kernel starch biosynthesis: amylose extender1 (ae1), brittle endosperm2 (bt2), shrunken1 (sh1), sh2, sugary1, and waxy1. Major kernel composition traits, such as protein, oil, and starch concentration, were assessed as well as important starch composition quality traits, including pasting properties and amylose levels. Overall, bt2, sh1, and sh2 showed significant associations for kernel composition traits, whereas ae1 and sh2 showed significant associations for starch pasting properties. ae1 and sh1 both associated with amylose levels. Additionally, haplotype analysis of sh2 suggested this gene is involved in starch viscosity properties and amylose content. Despite starch concentration being only moderately heritable for this particular panel of diverse maize inbreds, high resolution was achieved when evaluating these starch candidate genes, and diverse alleles for breeding and further molecular analysis were identified. PMID:15377761

  15. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins

    SciTech Connect

    Strebel, K.; Beck, E.; Strohmaier, K.; Schaller, H.

    1986-03-01

    Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambdaPL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in /sup 35/S-labeled extracts from foot-and-mouth disease virus-infected BHK cells. This allowed us to locate unequivocally all mature foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and mouth disease virus gene products not previously identified in vivo or in vitro.

  16. Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

    SciTech Connect

    Melis, Anastasios; Mitra, Mautusi

    2010-06-29

    The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

  17. Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

    SciTech Connect

    Napp, S.J.; Da Silva, N.A. )

    1993-04-01

    Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product [beta]-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and [beta]-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both [beta]-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and urid-k mutations were very stable; after more than 50 generation of growth in complex medium, stability values of 99-100% were measured.

  18. Improved assembly of multimeric genes for the biosynthetic production of protein polymers.

    PubMed

    Goeden-Wood, Nichole L; Conticello, Vincent P; Muller, Susan J; Keasling, Jay D

    2002-01-01

    We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the beta-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI-TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a beta-sheet structure in aqueous solution. PMID:12099837

  19. A Short cis-Acting Motif in the M112-113 Promoter Region Is Essential for IE3 To Activate M112-113 Gene Expression and Is Important for Murine Cytomegalovirus Replication

    PubMed Central

    Perez, Kareni J.; Martínez, Francisco Puerta; Cosme-Cruz, Ruth; Perez-Crespo, Neysa M.

    2013-01-01

    Immediate-early 3 (IE3) gene products are required to activate early (E)-stage gene expression of murine cytomegaloviruses (MCMV). The first early gene activated by IE3 is the M112-113 gene (also called E1), although a complete understanding of the activation mechanism is still lacking. In this paper, we identify a 10-bp cis-regulating motif upstream of the M112-113 TATA box as important for IE3 activation of M112-113 expression. Results from DNA affinity assays and chromatin immunoprecipitation assays show that the association of IE3 with the M112-113 gene promoter was eliminated by deletion of the 10-bp DNA sequence, now named IE3AM (for IE3 activating motif). In addition, IE3 interacts with TATA box binding protein (TBP), a core protein of TFIID (transcription initiation) complexes. Finally, we created an IE3AM-deleted MCMV (MCMVdIE3AM) using a bacterial artificial chromosome system. The mutant virus can still replicate in NIH 3T3 cells but at a significantly lower level. The defectiveness of the MCMVdIE3AM infection can be rescued in an M112-113-complemented cell line. Our results suggest that the interactions of IE3 with IE3AM and with TBP stabilize the TFIID complex at the M112-113 promoter such that M112-113 gene expression can be activated and/or enhanced. PMID:23255797

  20. Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor ?2 in Thyrotroph

    PubMed Central

    Matsunaga, Hideyuki; Sasaki, Shigekazu; Suzuki, Shingo; Matsushita, Akio; Nakamura, Hirotoshi; Nakamura, Hiroko Misawa; Hirahara, Naoko; Kuroda, Go; Iwaki, Hiroyuki; Ohba, Kenji; Morita, Hiroshi; Oki, Yutaka; Suda, Takafumi

    2015-01-01

    The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the ? subunit of TSH (TSH?). We previously reported that T3-dependent repression of the TSH? gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived T?T1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with T?T1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TR?2. The inhibition by T3-bound TR?2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TR?1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSH? gene. PMID:26571013

  1. A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila

    PubMed Central

    Enya, Sora; Ameku, Tomotsune; Igarashi, Fumihiko; Iga, Masatoshi; Kataoka, Hiroshi; Shinoda, Tetsuro; Niwa, Ryusuke

    2014-01-01

    In insects, the precise timing of moulting and metamorphosis is strictly guided by ecdysteroids that are synthesised from dietary cholesterol in the prothoracic gland (PG). In the past decade, several ecdysteroidogenic enzymes, some of which are encoded by the Halloween genes, have been identified and characterised. Here, we report a novel Halloween gene, noppera-bo (nobo), that encodes a member of the glutathione S-transferase family. nobo was identified as a gene that is predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knock-out mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo knock-down larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titres. Importantly, both embryonic and larval phenotypes were rescued by the administration of 20E or cholesterol. We also confirm that PG cells in nobo loss-of-function larvae abnormally accumulate cholesterol. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, our results raise the possibility that nobo plays a crucial role in regulating the behaviour of cholesterol in steroid biosynthesis in insects. PMID:25300303

  2. A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila.

    PubMed

    Enya, Sora; Ameku, Tomotsune; Igarashi, Fumihiko; Iga, Masatoshi; Kataoka, Hiroshi; Shinoda, Tetsuro; Niwa, Ryusuke

    2014-01-01

    In insects, the precise timing of moulting and metamorphosis is strictly guided by ecdysteroids that are synthesised from dietary cholesterol in the prothoracic gland (PG). In the past decade, several ecdysteroidogenic enzymes, some of which are encoded by the Halloween genes, have been identified and characterised. Here, we report a novel Halloween gene, noppera-bo (nobo), that encodes a member of the glutathione S-transferase family. nobo was identified as a gene that is predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knock-out mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo knock-down larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titres. Importantly, both embryonic and larval phenotypes were rescued by the administration of 20E or cholesterol. We also confirm that PG cells in nobo loss-of-function larvae abnormally accumulate cholesterol. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, our results raise the possibility that nobo plays a crucial role in regulating the behaviour of cholesterol in steroid biosynthesis in insects. PMID:25300303

  3. Studies of the human c-myb gene and its product in human acute leukemias.

    PubMed

    Slamon, D J; Boone, T C; Murdock, D C; Keith, D E; Press, M F; Larson, R A; Souza, L M

    1986-07-18

    The myb gene is the transforming oncogene of the avian myeloblastosis virus (AMV); its normal cellular homolog, c-myb, is conserved across a broad span of evolution. In humans, c-myb is expressed in malignant hematopoietic cell lines and in primary hematopoietic tumors. Partial complementary DNA clones were generated from blast cells of patients with acute myelogenous leukemia. The sequences of the clones were compared to the c-myb of other species, as well as the v-myb of AMV. In addition, the carboxyl terminal region of human c-myb was placed in an expression vector to obtain protein for the generation of antiserum, which was used to identify the human c-myb gene product. Like v-myb, this protein was found within the nucleus of leukemic cells where it was associated with the nuclear matrix. These studies provide further evidence that c-myb might be involved in human leukemia. PMID:3014652

  4. Human lens. gamma. -crystallins: isolation, identification, and characterization of the expressed gene products

    SciTech Connect

    Siezen, R.J.; Thomson, J.A.; Kaplan, E.D.; Benedek, G.B.

    1987-09-01

    The authors have isolated the individual ..gamma..-crystallins expressed in young human lenses and identified with which of the six known human ..gamma..-crystallin genes they each correspond. They find that at least 90% of the ..gamma..-crystallins synthesized in the young human lens are the products of genes ..gamma..G3 and ..gamma..G4. They demonstrate that ..gamma..G4-crystallin undergoes a temperature-dependent phase separation, and they have measured the low-concentration branch of its coexistence curve up to about 40 mg/ml. By comparison, they found no evidence of ..gamma..G3-crystallin phase separating, even at lower temperatures and higher concentrations. This is consistent with predictions based on sequence homology between human and rat ..gamma..-crystallins. The implications of these findings for human inherited and senile cataracts are considered.

  5. Increased biomass production of industrial bakers' yeasts by overexpression of Hap4 gene.

    PubMed

    Dueńas-Sánchez, Rafael; Codón, Antonio C; Rincón, Ana M; Benítez, Tahía

    2010-10-15

    HAP4 encodes a transcriptional activator of respiration-related genes and so, redirection from fermentation to respiration flux should give rise to an increase in biomass production in Saccharomyces cerevisiae transformants that overexpress HAP4. With this aim, three bakers' yeasts, that is, V1 used for lean doughs, its 2-deoxy-D-glucose resistant derivative DOG21, and V3 employed for sweet doughs, were transformed with integrative cassettes that carried HAP4 gene under the control of constitutive promoter pTEF2; in addition VTH, DTH and 3TH transformants were selected and characterized. Transformants showed increased expression of HAP4 and respiration-related genes such as QCR7 and QCR8 with regard to parental, and similar expression of SUC2 and MAL12; these genes are relevant in bakers' industry. Invertase (Suc2p) and maltase (Mal12p) activities, growth and sugar consumption rates in laboratory (YPD) or industrial media (MAB) were also comparable in bakers' strains and their transformants, but VTH, DTH and 3TH increased their final biomass production by 9.5, 5.0 and 5.0% respectively as compared to their parentals in MAB. Furthermore, V1 and its transformant VTH had comparable capacity to ferment lean doughs (volume increase rate and final volume) while V3 and its transformant 3TH fermented sweet doughs in a similar manner. Therefore transformants possessed increased biomass yield and appropriate characteristics to be employed in bakers' industry because they lacked drug resistant markers and bacterial DNA, and were genetically stable. PMID:20832886

  6. Metabolic engineering of Escherichia coli for ethanol production without foreign genes

    NASA Astrophysics Data System (ADS)

    Kim, Youngnyun

    Worldwide dependence on finite petroleum-based energy necessitates alternative energy sources that can be produced from renewable resources. A successful example of an alternative transportation fuel is bioethanol, produced by microorganisms, from corn starch that is blended with gasoline. However, corn, currently the main feedstock for bioethanol production, also occupies a significant role in human food and animal feed chains. As more corn is diverted to bioethanol, the cost of corn is expected to increase with an increase in the price of food, feed and ethanol. Using lignocellulosic biomass for ethanol production is considered to resolve this problem. However, this requires a microbial biocatalyst that can ferment hexoses and pentoses to ethanol. Escherichia coli is an efficient biocatalyst that can use all the monomeric sugars in lignocellulose, and recombinant derivatives of E. coli have been engineered to produce ethanol as the major fermentation product. In my study, ethanologenic E. coli strains were isolated from a ldhA-, pflB- derivative without introduction of foreign genes. These isolates grew anaerobically and produced ethanol as the main fermentation product. The mutation responsible for anaerobic growth and ethanol production was mapped in the lpdA gene and the mutation was identified as E354K in three of the isolates tested. Another three isolates carried an lpdA mutation, H352Y. Enzyme kinetic studies revealed that the mutated form of the dihydrolipoamide dehydrogenase (LPD) encoded by the lpdA was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity to NADH of the pyruvate dehydrogenase complex in strain SE2378. The net yield of 4 moles of NADH and 2 moles of acetyl-CoA per mole of glucose produced by a combination of glycolysis and PDH provided a logical basis to explain the production of 2 moles of ethanol per glucose. The development of E. coli provides a potential biocatalyst for conversion of pentoses derived from cellulosic biomass to biobased products without the introduction of new genes.

  7. Analysis of Tox5 gene expression in Gibberella pulicaris strains with different trichothecene production phenotypes.

    PubMed Central

    Hohn, T M; Desjardins, A E; McCormick, S P

    1993-01-01

    The Tox5 gene encodes trichodiene synthase, the first unique enzyme in the trichothecene biosynthetic pathway. In Gibberella pulicaris R-6380, the level of Tox5 mRNA was found to increase 47-fold in early stationary phase. Sequence analysis of the Tox5 promoter regions from geographically distinct strains of G. pulicaris revealed the existence of two Tox5 alleles (Tox5-1 and Tox5-2). All G. pulicaris strains that produce high levels of trichothecenes in liquid culture carry a 42-nucleotide (nt) tandem repeat sequence (Tox5-1) in the Tox5 promoter region, whereas strains that produce low levels of trichothecenes carry a single copy of this sequence (Tox5-2). A genetic cross between high- and low-level trichothecene producers resulted in the cosegregation of higher-level trichothecene production with the Tox5-1 allele. To determine the importance of the 42-nt repeat sequence in the regulation of Tox5 expression, reporter gene constructs carrying either the Tox5-1 or the Tox5-2 promoter region fused to the beta-galactosidase gene of Escherichia coli were introduced into the high-level-trichothecene-producing strain, R-6380. Expression of reporter gene activity in transformants was found to be regulated in a manner similar to Tox5 expression but appeared to be independent of the 42-nt sequence copy number. These results indicate that transcriptional controls play an important role in the regulation of Tox5 expression and that genes involved in trichothecene biosynthesis in G. pulicaris may be linked to Tox5. Images PMID:8368827

  8. Self-association of the WT1 tumor suppressor gene product

    SciTech Connect

    Bruening, W.; Nakagama, H.: Bardessy, N.

    1994-09-01

    Wilms` tumor (WT), an embryonal malignancy of the kidney, occurs most frequently in children under the age of 5 years, affecting {approximately}1 in 10,000 individuals. The WT1 tumor suppressor gene, residing at 11p13, is structurally altered in {approximately}10-15% of WT cases. Individuals with germline mutations within the WT1 gene suffer from predisposition to WT and developmental defects of the urogenital system. Patients with heterozygous deletions of the WT1 gene, or mutations predicted to cause inactivation of one WT1 allele, suffer relatively mild genital system defects (notably hypospadias and cryptorchidism in males) and a predisposition to WT. These results suggest that developing genital system development is sensitive to the absolute concentrations of the WT1 gene products. Patients with missense mutations within the WT1 gene, however, can suffer from a much more severe disorder known as Denys-Drash syndrome (DDS). This syndrome is characterized by intersex disorders, renal nephropathy, and a predisposition to WTs. The increased severity of the developmental defects associated with DDS, compared to those individuals with mild genital system anomalies and WTs, suggests that mutations defined in patients with DDS behave in a dominant-negative fashion. We have identified a novel WT1 mutation in a patient with DDS. This mutation, predicted to produce a truncated WT1 polypeptide encompassing exons 1, 2, and 3, defines a domain capable of behaving as an antimorph. We have also demonstrated that WT1 can self-associate in vivo using yeast two-hybrid systems. Deletion analysis have mapped the interacting domains to the amino terminus of the WT1 polypeptide, within exons 1 and 2. These results provide a molecular mechanism to explain how WT1 mutations can function in a dominant-negative fashion to eliminate wild-type WT1 activity, leading to DDS.

  9. High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model–based analyses of transposon-insertion sequencing data

    PubMed Central

    Chao, Michael C.; Pritchard, Justin R.; Zhang, Yanjia J.; Rubin, Eric J.; Livny, Jonathan; Davis, Brigid M.; Waldor, Matthew K.

    2013-01-01

    The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data. PMID:23901011

  10. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

    2015-03-01

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  11. DNA assembler: a synthetic biology tool for characterizing and engineering natural product gene clusters.

    PubMed

    Shao, Zengyi; Zhao, Huimin

    2012-01-01

    The majority of existing antibacterial and anticancer drugs are natural products or their derivatives. However, the characterization and engineering of these compounds are often hampered by limited ability to manipulate the corresponding biosynthetic pathways. Recently, we developed a genomics-driven, synthetic biology-based method, DNA assembler, for discovery, characterization, and engineering of natural product biosynthetic pathways (Shao, Luo, & Zhao, 2011). By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in individual hosts in a single-step manner. In this chapter, we describe the general guidelines for construct design. By using two distinct biosynthetic pathways, we demonstrate that DNA assembler can perform multiple tasks, including heterologous expression, introduction of single or multiple point mutations, scar-less gene deletion, generation of product derivatives, and creation of artificial gene clusters. As such, this method offers unprecedented flexibility and versatility in pathway manipulations. PMID:23084940

  12. Mutations in ZMYND10, a gene essential for proper axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia.

    PubMed

    Moore, Daniel J; Onoufriadis, Alexandros; Shoemark, Amelia; Simpson, Michael A; zur Lage, Petra I; de Castro, Sandra C; Bartoloni, Lucia; Gallone, Giuseppe; Petridi, Stavroula; Woollard, Wesley J; Antony, Dinu; Schmidts, Miriam; Didonna, Teresa; Makrythanasis, Periklis; Bevillard, Jeremy; Mongan, Nigel P; Djakow, Jana; Pals, Gerard; Lucas, Jane S; Marthin, June K; Nielsen, Kim G; Santoni, Federico; Guipponi, Michel; Hogg, Claire; Antonarakis, Stylianos E; Emes, Richard D; Chung, Eddie M K; Greene, Nicholas D E; Blouin, Jean-Louis; Jarman, Andrew P; Mitchison, Hannah M

    2013-08-01

    Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects. PMID:23891471

  13. Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

    PubMed Central

    Nakai, T; Yasuhara, T; Fujiki, Y; Ohashi, A

    1995-01-01

    Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function. PMID:7623837

  14. Uncovering Divergence of Rice Exon Junction Complex Core Heterodimer Gene Duplication Reveals Their Essential Role in Growth, Development, and Reproduction1[W

    PubMed Central

    Gong, Pichang; He, Chaoying

    2014-01-01

    The exon junction complex (EJC) plays important developmental roles in animals; however, its role in plants is not well known. Here, we show various aspects of the divergence of each duplicated MAGO NASHI (MAGO) and Y14 gene pair in rice (Oryza sativa) encoding the putative EJC core subunits that form the obligate MAGO-Y14 heterodimers. OsMAGO1, OsMAGO2, and OsY14a were constitutively expressed in all tissues, while OsY14b was predominantly expressed in embryonic tissues. OsMAGO2 and OsY14b were more sensitive to different stresses than OsMAGO1 and OsY14a, and their encoded protein pair shared 93.8% and 46.9% sequence identity, respectively. Single MAGO down-regulation in rice did not lead to any phenotypic variation; however, double gene knockdowns generated short rice plants with abnormal flowers, and the stamens of these flowers showed inhibited degradation and absorption of both endothecium and tapetum, suggesting that OsMAGO1 and OsMAGO2 were functionally redundant. OsY14a knockdowns phenocopied OsMAGO1OsMAGO2 mutants, while down-regulation of OsY14b failed to induce plantlets, suggesting the functional specialization of OsY14b in embryogenesis. OsMAGO1OsMAGO2OsY14a triple down-regulation enhanced the phenotypes of OsMAGO1OsMAGO2 and OsY14a down-regulated mutants, indicating that they exert developmental roles in the MAGO-Y14 heterodimerization mode. Modified gene expression was noted in the altered developmental pathways in these knockdowns, and the transcript splicing of UNDEVELOPED TAPETUM1 (OsUDT1), a key regulator in stamen development, was uniquely abnormal. Concomitantly, MAGO and Y14 selectively bound to the OsUDT1 premessenger RNA, suggesting that rice EJC subunits regulate splicing. Our work provides novel insights into the function of the EJC locus in growth, development, and reproduction in angiosperms and suggests a role for these genes in the adaptive evolution of cereals. PMID:24820023

  15. Re-examination of regulatory opinions in Europe: possible contribution for the approval of the first gene therapy product Glybera.

    PubMed

    Watanabe, Natsumi; Yano, Kazuo; Tsuyuki, Kenichiro; Okano, Teruo; Yamato, Masayuki

    2015-01-01

    The first commercially approved human gene therapy in the Western world is Glybera (alipogene tiparvovec), which is an adenoassociated viral vector encoding the lipoprotein lipase gene. Glybera was recommended for marketing authorization by the European Medicines Agency in 2012. The European Medicines Agency had only ever reviewed three marketing authorization applications for gene therapy medicinal products. Unlike in the case of Glybera, the applications of the first two products, Cerepro and Contusugene Ladenovec Gendux/Advexin, both of which were for cancer diseases, were withdrawn. In this report, we studied the European public assessment reports of the three gene therapy products. During the assessment process, Glybera was re-examined and reviewed for a fourth time. We therefore researched the re-examination procedure of the European Union regulatory process. Approximately 25% of the new medicinal products initially given negative opinions from the Committee for Medicinal Products for Human Use were ultimately approved after re-examination from 2009 to 2013. The indications of most medicines were changed during the re-examination procedure, and the products were later approved with a mode of approval. These results suggested that the re-examination system in the European Union contributed to the approval of both several new drugs and the first gene therapy product. PMID:26052534

  16. The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast

    PubMed Central

    1991-01-01

    We recently described the identification of BOS1 (Newman, A., J. Shim, and S. Ferro-Novick. 1990. Mol. Cell. Biol. 10:3405-3414.). BOS1 is a gene that in multiple copy suppresses the growth and secretion defect of bet1 and sec22, two mutants that disrupt transport from the ER to the Golgi complex in yeast. The ability of BOS1 to specifically suppress mutants blocked at a particular stage of the secretory pathway suggested that this gene encodes a protein that functions in this process. The experiments presented in this study support this hypothesis. Specifically, the BOS1 gene was found to be essential for cellular growth. Furthermore, cells depleted of the Bos1 protein fail to transport pro-alpha-factor and carboxypeptidase Y (CPY) to the Golgi apparatus. This defect in export leads to the accumulation of an extensive network of ER and small vesicles. DNA sequence analysis predicts that Bos1 is a 27-kD protein containing a putative membrane- spanning domain. This prediction is supported by differential centrifugation experiments. Thus, Bos1 appears to be a membrane protein that functions in conjunction with Bet1 and Sec22 to facilitate the transport of proteins at a step subsequent to translocation into the ER but before entry into the Golgi apparatus. PMID:2007627

  17. SIL1, a causative cochaperone gene of Marinesco-Sjögren syndrome, plays an essential role in establishing the architecture of the developing cerebral cortex

    PubMed Central

    Inaguma, Yutaka; Hamada, Nanako; Tabata, Hidenori; Iwamoto, Ikuko; Mizuno, Makoto; Nishimura, Yoshiaki V; Ito, Hidenori; Morishita, Rika; Suzuki, Motomasa; Ohno, Kinji; Kumagai, Toshiyuki; Nagata, Koh-ichi

    2014-01-01

    Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessively inherited disorder with mental retardation (MR). Recently, mutations in the SIL1 gene, encoding a co-chaperone which regulates the chaperone HSPA5, were identified as a major cause of MSS. We here examined the pathophysiological significance of SIL1 mutations in abnormal corticogenesis of MSS. SIL1-silencing caused neuronal migration delay during corticogenesis ex vivo. While RNAi-resistant SIL1 rescued the defects, three MSS-causing SIL1 mutants tested did not. These mutants had lower affinities to HSPA5 in vitro, and SIL1-HSPA5 interaction as well as HSPA5 function was found to be crucial for neuronal migration ex vivo. Furthermore time-lapse imaging revealed morphological disorganization associated with abnormal migration of SIL1-deficient neurons. These results suggest that the mutations prevent SIL1 from interacting with and regulating HSPA5, leading to abnormal neuronal morphology and migration. Consistent with this, when SIL1 was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed. Taken together, abnormal neuronal migration and interhemispheric axon development may contribute to MR in MSS. Subject Categories Genetics; Gene Therapy & Genetic Disease; PMID:24473200

  18. The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisin production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in synthesis of mycotoxins and other secondary met...

  19. Diversity, Distribution and Quantification of Antibiotic Resistance Genes in Goat and Lamb Slaughterhouse Surfaces and Meat Products

    PubMed Central

    Lavilla Lerma, Leyre; Benomar, Nabil; Knapp, Charles W.; Correa Galeote, David; Gálvez, Antonio; Abriouel, Hikmate

    2014-01-01

    The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated ‘hot spots.’ The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination. PMID:25479100

  20. Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

    PubMed

    Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

    2015-02-01

    Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H?O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P??. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. PMID:25644955

  1. Virulence genes in a probiotic E. coli product with a recorded long history of safe use

    PubMed Central

    Zschüttig, Anke; Beimfohr, Claudia; Geske, Thomas; Auerbach, Christian; Cook, Helen; Zimmermann, Kurt; Gunzer, Florian

    2015-01-01

    The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli. PMID:25883796

  2. Expression study on D123 gene product: evidence for high positivity in testis.

    PubMed

    Onisto, M; Zeilante, P; Scannapieco, P; Pellati, D; Pozza, M; Caenazzo, C; Negro, A; Garbisa, S

    1998-08-01

    A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared with D123, three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in alpha-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified. PMID:9683532

  3. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  4. c-erbB-2 gene product directly associates with beta-catenin and plakoglobin.

    PubMed

    Kanai, Y; Ochiai, A; Shibata, T; Oyama, T; Ushijima, S; Akimoto, S; Hirohashi, S

    1995-03-28

    Association of the c-erbB-2 oncogene product with the cadherin-catenin complex has been demonstrated in human cancer cell lines. Although beta-catenin and plakoglobin have been proven to be crucial for the association, no previous study has shown whether the interactions are direct or indirect. In the present study, the c-erbB-2 gene product was shown by far-Western blotting analysis to associate directly with both beta-catenin and plakoglobin through its cytoplasmic domain core region, which showed extensive homology with epidermal growth factor receptor. These data suggest that c-erbB-2-induced signaling is also directly liked to the cadherin-mediated cell adhesion and "invasion-suppressor" system through beta-catenin and plakoglobin in cancers. PMID:7702605

  5. AMPK? is essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle.

    PubMed

    Fentz, Joachim; Kjřbsted, Rasmus; Kristensen, Caroline Maag; Hingst, Janne Rasmus; Birk, Jesper Bratz; Gudiksen, Anders; Foretz, Marc; Schjerling, Peter; Viollet, Benoit; Pilegaard, Henriette; Wojtaszewski, Jřrgen F P

    2015-12-01

    Exercise training increases skeletal muscle expression of metabolic proteins improving the oxidative capacity. Adaptations in skeletal muscle by pharmacologically induced activation of 5'-AMP-activated protein kinase (AMPK) are dependent on the AMPK?2 subunit. We hypothesized that exercise training-induced increases in exercise capacity and expression of metabolic proteins, as well as acute exercise-induced gene regulation, would be compromised in muscle-specific AMPK?1 and -?2 double-knockout (mdKO) mice. An acute bout of exercise increased skeletal muscle mRNA content of cytochrome c oxidase subunit I, glucose transporter 4, and VEGF in an AMPK-dependent manner, whereas cluster of differentiation 36 and fatty acid transport protein 1 mRNA content increased similarly in AMPK? wild-type (WT) and mdKO mice. During 4 wk of voluntary running wheel exercise training, the AMPK? mdKO mice ran less than WT. Maximal running speed was lower in AMPK? mdKO than in WT mice but increased similarly in both genotypes with exercise training. Exercise training increased quadriceps protein content of ubiquinol-cytochrome c reductase core protein 1 (UQCRC1), cytochrome c, hexokinase II, plasma membrane fatty acid-binding protein, and citrate synthase activity more in AMPK? WT than in mdKO muscle. However, analysis of a subgroup of mice matched for running distance revealed that only UQCRC1 protein content increased more in WT than in mdKO mice with exercise training. Thus, AMPK?1 and -?2 subunits are important for acute exercise-induced mRNA responses of some genes and may be involved in regulating basal metabolic protein expression but seem to be less important in exercise training-induced adaptations in metabolic proteins. PMID:26419588

  6. 5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene

    SciTech Connect

    Van Der Werf, M.J.; Zeikus, J.G. |

    1996-10-01

    The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinate (ALA) synthase. This enzyme catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine-forming ALA. The R. sphaeroides hemA gene in the pUC18/19 vector system was transformed into Escherichia coli. The effects of both genetic and physiological factors on the expression of ALA synthase and the production of ALA were studied. ALA synthase activity levels were maximal when hemA had the same transcription direction as the lac promoter. The distance between the lac promoter and hemA affected the expression of ALA synthase on different growth substrates. The E. coli host strain used had an enormous effect on the ALA synthase activity level and on the production of ALA, with E. coli DH1 being best suited. The ALA synthase activity level was also dependent on the carbon source. Succinate, L-malate, fumarate, and L-aspartate gave the highest levels of ALA synthase activity, while the use of lactose as a carbon source resulted in a repression of ALA synthase. After growth on succinate, ALA synthase represented {approx}5% of total cellular protein. The ALA synthase activity level was also dependent on the pH of the medium, with maximal activity occurring at pH 6.5. ALA production by whole cells was limited by the availability of glycine, and the addition of 2 g of glycine per liter to the growth medium increased the production of ALA fivefold, to 2.25 mM. In recombinant E. coli extracts, up to 22 mM ALA was produced from succinate, glycine, and ATP. 58 refs., 4 figs., 7 tabs.

  7. Nucleotide and protein sequences for dog masticatory tropomyosin identify a novel Tpm4 gene product.

    PubMed

    Brundage, Elizabeth A; Biesiadecki, Brandon J; Reiser, Peter J

    2015-10-01

    Jaw-closing muscles of several vertebrate species, including members of Carnivora, express a unique, "masticatory", isoform of myosin heavy chain, along with isoforms of other myofibrillar proteins that are not expressed in most other muscles. It is generally believed that the complement of myofibrillar isoforms in these muscles serves high force generation for capturing live prey, breaking down tough plant material and defensive biting. A unique isoform of tropomyosin (Tpm) was reported to be expressed in cat jaw-closing muscle, based upon two-dimensional gel mobility, peptide mapping, and immunohistochemistry. The objective of this study was to obtain protein and gene sequence information for this unique Tpm isoform. Samples of masseter (a jaw-closing muscle), tibialis (predominantly fast-twitch fibers), and the deep lateral gastrocnemius (predominantly slow-twitch fibers) were obtained from adult dogs. Expressed Tpm isoforms were cloned and sequencing yielded cDNAs that were identical to genomic predicted striated muscle Tpm1.1St(a,b,b,a) (historically referred to as ?Tpm), Tpm2.2St(a,b,b,a) (?Tpm) and Tpm3.12St(a,b,b,a) (?Tpm) isoforms (nomenclature reflects predominant tissue expression ("St"-striated muscle) and exon splicing pattern), as well as a novel 284 amino acid isoform observed in jaw-closing muscle that is identical to a genomic predicted product of the Tpm4 gene (?Tpm) family. The novel isoform is designated as Tpm4.3St(a,b,b,a). The myofibrillar Tpm isoform expressed in dog masseter exhibits a unique electrophoretic mobility on gels containing 6 M urea, compared to other skeletal Tpm isoforms. To validate that the cloned Tpm4.3 isoform is the Tpm expressed in dog masseter, E. coli-expressed Tpm4.3 was electrophoresed in the presence of urea. Results demonstrate that Tpm4.3 has identical electrophoretic mobility to the unique dog masseter Tpm isoform and is of different mobility from that of muscle Tpm1.1, Tpm2.2 and Tpm3.12 isoforms. We conclude that the unique Tpm isoform in dog masseter is a product of the Tpm4 gene and that the 284 amino acid protein product of this gene represents a novel myofibrillar Tpm isoform never before observed to be expressed in striated muscle. PMID:26400443

  8. Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum.

    PubMed Central

    Weaver, C H; Sheehan, K C; Keath, E J

    1996-01-01

    A yeast-phase-specific gene, yps-3, has been identified in the virulent Histoplasma capsulatum strain, G217B. Although DNA sequencing of the genomic yps-3 gene from G217B failed to detect homologies with known proteins, the 5' end of a yps-3 cDNA contained a consensus signal sequence. A 519-bp fragment of the cDNA containing the translational stop codon was linker modified and inserted into the bacterial expression vector, pATH 1. Escherichia coli extracts containing the pATH 1 vector alone expressed a major 34-kDa TrpE polypeptide following induction with indoleacrylic acid, while the pATH 1/yps-3 construct produced a predominant 54-kDa TrpE/yps-3 fusion protein. Polyclonal rabbit sera directed against G217B reacted exclusively with the 54-kDa fusion protein in Western blots (immunoblots); serum samples from three patients with acute pulmonary or disseminated histoplasmosis were also positive. To localize the yps-3 protein within G217B, a monoclonal antibody (MAb 7.1) which recognized the yps-3 portion of the fusion protein was generated. A 17.4-kDa protein was detected with MAb 7.1 in Western blots prepared from cell wall fractions of G217B; cytoplasmic fractions were unreactive. No yps-3 antigen was detected in either fraction of the Downs strain, which fails to express the yps-3 gene. MAb 7.1 also detected a 17.4-kDa antigen in ethanol-precipitated culture supernatants derived from G217B. These findings localize the yps-3 gene product to the cell wall and culture supernatants, where the protein may influence the phase transition or the maintenance of the yeast state. PMID:8757832

  9. Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

    PubMed

    Valera, Maria José; Torija, Maria Jesús; Mas, Albert; Mateo, Estibaliz

    2015-02-01

    The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose. PMID:25381910

  10. Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep

    PubMed Central

    Vitezica, Zulma G; Moreno, Carole R; Lantier, Frederic; Lantier, Isabelle; Schibler, Laurent; Roig, Anne; François, Dominique; Bouix, Jacques; Allain, Daniel; Brunel, Jean-Claude; Barillet, Francis; Elsen, Jean-Michel

    2007-01-01

    In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapiničre INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance) and production (wool and carcass) traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL) detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes. PMID:17612481

  11. Effect of retS gene on antibiotics production in Pseudomonas fluorescens FD6.

    PubMed

    Zhang, Qingxia; Xiao, Qi; Xu, Jingyou; Tong, Yunhui; Wen, Jia; Chen, Xijun; Wei, Lihui

    2015-11-01

    A hybrid sensor kinase termed RetS (regulator of exopolysaccharide and Type III secretion) controls expression of numerous genes in Pseudomonas aeruginosa. To investigate the function of RetS in P. fluorescens FD6, the retS gene was disrupted. Genetic inactivation of retS resulted in enhanced production of 2, 4-diacetylphloroglucinol, pyrrolnitrin, and pyoluteorin. The retS mutant also exhibited significant increase in phlA-lacZ, prnA-lacZ, and pltA-lacZ transcription levels, influencing expression levels of the small regulatory RNAs RsmX and RsmZ. In the gacSretS double mutant, all the phenotypic changes caused by the retS deletion were reversed to the level of gacS single mutant. Furthermore, the retS mutation drastically elevated biofilm formation and improved the colonization ability of strain FD6 on wheat rhizospheres. Based on these results, we proposed that RetS negatively controlled the production of antibiotics through the Gac/Rsm pathway in P. fluorescens FD6. PMID:26505308

  12. Effects of Rosmarinus officinalis?L. essential oils supplementation on digestion, colostrum production of dairy ewes and lamb mortality and growth.

    PubMed

    Smeti, Samir; Joy, Margalida; Hajji, Hadhami; Alabart, José Luis; Muńoz, Fernando; Mahouachi, Mokhtar; Atti, Naziha

    2015-07-01

    The aim of this study was to evaluate the effect of rosemary essential oils (REO) and the forage nature on ewes' performances, immune response and lambs' growth and mortality. Forty-eight dairy ewes (Sicilo-Sarde) were fed oat-hay or oat-silage supplemented with 400?g of concentrate during pregnancy and 600?g during postpartum. The experimental concentrate contained the same mixture as the control (barley, soybean meal and mineral vitamin supplement) more 0.6?g/kg of REO. Two groups were