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Sample records for gene product trans-activates

  1. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    SciTech Connect

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  2. The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

    PubMed

    Scholthof, H B; Gowda, S; Wu, F C; Shepherd, R J

    1992-05-01

    Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes. PMID:1560539

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

    SciTech Connect

    Takada, Shinako; Koike, Katsuro )

    1990-08-01

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

  4. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  5. Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

    PubMed

    Yu, Zhiyuan; Li, Mei; Zhang, Dongyu; Xu, William; Kone, Bruce C

    2009-07-01

    The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro. PMID:19420113

  6. Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus

    SciTech Connect

    Davis, M.G.; Kenney, S.C.; Kamine, J.; Pagano, J.S.; Huang, E.S.

    1987-12-01

    Almost all homosexual patients with acquired immunodeficiency syndrome are also actively infected with human cytomegalovirus (HCMV). The authors have hypothesized that an interaction between HCMV and human immunodeficiency virus (HIV), the agent that causes acquired immunodeficiency syndrome, may exist at a molecular level and contribute to the manifestations of HIV infection. In this report, they demonstrate that the immediate-early gene region of HCMV, in particular immediate-early region 2, trans-activates the expression of the bacterial gene chloramphenicol acetyltransferase that is fused to the HIV long terminal repeat and carried by plasmid pHIV-CAT. The HCMV immediate-early trans-activator increases the level of mRNA from the plamid pHIV-CAT. The sequences of HIV that are responsive to trans-activation by the HDMV immediate-early region are distinct from HIV sequences that are required for response to the HIV tat. The stimulation of HIV gene expression by HDMV gene functions could enhance the consequences of HIV infection in persons with previous or concurrent HCMV infection.

  7. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events

    SciTech Connect

    Hauber, J.; Perkins, A.; Heimer, E.P.; Cullen, B.R.

    1987-09-01

    Human immunodeficiency virus encodes a gene product termed tat that is able to activate viral gene expression when present in trans. The mechanism of action of the tat gene product appears to be bimodal, resulting in both an increase in the steady-state level of viral mRNA and the enhanced translation of that RNA. In this report, the authors have examined the mechanism by which tat elevates viral mRNA levels. Data are presented demonstrating that tat acts by increasing the rate of viral transcription, rather than by modulating the stability of viral mRNA. Indirect immunofluorescence was used to show that tat is predominantly localized in the nucleus of expressing cells, a location consistent with a role in the regulation of viral transcription. These results suggest that tat could play a role in human immunodeficiency virus replication essentially similar to that proposed for the trans-acting nuclear gene products described for several other virus species.

  8. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    PubMed

    Alwine, J C

    1985-05-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. PMID:2987671

  9. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    PubMed Central

    Alwine, J C

    1985-01-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. Images PMID:2987671

  10. Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

    PubMed

    Sampey, Gavin C; Saifuddin, Mohammed; Schwab, Angela; Barclay, Robert; Punya, Shreya; Chung, Myung-Chul; Hakami, Ramin M; Zadeh, Mohammad Asad; Lepene, Benjamin; Klase, Zachary A; El-Hage, Nazira; Young, Mary; Iordanskiy, Sergey; Kashanchi, Fatah

    2016-01-15

    HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART. PMID:26553869

  11. Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.

    PubMed Central

    Knust, B; Brüggemann, U; Doerfler, W

    1989-01-01

    In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene. Images PMID:2473219

  12. Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.

    PubMed Central

    Guarino, L A; Summers, M D

    1986-01-01

    The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1. Images PMID:3944847

  13. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    PubMed Central

    Geng, Deyu; Zhang, Zhixia; Guo, Huarong

    2012-01-01

    p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. PMID:25585933

  14. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum (Dendranthema vestitum).

    PubMed

    Liu, Liqing; Zhu, Kai; Yang, Yanfang; Wu, Jian; Chen, Fadi; Yu, Deyue

    2008-03-01

    Dehydration responsive element binding (DREB) proteins are important transcription factors in plant stress response and signal transduction. In this study, a DREB homolog gene, DvDREB2A, was isolated from chrysanthemum (Dendranthema vestitum) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. It contained an open reading frame (ORF) of 1,471 bp encoding 366 amino acid residues and was classified as a DREB2 subfamily member based on multiple sequence alignment. The predicted protein sequence contained a typical AP2/EREBP DNA-binding domain near the N-terminal region. In yeast one-hybrid analysis DvDREB2A protein was specifically bound to DRE elements (core sequence, A/GCCGAC) and activated the expression of the reporter HIS3 and LacZ. Transient expression experiment suggested that DvDREB2A protein was localized to the nucleus of onion epidermis cells. Quantitative real-time PCR (QRT-PCR) experiments showed that expression level of DvDREB2A was significantly affected by heat, low temperature, drought, abscisic acid (ABA) and high salinity treatments. These results indicated that the DvDREB2A gene is a new member of the DREB transcription factors, which may play an important role in providing tolerance to environmental stresses. PMID:18224275

  15. Cotton leaf curl Multan betasatellite as a plant gene delivery vector trans-activated by taxonomically diverse geminiviruses.

    PubMed

    Kharazmi, S; Behjatnia, S A A; Hamzehzarghani, H; Niazi, A

    2012-07-01

    Cotton leaf curl Multan betasatellite (CLCuMB) replicates in tobacco, tomato and datura plants in the presence of the helper viruses tomato leaf curl virus-Australia, Iranian isolates of tomato yellow leaf curl virus, tomato leaf curl Karnataka virus, and beet severe curly top virus (BSCTV). Infectious recombinant CLCuMB constructs were made in which segments of either the CaMV 35S or the petunia ChsA promoter replaced the CLCuMB βC1 ORF, and these were designated pBinβΔC1-35S and pBinβΔC1-ChsA, respectively. Inoculation of tobacco plants containing a functional 35S-GUS transgene with pBinβΔC1-35S, and normal petunia plants with pBinβΔC1-ChsA, in the presence of helper viruses resulted in silencing of GUS and ChsA activities in transgenic tobacco and non-transgenic petunia plants, respectively. Replication of CLCuMB with different geminiviruses, especially BSCTV, a curtovirus with a broad host range, makes it a valuable gene delivery vector to the large number of host plant species of geminiviruses that support CLCuMB. PMID:22476203

  16. CDC42-Interacting Protein 4 Gene Is Down Trans-Regulated by HBV DNA polymerase Trans Activated Protein 1

    PubMed Central

    LUN, Yongzhi; XU, Chongbo; CHI, Qing; WANG, Xuelei; SUI, Wen; JIANG, Sujuan

    2014-01-01

    Abstract Background Hepatitis B Virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein transactivated by HBV DNA polymerase, screened by suppression subtractive hybridization technique (GenBank accession no: AY450389). The biological function of HBVDNAPTP1 was investigated in this study. Methods We constructed a vector pcDNA3.1 (-)/myc-His A-HBVDNAPTP1 and used it to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by western blot analysis in the cells. A cDNA library of genes transactivated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using suppression subtractive hybridization (SSH). The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank. Results Some sequences, such as CIP4, might be involved in apoptosis development. mRNA and protein expression of CIP4 was identified by Real time RT-PCR and western blot in THP-1 cells. HBVDNAPTP1 could down-regulate the expression of CIP4 at both transcription and translation levels. Conclusion HBVDNAPTP1 may be involved in the positive regulation on the initiation of monocyte apoptosis. The result contribute to reveal the HBVDNAPTP1 biological functions and provide new evidences for further exploration of the regulatory mechanism of HBVDNAPTP1. PMID:25988087

  17. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    SciTech Connect

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O'Callaghan, Dennis J. . E-mail: docall@lsuhsc.edu

    2007-06-20

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed.

  18. Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation.

    PubMed

    Rawls, J A; Pusztai, R; Green, M

    1990-12-01

    The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F. PMID:2173783

  19. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

    PubMed Central

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C.-K.

    2015-01-01

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

  20. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene.

    PubMed

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C-K

    2015-12-15

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

  1. Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat.

    PubMed Central

    Sherman, L; Gazit, A; Yaniv, A; Kawakami, T; Dahlberg, J E; Tronick, S R

    1988-01-01

    We used the Escherichia coli chloramphenicol acetyltransferase gene (cat) to study sequences that influence expression of the equine infectious anemia virus (EIAV) genome. The EIAV long terminal repeat (LTR) directed CAT activity in a canine cell line, but at levels much lower than those achieved with other eucaryotic viral promoters. In the same cells infected with EIAV or cotransfected with molecularly cloned EIAV genomic DNA, LTR-directed activity was markedly enhanced. Comparison of cat mRNA and protein levels in these cells indicated that this trans-activating effect could be accounted for by a bimodal mechanism in which both transcriptional and posttranscriptional events are enhanced. trans-Activation but not promoter activity was abolished by deletion of the R-U5 region of the EIAV LTR. EIAV sequences responsible for the trans-activating function could be localized to a region encompassing the 3' and 5' termini of the pol and env genes, respectively (nucleotides 4474 to 5775). Interestingly, this stretch harbors a short open reading frame with some amino acid sequence similarity to the human immunodeficiency virus type I tat gene product. Images PMID:2824840

  2. EPAS1 trans-activation during hypoxia requires p42/p44 MAPK.

    PubMed

    Conrad, P W; Freeman, T L; Beitner-Johnson, D; Millhorn, D E

    1999-11-19

    Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-alpha/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-alpha (HIF1-alpha). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-alpha activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-induced trans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for the trans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation. PMID:10559262

  3. Analysis of DNA sequences which regulate the transcription of herpes simplex virus immediate early gene 3: DNA sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide.

    PubMed Central

    Bzik, D J; Preston, C M

    1986-01-01

    The far upstream region of herpes simplex virus (HSV) immediate early (IE) gene 3 has previously been shown to increase gene expression in an enhancer-like manner, and to contain sequences which respond to stimulation of transcription by a virion polypeptide, Vmw65. To analyse the specific DNA sequences which mediate these functions, sequential deletions from each end of the far upstream region were made. The effects of the deletions on transcription in the absence or presence of the Vmw65 were measured by use of a transient expression assay. The enhancer-like activity was due to three separable elements, whereas two additional DNA regions were involved in the response to Vmw65. One of the responding elements corresponded to an AT-rich consensus (TAATGARATTC, where R = purine) present in all IE gene far upstream regions, and the other was a GA-rich sequence also present in IE genes 2 and 4/5. The TAATGARATTC element could mediate responsiveness to Vmw65 but it was fully active only in the presence of the GA-rich element. The GA-rich element was unable to confer a strong response alone but could activate an otherwise nonfunctional homologue of TAATGARATTC. PMID:3003700

  4. Analysis of DNA sequences which regulate the transcription of herpes simplex virus immediate early gene 3: DNA sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide.

    PubMed

    Bzik, D J; Preston, C M

    1986-01-24

    The far upstream region of herpes simplex virus (HSV) immediate early (IE) gene 3 has previously been shown to increase gene expression in an enhancer-like manner, and to contain sequences which respond to stimulation of transcription by a virion polypeptide, Vmw65. To analyse the specific DNA sequences which mediate these functions, sequential deletions from each end of the far upstream region were made. The effects of the deletions on transcription in the absence or presence of the Vmw65 were measured by use of a transient expression assay. The enhancer-like activity was due to three separable elements, whereas two additional DNA regions were involved in the response to Vmw65. One of the responding elements corresponded to an AT-rich consensus (TAATGARATTC, where R = purine) present in all IE gene far upstream regions, and the other was a GA-rich sequence also present in IE genes 2 and 4/5. The TAATGARATTC element could mediate responsiveness to Vmw65 but it was fully active only in the presence of the GA-rich element. The GA-rich element was unable to confer a strong response alone but could activate an otherwise nonfunctional homologue of TAATGARATTC. PMID:3003700

  5. A versatile cis-blocking and trans-activation strategy for ribozyme characterization

    PubMed Central

    Kennedy, Andrew B.; Liang, Joe C.; Smolke, Christina D.

    2013-01-01

    Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices. PMID:23155065

  6. Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain.

    PubMed Central

    Garrett, E D; He, F; Bogerd, H P; Cullen, B R

    1993-01-01

    The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1. Images PMID:8411385

  7. Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

    PubMed Central

    Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

    1990-01-01

    Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

  8. FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions.

    PubMed Central

    Nilsson, L; Verbeek, H; Vijgenboom, E; van Drunen, C; Vanet, A; Bosch, L

    1992-01-01

    In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is

  9. FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions.

    PubMed

    Nilsson, L; Verbeek, H; Vijgenboom, E; van Drunen, C; Vanet, A; Bosch, L

    1992-02-01

    In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is

  10. Member of the CREB/ATF protein family, but not CREB alpha plays an active role in BLV tax trans activation in vivo.

    PubMed Central

    Kiss-Toth, E; Paca-uccaralertkun, S; Unk, I; Boros, I

    1993-01-01

    The trans activator protein of Bovine Leukaemia Virus (tax) increases the rate of transcription from the virus promoter through 21 bp sequences located in three tandem copies in the virus LTR. Based on data obtained by three different experimental approaches we concluded that the central CRE-like motif found in each of the BLV 21 bp repeats plays an important and indispensable role in tax mediated trans activation. These include (i) in vivo analysis of the function of mutant 21 bp sequences in transient transfection, (ii) gel mobility shift assay to show that CREB binds to BLV 21 bp repeats in vitro and (iii) the demonstration that the production of antisense CREB mRNA inhibits tax trans activation. Further studies with different deletion mutant CREB proteins suggest that although CREB alpha can interact with factors involved in BLV trans activation, it does not promote transcription initiation; consequently some other member/s of the CREB/ATF family must be involved. Images PMID:8396235

  11. Subnuclear localization of the trans-activating protein of human T-cell leukemia virus type I

    SciTech Connect

    Slamon, D.J.; Keith, D.E.; Golde, D.W. ); Boyle, W.J. ); Press, M.F. ); Souza, L.M. )

    1988-03-01

    Human T-cell leukemia virus type I is associated with human lymphoid malignancies. The p40{sup xI} protein encoded by the x gene of this virus is believed to play some role in virally mediated transformation. This gene is known to encode a transcriptional trans activator which previous studies have shown to be a nuclear protein. Further characterization of the intracellular kinetics of this protein showed that it migrated into the nucleus very soon after synthesis. Within the nucleus, p40{sup xI} was distributed almost equally between the nucleoplasm and the nuclear matrix. Given the proposed role of the nuclear matrix in RNA transcription, the association of p40{sup xI} with the matrix places it in an appropriate cellular compartment to exercise an effect on transcription.

  12. Subnuclear localization of the trans-activating protein of human T-cell leukemia virus type I.

    PubMed Central

    Slamon, D J; Boyle, W J; Keith, D E; Press, M F; Golde, D W; Souza, L M

    1988-01-01

    Human T-cell leukemia virus type I is associated with human lymphoid malignancies. The p40xI protein encoded by the x gene of this virus is believed to play some role in virally mediated transformation. This gene is known to encode a transcriptional trans activator which previous studies have shown to be a nuclear protein. Further characterization of the intracellular kinetics of this protein showed that it migrated into the nucleus very soon after synthesis. Within the nucleus, p40xI was distributed almost equally between the nucleoplasm and the nuclear matrix. Given the proposed role of the nuclear matrix in RNA transcription, the association of p40xI with the matrix places it in an appropriate cellular compartment to exercise an effect on transcription. Images PMID:2828664

  13. trans Activation of the simian virus 40 enhancer.

    PubMed Central

    Robbins, P D; Rio, D C; Botchan, M R

    1986-01-01

    We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth. Images PMID:3023880

  14. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

    PubMed

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-07-01

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

  15. Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant

    PubMed Central

    Arazi, Tzahi

    2014-01-01

    DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato. PMID:24376253

  16. A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator

    SciTech Connect

    Jakobovits, A.; Smith, D.H.; Jakobovits, E.B.; Capon, D.J.

    1988-06-01

    An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. The authors' previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using in an vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

  17. Effect of p40tax trans-activator of human T cell lymphotropic virus type I on expression of autoantigens.

    PubMed

    Banki, K; Ablonczy, E; Nakamura, M; Perl, A

    1994-03-01

    The possibility of a retroviral etiology has long been raised in a number of autoimmune disorders. More recently, Sjögren's syndrome and rheumatoid arthritis were noted in transgenic mice carrying the tax gene of human T cell leukemia virus type I (HTLV-I). To evaluate the involvement of HTLV-I Tax in autoimmunity, its effect on expression of autoantigens was investigated. A metallothionein promoter-driven p40tax expression plasmid, pMAXRHneo-1, was stably transfected into Molt4 and Jurkat cells and the p40tax protein was induced with CdCl2. trans-Activation or trans-repression of autoantigens by HTLV-I Tax was studied by Western blot analysis utilizing autoantigen-specific murine monoclonal and rabbit polyvalent antibodies as well as sera from 161 autoimmune patients. Induction of p40tax of HTLV-I had no significant effect on levels of expression of common autoantigens U1 snRNP, Sm, Ro, La, HSP-70, topoisomerase I/Scl70, PCNA, and HRES-1. Expression of two potentially novel autoantigens, 44 and 46 kDa, was induced by p40tax as detected by sera of progressive systemic sclerosis patients, BAK and VAR. By contrast, expression of 24- and 34-kDa proteins was suppressed in response to induction of p40tax as detected by sera of systemic lupus erythematosus patients PUS and HOR. Because none of these patients were infected by HTLV-I, a protein functionally similar to p40tax may be involved in eliciting autoantigen expression and a subsequent autoantibody response in a minority of patients with PSS and SLE. Sera of autoimmune patients may also be utilized to detect novel proteins trans-activated or trans-repressed by p40tax of HTLV-I. PMID:8018391

  18. Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages.

    PubMed Central

    Murphy, K M; Sweet, M J; Ross, I L; Hume, D A

    1993-01-01

    The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells

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

    PubMed

    Engebrecht, J; Silverman, M

    1984-07-01

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

  20. 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.

  1. Endosomolytic activity of cationic liposomes enhances the delivery of human immunodeficiency virus-1 trans-activator protein (TAT) to mammalian cells.

    PubMed

    Huang, L; Farhood, H; Serbina, N; Teepe, A G; Barsoum, J

    1995-12-26

    We have explored the use of cationic liposomes to deliver the human immunodeficiency virus-1 trans-activator protein tat using a reporter gene expression assay. The human epidermoid carcinoma cell A431 stably transfected with a reporter gene under the control of human immunodeficiency virus-1 promoter was used as a target cell. Phosphatidylcholine-containing cationic liposomes had no detectable tat delivery activity. In contrast, delivery of tat was enhanced by up to 150-fold using cationic liposomes enriched with dioleoyl phosphatidylethanolamine (DOPE), a lipid which readily transforms a bilayer into a nonbilayer structure. Enhanced delivery of tat by DOPE-containing liposomes was most likely the result of the endosomolytic activity of the liposome. This phospholipid-rich formulation showed no toxicity at concentrations sufficient for maximal delivery of tat. A variety of cationic liposome formulations which contain DOPE were tested successfully for tat delivery. PMID:8554596

  2. APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

    PubMed

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-07-29

    Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

  3. APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

    PubMed Central

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-01-01

    Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

  4. Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons.

    PubMed

    Lee, Dong Woo; Cho, Pyung Sun; Lee, Han Kyu; Lee, Sang Hoon; Jung, Sung Jun; Oh, Seog Bae

    2015-10-01

    TRPV1, a ligand-gated ion channel expressed in nociceptive sensory neurons is modulated by a variety of intracellular signaling pathways. Dopamine is a neurotransmitter that plays important roles in motor control, cognition, and pain modulation in the CNS, and acts via a variety of dopamine receptors (D1R-D5R), a class of GPCRs. Although nociceptive sensory neurons express D1-like receptors, very little is known about the effect of dopamine on TRPV1 in the peripheral nervous system. Therefore, in this study, we examined the effects of D1R activation on TRPV1 in mouse DRG neurons using Ca(2+) imaging and immunohistochemical analysis. The D1R agonist SKF-38393 induced reproducible Ca(2+) responses via Ca(2+) influx through TRPV1 rather than Ca(2+) mobilization from intracellular Ca(2+) stores. Immunohistochemical analysis revealed co-expression of D1R and TRPV1 in mouse DRG neurons. The PLC-specific inhibitor blocked the SKF-38393-induced Ca(2+) response, whereas the PKC, DAG lipase, AC, and PKA inhibitors had no effect on the SKF-38393-induced Ca(2+) response. Taken together, our results suggest that the SKF-38393-induced Ca(2+) response results from the direct activation of TRPV1 by a PLC/DAG-mediated membrane-delimited pathway. These results provide evidence that the trans-activation of TRPV1 following D1R activation may contribute to the modulation of pain signaling in nociceptive sensory neurons. PMID:26319554

  5. Brain-Targeted Delivery of Trans-Activating Transcriptor-Conjugated Magnetic PLGA/Lipid Nanoparticles

    PubMed Central

    Zhang, Yifang; Sun, Tingting; Zhang, Fang; Wu, Jian; Fu, Yanyan; Du, Yang; Zhang, Lei; Sun, Ying; Liu, YongHai; Ma, Kai; Liu, Hongzhi; Song, Yuanjian

    2014-01-01

    Magnetic poly (D,L-lactide-co-glycolide) (PLGA)/lipid nanoparticles (MPLs) were fabricated from PLGA, L-α-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino (polyethylene glycol) (DSPE-PEG-NH2), and magnetic nanoparticles (NPs), and then conjugated to trans-activating transcriptor (TAT) peptide. The TAT-MPLs were designed to target the brain by magnetic guidance and TAT conjugation. The drugs hesperidin (HES), naringin (NAR), and glutathione (GSH) were encapsulated in MPLs with drug loading capacity (>10%) and drug encapsulation efficiency (>90%). The therapeutic efficacy of the drug-loaded TAT-MPLs in bEnd.3 cells was compared with that of drug-loaded MPLs. The cells accumulated higher levels of TAT-MPLs than MPLs. In addition, the accumulation of QD-loaded fluorescein isothiocyanate (FITC)-labeled TAT-MPLs in bEnd.3 cells was dose and time dependent. Our results show that TAT-conjugated MPLs may function as an effective drug delivery system that crosses the blood brain barrier to the brain. PMID:25187980

  6. The role of FIS in trans activation of stable RNA operons of E. coli.

    PubMed

    Nilsson, L; Vanet, A; Vijgenboom, E; Bosch, L

    1990-03-01

    The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium. PMID:1690124

  7. trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1.

    PubMed Central

    Casaz, P; Sundseth, R; Hansen, U

    1991-01-01

    Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein. Images PMID:1658359

  8. Trans-activity of plasma membrane-associated ganglioside sialyltransferase in mammalian cells.

    PubMed

    Vilcaes, Aldo A; Demichelis, Vanina Torres; Daniotti, Jose L

    2011-09-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

  9. Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells*

    PubMed Central

    Vilcaes, Aldo A.; Demichelis, Vanina Torres; Daniotti, Jose L.

    2011-01-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

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

    SciTech Connect

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

    2007-03-01

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

  11. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

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

  12. 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.

  13. 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

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

    SciTech Connect

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

    2006-05-28

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

  15. 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.

  16. Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

    PubMed Central

    Turner, John J.; Arzumanov, Andrey A.; Gait, Michael J.

    2005-01-01

    Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R9F2 was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R9 conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R9F2 and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide. PMID:15640444

  17. Gene analogue finder: a GRID solution for finding functionally analogous gene products

    PubMed Central

    Tulipano, Angelica; Donvito, Giacinto; Licciulli, Flavio; Maggi, Giorgio; Gisel, Andreas

    2007-01-01

    Background To date more than 2,1 million gene products from more than 100000 different species have been described specifying their function, the processes they are involved in and their cellular localization using a very well defined and structured vocabulary, the gene ontology (GO). Such vast, well defined knowledge opens the possibility of compare gene products at the level of functionality, finding gene products which have a similar function or are involved in similar biological processes without relying on the conventional sequence similarity approach. Comparisons within such a large space of knowledge are highly data and computing intensive. For this reason this project was based upon the use of the computational GRID, a technology offering large computing and storage resources. Results We have developed a tool, GENe AnaloGue FINdEr (ENGINE) that parallelizes the search process and distributes the calculation and data over the computational GRID, splitting the process into many sub-processes and joining the calculation and the data on the same machine and therefore completing the whole search in about 3 days instead of occupying one single machine for more than 5 CPU years. The results of the functional comparison contain potential functional analogues for more than 79000 gene products from the most important species. 46% of the analyzed gene products are well enough described for such an analysis to individuate functional analogues, such as well-known members of the same gene family, or gene products with similar functions which would never have been associated by standard methods. Conclusion ENGINE has produced a list of potential functionally analogous relations between gene products within and between species using, in place of the sequence, the gene description of the GO, thus demonstrating the potential of the GO. However, the current limiting factor is the quality of the associations of many gene products from non-model organisms that often have

  18. 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

  19. Efficient translation of distal cistrons of a polycistronic mRNA of a plant pararetrovirus requires a compatible interaction between the mRNA 3' end and the proteinaceous trans-activator.

    PubMed

    Edskes, H K; Kiernan, J M; Shepherd, R J

    1996-10-15

    Caulimoviruses, a type of plant pararetrovirus, employ a highly unusual mechanism to express the multiple cistrons of their pregenomic RNA. It involves translation of a polycistronic mRNA utilizing cis-acting viral RNA sequences and a transacting virus-encoded protein (P6). In addition to its role in polycistronic translation, the translational trans-activator protein P6 also activates its own expression from a monocistronic subgenomic RNA. Using Nicotiana Edwardsonii cell suspension protoplasts, we analyzed the ability of P6 proteins from three different caulimoviruses to activate viral RNA-based reporter constructs. Cis-acting elements present in figwort mosaic caulimovirus (FMV) are functional not only in the presence of the cognate P6 activator protein, but also in the presence of the heterologous activators from cauliflower mosaic caulimovirus (CaMV) and peanut chlorotic streak caulimovirus (PCISV). However, when 3' cis-acting elements essential for efficient polycistronic expression of FMV are replaced by their counterparts from PCISV, reporter gene expression is only observed in the presence of PCISV P6. Derepression of monocistronic reporter constructs tailed with FMV or CaMV 3' proximal sequences is less efficient in the presence of PCISV P6 than with either FMV or CaMV P6, but more efficient when the constructs contain a cognate PCISV 3' cis-element. Efficient expression of polycistronic and monocistronic caulimovirus mRNAs in plant cells thus requires compatible interactions between P6, a translational trans-activator, and its cognate cis-element at the 3' end of the mRNA. PMID:8874519

  20. Natural Product Biosynthetic Gene Diversity in Geographically Distinct Soil Microbiomes

    PubMed Central

    Reddy, Boojala Vijay B.; Kallifidas, Dimitris; Kim, Jeffrey H.; Charlop-Powers, Zachary; Feng, Zhiyang

    2012-01-01

    The number of bacterial species estimated to exist on Earth has increased dramatically in recent years. This newly recognized species diversity has raised the possibility that bacterial natural product biosynthetic diversity has also been significantly underestimated by previous culture-based studies. Here, we compare 454-pyrosequenced nonribosomal peptide adenylation domain, type I polyketide ketosynthase domain, and type II polyketide ketosynthase alpha gene fragments amplified from cosmid libraries constructed using DNA isolated from three different arid soils. While 16S rRNA gene sequence analysis indicates these cloned metagenomes contain DNA from similar distributions of major bacterial phyla, we found that they contain almost completely distinct collections of secondary metabolite biosynthetic gene sequences. When grouped at 85% identity, only 1.5% of the adenylation domain, 1.2% of the ketosynthase, and 9.3% of the ketosynthase alpha sequence clusters contained sequences from all three metagenomes. Although there is unlikely to be a simple correlation between biosynthetic gene sequence diversity and the diversity of metabolites encoded by the gene clusters in which these genes reside, our analysis further suggests that sequences in one soil metagenome are so distantly related to sequences in another metagenome that they are, in many cases, likely to arise from functionally distinct gene clusters. The marked differences observed among collections of biosynthetic genes found in even ecologically similar environments suggest that prokaryotic natural product biosynthesis diversity is, like bacterial species diversity, potentially much larger than appreciated from culture-based studies. PMID:22427492

  1. Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes.

    PubMed Central

    Bartlett, D H; Matsumura, P

    1984-01-01

    Region III flagellar genes in Escherichia coli are involved with the assembly and rotation of the flagella, as well as taxis. We subcloned the flaB operon from a lambda fla transducing phage onto plasmid pMK2004. Two additional genes were found at the flaB locus, and we subdivided the flaB gene into flaB1, flaBII, and flaBIII. The cheY suppressor mutations which have previously been mapped to flaB were further localized to flaB11 (Parkinson et al., J. Bacteriol. 155:265-274, 1983). Until now, gene product identification has not been possible for these genes because of their low levels of gene expression. Overexpression of the flagellar genes was accomplished by placing the flaB operon under the control of the lacUV5 or tac promoters. Plasmid-encoded proteins were examined in a minicell expression system. By correlating various deletions and insertions in the flaB operon with the ability to complement specific flagellar mutants and code for polypeptides, we made the following gene product assignments: flaB 1, 60 kilodaltons; flaB 11, 38 kilodaltons; flaB111, 28 kilodaltons; flaC, 56 kilodaltons; fla0, 16 kilodaltons; and flaE, 54 kilodaltons. Images PMID:6094477

  2. Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.

    PubMed Central

    Bergqvist, A; Nilsson, M; Bondeson, K; Magnusson, G

    1990-01-01

    A putative zinc finger in polyomavirus large T-antigen was investigated. We were unable to demonstrate unequivocally a requirement for zinc in specific DNA-binding using the chelating agent 1, 10-phenanthroline. An involvement of the putative zinc finger in specific DNA-binding was nevertheless suggested by the properties of a mutant protein with a cys----ser replacement in the finger motif. Probably as a result of the defective DNA-binding, the mutant protein had lost its activity in initiation of viral DNA-replication and in negative regulation of viral early transcription. However, the trans-activation of the viral late promoter was normal. The analysis also revealed a previously unrecognized activity of large T-antigen. The mutant protein trans-activated the viral early promoter. In the wild-type protein this activity is probably concealed by the separate, negative regulatory function. Images PMID:2160069

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

    SciTech Connect

    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.

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

    Giri, C C; Vijaya Laxmi, G

    2000-12-01

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

  6. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene.

    PubMed Central

    Totten, P A; Lara, J C; Lory, S

    1990-01-01

    The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism. Images FIG. 1 FIG. 2 PMID:2152909

  7. Preclinical development strategies for novel gene therapeutic products.

    PubMed

    Pilaro, A M; Serabian, M A

    1999-01-01

    With over 220 investigational new drug applications currently active, gene therapy represents one of the fastest growing areas in biotherapeutic research. Initially conceived for replacing defective genes in diseases such as cystic fibrosis or inborn errors of metabolism with genes encoding the normal, or wild-type, gene product, gene therapy has expanded into other novel applications such as treatment of cancer or cardiovascular disease, where the risk:benefit profiles may be more acceptable in relation to the severity of the disease. Different types of vectors, including modified retroviruses, adenoviruses, adenovirus-associated viruses, and herpesviruses and plasmid DNA, are used to transfer foreign genetic material into patients' cells or tissues. Developing a toxicology program to determine the safety of these agents, therefore, requires a modified approach that encompasses the pharmacology and toxicity of both the gene product itself and the vector system used for delivery in the context of the application for the clinical trial. In general, the issues involved in designing and developing appropriate preclinical testing to determine the safety of these products are similar to those encountered for other recombinant molecules, including protein biotherapeutics. Limitations to some of the typical toxicology studies conducted for a traditional drug development program may exist for these agents, and nontraditional approaches may be required to demonstrate their safety. Many factors may affect the safety and clinical activity of these agents, including the route, frequency, and duration of exposure and the type of vector employed. Other safety considerations include quantitation of the duration and degree of expression of the vector in target and other tissues, the effects of gene expression on organ pathology and/or histology, evaluation of trafficking of gene-transduced cells or vector after injection, and interactions of the host immune system with the

  8. Use of Galerina marginata genes and proteins for peptide production

    DOEpatents

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

    2016-03-01

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  9. 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

  10. 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

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

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

  13. Technical development for production of gene-modified laboratory rats.

    PubMed

    Hirabayashi, Masumi

    2008-04-01

    Transgenic rats have been used as model animals for human diseases and organ transplantation and as animal bioreactors for protein production. In general, transgenic rats are produced by pronuclear microinjection of exogenous DNA. Improvement of post-injection survival has been achieved by micro-vibration of the injection pipette. The promoter region, structural gene, chain length and strand ends of the exogenous DNA are not involved in the production efficiency of transgenic rats. Exogenous DNA prepared at 5 microg/ml seemed to be better integrated than lower and higher concentrations. Intracytoplasmic sperm injection (ICSI) has been successfully achieved in rats using a piezo-driven injection pipette. The ICSI technique has not only been applied to rescue infertile male strains but also to produce transgenic rats. The optimal DNA concentration for the ICSI-tg method (0.1 to 0.5 microg/ml) is lower than that for the conventional pronuclear microinjection. Production efficiency was improved when the membrane structure of the sperm head was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. For successful production of transgenic rats with a modified endogenous gene, establishment of embryonic stem cell lines or alternatively male germline stem cell lines and technical development of somatic cell nuclear transfer are still necessary for this species. PMID:18446007

  14. 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 (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate. PMID:26241189

  15. dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

    PubMed Central

    Henrich, B; Becker, S; Schroeder, U; Plapp, R

    1993-01-01

    Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties. Images PMID:8226676

  16. GOChase-II: correcting semantic inconsistencies from Gene Ontology-based annotations for gene products

    PubMed Central

    2011-01-01

    Background The Gene Ontology (GO) provides a controlled vocabulary for describing genes and gene products. In spite of the undoubted importance of GO, several drawbacks associated with GO and GO-based annotations have been introduced. We identified three types of semantic inconsistencies in GO-based annotations; semantically redundant, biological-domain inconsistent and taxonomy inconsistent annotations. Methods To determine the semantic inconsistencies in GO annotation, we used the hierarchical structure of GO graph and tree structure of NCBI taxonomy. Twenty seven biological databases were collected for finding semantic inconsistent annotation. Results The distributions and possible causes of the semantic inconsistencies were investigated using twenty seven biological databases with GO-based annotations. We found that some evidence codes of annotation were associated with the inconsistencies. The numbers of gene products and species in a database that are related to the complexity of database management are also in correlation with the inconsistencies. Consequently, numerous annotation errors arise and are propagated throughout biological databases and GO-based high-level analyses. GOChase-II is developed to detect and correct both syntactic and semantic errors in GO-based annotations. Conclusions We identified some inconsistencies in GO-based annotation and provided software, GOChase-II, for correcting these semantic inconsistencies in addition to the previous corrections for the syntactic errors by GOChase-I. PMID:21342572

  17. 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.

  18. 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

  19. 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.

  20. Gas-inducible product gene expression in bioreactors.

    PubMed

    Weber, Wilfried; Rimann, Markus; de Glutz, François-Nicolas; Weber, Eric; Memmert, Klaus; Fussenegger, Martin

    2005-05-01

    Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals. PMID:15885616

  1. 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

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

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

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

  3. 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.

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

    DOEpatents

    Wohlbach, Dana J.; Gasch, Audrey P.

    2014-08-05

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

  5. Control of adenovirus early gene expression: Posttranscriptional control mediated by both viral and cellular gene products

    SciTech Connect

    Katze, M.G.; Persson, H.; Philipson, L.

    1981-09-01

    An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

  6. Production and clinical development of nanoparticles for gene delivery

    PubMed Central

    Chen, Jie; Guo, Zhaopei; Tian, Huayu; Chen, Xuesi

    2016-01-01

    Gene therapy is a promising strategy for specific treatment of numerous gene-associated human diseases by intentionally altering the gene expression in pathological cells. A successful clinical application of gene-based therapy depends on an efficient gene delivery system. Many efforts have been attempted to improve the safety and efficiency of gene-based therapies. Nanoparticles have been proved to be the most promising vehicles for clinical gene therapy due to their tunable size, shape, surface, and biological behaviors. In this review, the clinical development of nanoparticles for gene delivery will be particularly highlighted. Several promising candidates, which are closest to clinical applications, will be briefly reviewed. Then, the recent developments of nanoparticles for clinical gene therapy will be identified and summarized. Finally, the development of nanoparticles for clinical gene delivery in future will be prospected. PMID:27088105

  7. 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

  8. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    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

  9. Sterol-dependent nuclear import of ORP1S promotes LXR regulated trans-activation of apoE

    SciTech Connect

    Lee, Sungsoo; Wang, Ping-Yuan; Jeong, Yangsik; Mangelsdorf, David J.; Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041 ; Anderson, Richard G.W.; Michaely, Peter

    2012-10-01

    Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to the nucleus and promotes LXR-dependent gene transcription through select enhancer elements. -- Highlights: Black-Right-Pointing-Pointer ORP1S translocates to the nucleus in response to sterol binding. Black-Right-Pointing-Pointer The sterols that best promote nuclear import of ORP1S are LXR agonists. Black-Right-Pointing-Pointer ORP1S binds to LXRs, enhances binding of LXRs to LXREs and promotes LXR-dependent transcription of apoE.

  10. Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.

    PubMed

    Qin, Li-Na; Cai, Fu-Rong; Dong, Xin-Rui; Huang, Zhen-Bang; Tao, Yong; Huang, Jian-Zhong; Dong, Zhi-Yang

    2012-04-01

    A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference. PMID:22305540

  11. Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2′-O-methyl G-clamp ribonucleoside analogues

    PubMed Central

    Holmes, Stephen C.; Arzumanov, Andrey A.; Gait, Michael J.

    2003-01-01

    We report the synthesis of a novel 2′-O-methyl (OMe) riboside phosphoramidite derivative of the G-clamp tricyclic base and incorporation into a series of small steric blocking OMe oligonucleotides targeting the apical stem–loop region of human immunodeficiency virus type 1 (HIV-1) trans- activation-responsive (TAR) RNA. Binding to TAR RNA is substantially enhanced for certain single site substitutions in the centre of the oligonucleotide, and doubly substituted anti-TAR OMe 9mers or 12mers exhibit remarkably low binding constants of <0.1 nM. G-clamp-containing oligomers achieved 50% inhibition of Tat-dependent in vitro transcription at ∼25 nM, 4-fold lower than for a TAR 12mer OMe oligonucleotide and better than found for any other oligonucleotide tested to date. Addition of one or two OMe G-clamps did not impart cellular trans-activation inhibition activity to cellularly inactive OMe oligonucleotides. Addition of an OMe G-clamp to a 12mer OMe–locked nucleic acid chimera maintained, but did not enhance, inhibition of Tat-dependent in vitro transcription and cellular trans-activation in HeLa cells. The results demonstrate clearly that an OMe G-clamp has remarkable RNA-binding enhancement ability, but that oligonucleotide effectiveness in steric block inhibition of Tat-dependent trans-activation both in vitro and in cells is governed by factors more complex than RNA-binding strength alone. PMID:12771202

  12. trans activation of the tumor necrosis factor alpha promoter by the human T-cell leukemia virus type I Tax1 protein.

    PubMed Central

    Albrecht, H; Shakhov, A N; Jongeneel, C V

    1992-01-01

    In a cotransfection assay, the human T-cell leukemia virus type I Tax1 gene product specifically activated transcription from the mouse tumor necrosis factor alpha promoter. The activation patterns of 5' deletion mutants, artificial enhancer constructs, and point mutations in the promoter indicate that the major Tax1-responsive element is a site at position -655 which binds the NF-kappa B/rel and NF-GMa transcription factors. Images PMID:1527856

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

    PubMed

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

    2015-01-01

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

  14. 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

  15. 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

  16. Characterizing Milk Production Related Genes in Holstein Using RNA-seq.

    PubMed

    Seo, Minseok; Lee, Hyun-Jeong; Kim, Kwondo; Caetano-Anolles, Kelsey; Jeong, Jin Young; Park, Sungkwon; Oh, Young Kyun; Cho, Seoae; Kim, Heebal

    2016-03-01

    Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, only a few studies have attempted to characterize milk-synthesizing genes using RNA-seq data. RNA-seq data was collected from 21 Holstein samples, along with group information about milk production ability; milk yield; and protein, fat, and solid contents. Meta-analysis was employed in order to generally characterize genes related to milk production. In addition, we attempted to investigate the relationship between milk related traits, parity, and lactation period. We observed that milk fat is highly correlated with lactation period; this result indicates that this effect should be considered in the model in order to accurately detect milk production related genes. By employing our developed model, 271 genes were significantly (false discovery rate [FDR] adjusted p-value<0.1) detected as milk production related differentially expressed genes. Of these genes, five (albumin, nitric oxide synthase 3, RNA-binding region (RNP1, RRM) containing 3, secreted and transmembrane 1, and serine palmitoyltransferase, small subunit B) were technically validated using quantitative real-time polymerase chain reaction (qRT-PCR) in order to check the accuracy of RNA-seq analysis. Finally, 83 gene ontology biological processes including several blood vessel and mammary gland development related terms, were significantly detected using DAVID gene-set enrichment analysis. From these results, we observed that detected milk production related genes are highly enriched in the circulation system process and mammary gland related biological functions. In addition, we observed that detected genes including caveolin 1, mammary serum amyloid A3.2, lingual antimicrobial peptide, cathelicidin 4 (CATHL4), cathelicidin 6 (CATHL6) have been reported in other species as milk production related gene. For this reason, we concluded that our detected 271 genes would be strong candidates for

  17. Characterizing Milk Production Related Genes in Holstein Using RNA-seq

    PubMed Central

    Seo, Minseok; Lee, Hyun-Jeong; Kim, Kwondo; Caetano-Anolles, Kelsey; Jeong, Jin Young; Park, Sungkwon; Oh, Young Kyun; Cho, Seoae; Kim, Heebal

    2016-01-01

    Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, only a few studies have attempted to characterize milk-synthesizing genes using RNA-seq data. RNA-seq data was collected from 21 Holstein samples, along with group information about milk production ability; milk yield; and protein, fat, and solid contents. Meta-analysis was employed in order to generally characterize genes related to milk production. In addition, we attempted to investigate the relationship between milk related traits, parity, and lactation period. We observed that milk fat is highly correlated with lactation period; this result indicates that this effect should be considered in the model in order to accurately detect milk production related genes. By employing our developed model, 271 genes were significantly (false discovery rate [FDR] adjusted p-value<0.1) detected as milk production related differentially expressed genes. Of these genes, five (albumin, nitric oxide synthase 3, RNA-binding region (RNP1, RRM) containing 3, secreted and transmembrane 1, and serine palmitoyltransferase, small subunit B) were technically validated using quantitative real-time polymerase chain reaction (qRT-PCR) in order to check the accuracy of RNA-seq analysis. Finally, 83 gene ontology biological processes including several blood vessel and mammary gland development related terms, were significantly detected using DAVID gene-set enrichment analysis. From these results, we observed that detected milk production related genes are highly enriched in the circulation system process and mammary gland related biological functions. In addition, we observed that detected genes including caveolin 1, mammary serum amyloid A3.2, lingual antimicrobial peptide, cathelicidin 4 (CATHL4), cathelicidin 6 (CATHL6) have been reported in other species as milk production related gene. For this reason, we concluded that our detected 271 genes would be strong candidates for

  18. 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

  19. Comparison of Bacillus monooxygenase genes for unique fatty acid production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reviews Bacillus genes encoding monooxygenase enzymes producing unique fatty acid metabolites. Specifically, it examines standard monooxygenase electron transfer schemes and related domain structures of these fused domain enzymes on route to understanding the observed oxygenase activiti...

  20. C-terminal trans-activation sub-region of VP16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells.

    PubMed

    Danaher, Robert J; Cook, Ross K; Wang, Chunmei; Triezenberg, Steven J; Jacob, Robert J; Miller, Craig S

    2013-02-01

    The HSV-1 tegument protein VP16 contains a trans-activation domain (TAD) that is required for induction of immediate early (IE) genes during lytic infection and induced reactivation from latency. Here we report the differential contributions of the two sub-regions of the TAD in neuronal and non-neuronal cells during activation of IE gene expression, virus replication, and reactivation from quiescently infected (QIF)-PC12 cells. Our studies show that VP16- and chemical (hexamethylenebisacetamide)-induced IE gene activation is attenuated in neuronal cells. Irrespective of neuronal or non-neuronal cell backgrounds, IE gene activation demonstrated a greater requirement for the N-terminal sub-region of VP16 TAD (VP16N) than the C-terminal sub-region (VP16C). In surprising contrast to these findings, a recombinant virus (RP4) containing the VP16N deletion was capable of modest forskolin-induced reactivation whereas a recombinant (RP3) containing a deletion of VP16C was incapable of stress-induced reactivation from QIF-PC12 cells. These unique process-dependent functions of the VP16 TAD sub-regions may be important during particular stages of the virus life cycle (lytic, entrance, and maintenance of a quiescent state and reactivation) when viral DNA would be expected to be differentially modified. PMID:23192733

  1. 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.

  2. Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes.

    PubMed Central

    Jørgensen, S; Skov, K W; Diderichsen, B

    1991-01-01

    The lipA gene encoding an extracellular lipase from Pseudomonas cepacia was cloned and sequenced. Downstream from the lipase gene an open reading frame was identified, and the corresponding gene was named limA. lipA was well expressed only in the presence of limA. limA exerts its effect both in cis and in trans and therefore produces a diffusible gene product, presumably a protein of 344 amino acids. Replacement of the lipA expression signals (promoter, ribosome-binding site, and signal peptide-coding sequences) by heterologous signals from gram-positive bacteria still resulted in limA-dependent lipA expression in Escherichia coli, Bacillus subtilis, and Streptomyces lividans. Images PMID:1987151

  3. Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis.

    PubMed

    Leong, Wan Yee; Guo, Hong; Ma, Ou; Huang, Hui; Cantor, Alan B; Friedman, Alan D

    2016-01-01

    Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow. PMID:26598521

  4. Escherichia coli genes whose products are involved in selenium metabolism

    SciTech Connect

    Leinfelder, W.; Forchhammer, K.; Zinoni, F.; Sawers, G.; Mandrand-Berthelot, M.A.; Boeck, A.

    1988-02-01

    Mutants of Escherichia coli were isolated which were affected in the formation of both formate dehydrogenase N (phenazine methosulfate reducing) (FDN/sub N/) and formate dehydrogenase H (benzylviologen reducing) (FDH/sub H/). They were analyzed, together with previously characterized pleiotropic fdh mutants (fdhA, fdhB, and fdhC), for their ability to incorporate selenium into the selenopolypeptide subunits of FDH/sub N/ and FDH/sub H/. Results of this study support the notion that the pleiotropic fdh mutants analyzed possess a lesion in the gene(s) encoding the biosynthesis or the incorporation of selenocysteine. The gene complementing the defect in one of the isolated mutants was cloned from a cosmid library. Subclones were tested for complementation of other pleiotropic fdh mutants. The results revealed that the mutations in the eight isolates fell into two complementation groups, one of them containing the fdhA mutation. fdhB, fdhC, and two of the new fdh isolates do not belong to these complementation groups. A new nomenclature (sel) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at the fdhA locus), selC (previously fdhC), and selD (previously fdhB).

  5. 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

    ..., 2008 (73 FR 59635), FDA announced the availability of the draft guidance of the same title. FDA... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Potency Tests for Cellular and Gene... Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance...

  6. Mobile antibiotic resistance - the spread of genes determining the resistance of bacteria through food products.

    PubMed

    Godziszewska, Jolanta; Guzek, Dominika; Głąbski, Krzysztof; Wierzbicka, Agnieszka

    2016-01-01

    In recent years, more and more antibiotics have become ineffective in the treatment of bacterial nfections. The acquisition of antibiotic resistance by bacteria is associated with circulation of genes in the environment. Determinants of antibiotic resistance may be transferred to pathogenic bacteria. It has been shown that conjugation is one of the key mechanisms responsible for spread of antibiotic resistance genes, which is highly efficient and allows the barrier to restrictions and modifications to be avoided. Some conjugative modules enable the transfer of plasmids even between phylogenetically distant bacterial species. Many scientific reports indicate that food is one of the main reservoirs of these genes. Antibiotic resistance genes have been identified in meat products, milk, fruits and vegetables. The reason for such a wide spread of antibiotic resistance genes is the overuse of antibiotics by breeders of plants and animals, as well as by horizontal gene transfer. It was shown, that resistance determinants located on mobile genetic elements, which are isolated from food products, can easily be transferred to another niche. The antibiotic resistance genes have been in the environment for 30 000 years. Their removal from food products is not possible, but the risks associated with the emergence of multiresistant pathogenic strains are very large. The only option is to control the emergence, selection and spread of these genes. Therefore measures are sought to prevent horizontal transfer of genes. Promising concepts involve the combination of developmental biology, evolution and ecology in the fight against the spread of antibiotic resistance. PMID:27383577

  7. 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

  8. Sequence-specific and general transcriptional activation by the bovine papillomavirus-1 E2 trans-activator require an N-terminal amphipathic helix-containing E2 domain.

    PubMed

    Haugen, T H; Turek, L P; Mercurio, F M; Cripe, T P; Olson, B J; Anderson, R D; Seidl, D; Karin, M; Schiller, J

    1988-12-20

    The sequence-specific trans-activator protein of bovine papillomavirus (BPV)-1, E2, strongly increases transcription at promoters containing papillomaviral ACCG(N)4CGGT (E2P) cis motifs, but can also activate a wide range of co-transfected promoters without E2P cores to a lower extent. Analysis of multiple E2 mutants in transfected cells revealed that the C-terminal DNA binding E2 domain binds to the E2P cis sequences in the form of pre-existing nuclear dimers. The DNA binding function of E2 was required for specific trans-activation of the E2P elements, as well as for the function of the previously described C-terminal 'short E2' transrepressor. In addition to the C terminus, specific trans-activation also required an intact N-terminal half of the E2 protein. When expressed alone, the N-terminal E2 domain was found to activate heterologous promoters without E2P elements to an extent comparable to wild-type E2, and therefore represents the functional transcription activation domain of the E2 factor. In contrast to other DNA-binding activator proteins described to date, the transcriptional activation by the E2 factor can occur without specific DNA binding. Its mechanism may thus involve protein--protein interactions between common transcription factors and the N-terminal E2 domain which contains amphipathic helix motifs. PMID:2854060

  9. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1991--May 31, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-05-01

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

  10. The product of the bovine papillomavirus type 1 modulator gene (M) is a phosphoprotein.

    PubMed Central

    Thorner, L; Bucay, N; Choe, J; Botchan, M

    1988-01-01

    The M gene of bovine papillomavirus type 1 has been genetically defined as encoding a trans-acting product which negatively regulates bovine papillomavirus type 1 replication and is important for establishment of stable plasmids in transformed cells. The gene for this regulatory protein has been mapped in part to the 5' portion of the largest open reading frame (E1) in the virus. We constructed a trpE-E1 fusion gene and expressed this gene in Escherichia coli. Rabbits were immunized with purified fusion protein, and antisera directed against the product were used to identify the M gene product in virus-transformed cells. In this way a polypeptide with an apparent molecular mass of 23 kilodaltons was detected. The virus-encoded product is phosphorylated and can be readily detected by immunoprecipitation assays from cells transformed by the virus. Cells that harbor viral DNA without M as integrated copies do not produce this protein, whereas cells that harbor integrated viral genomes which are defective for another E1 viral gene important for plasmid replication, R, do produce this protein. The protein has an anomalously low electrophoretic mobility. An in vitro translation product of an SP6 RNA product of a sequenced cDNA predicts a molecular mass of 16 kilodaltons for the protein, and this in vitro translation product has an electrophoretic mobility identical to that of the in vivo immunoprecipitated protein. The results of these studies confirm our previous genetic studies which indicated that part of the E1 open reading frame defined a discrete gene product distinct from other putative products which may be encoded by this open reading frame. Images PMID:2836626

  11. 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.

  12. Regulation of the human stress response gene GADD153 expression: role of ETS1 and FLI-1 gene products.

    PubMed

    Seth, A; Giunta, S; Franceschil, C; Kola, I; Venanzoni, M C

    1999-09-01

    We have previously shown that ETS transcription factors, regulate cell growth and differentiation, and ETS1 and ETS2 are able to transcriptionally regulate wt p53 gene expression. In the present study we show that the ETS transcription factors also play a role in regulating expression of GADD153, a wt p53 inducible gene, which induces growth arrest and apoptosis in response to stress signals or DNA damage. We report the presence of a single EBS in the human GADD153 promoter, and that the GADD45 gene promoter lacks EBSs. The GADD153 promoter EBS shows a very high affinity for ETS1 and FLI-1 gene products. In addition, our data show that both ETS1 and FLI-1 strongly activate transcription of the GADD153 EBS linked to the CAT reporter gene. Our results also demonstrate how ETS1 and FLI-1 specifically regulate GADD153 expression. In addition, ectopic ETS2 protein expression resulted in only a weak induction of the same CAT reporter construct. The ETS1 and FLI-1 proteins provide a novel mechanism of activation for GADD153, allowing these two ETS genes to control its expression during cell growth and differentiation, rather than in response to oxidative stress. PMID:10510472

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

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    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.

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

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    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.

  15. Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

    NASA Astrophysics Data System (ADS)

    Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O’Dwyer, Karen; Spence, David W.; Foster, Gary D.

    2016-05-01

    Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

  16. Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

    PubMed Central

    Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O’Dwyer, Karen; Spence, David W.; Foster, Gary D.

    2016-01-01

    Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi. PMID:27143514

  17. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    SciTech Connect

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

    2014-09-01

    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, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα 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. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  18. Production of the Ramoplanin Activity Analogue by Double Gene Inactivation

    PubMed Central

    Han, Jungang; Chen, Junsheng; Shao, Lei; Zhang, Junliang; Dong, Xiaojing; Liu, Pengyu; Chen, Daijie

    2016-01-01

    Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin. PMID:27149627

  19. 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

  20. Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes.

    PubMed

    Sindelar, Georg; Wendisch, Volker F

    2007-09-01

    For the biotechnological production of L: -lysine, mainly strains of Corynebacterium glutamicum are used, which have been obtained by classical mutagenesis and screening or selection or by metabolic engineering. Gene targets for the amplification and deregulation of the lysine biosynthesis pathway, for the improvement of carbon precursor supply and of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) regeneration, are known. To identify novel target genes to improve lysine production, the transcriptomes of the classically obtained lysine producing strain MH20-22B and several other C. glutamicum strains were compared. As lysine production by the classically obtained strain, which possesses feedback-resistant aspartokinase and is leucine auxotrophic, exceeds that of a genetically defined leucine auxotrophic wild-type derivative possessing feedback-resistant aspartokinase, additional traits beneficial for lysine production are present. NCgl0855, putatively encoding a methyltransferase, and the amtA-ocd-soxA operon, encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized enzyme, were among the genes showing increased expression in the classically obtained strain irrespective of the presence of feedback-resistant aspartokinase. Lysine production could be improved by about 40% through overexpression of NCgl0855 or the amtA-ocd-soxA operon. Thus, novel target genes for the improvement of lysine production could be identified in a discovery-driven approach based on global gene expression analysis. PMID:17364200

  1. Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

    PubMed

    Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

    2016-03-28

    Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes. PMID:26822930

  2. Lack of feedback inhibition of V kappa gene rearrangement by productively rearranged alleles.

    PubMed

    Harada, K; Yamagishi, H

    1991-02-01

    Circular DNAs excised by immunoglobulin kappa chain gene rearrangements were cloned and characterized. 16 of 17 clones examined were double recombination products containing a V kappa-J kappa rearrangement (coding joint) as well as the reciprocal element (signal joint) of another V kappa-J kappa rearrangement. These products suggested multiple recombination, primary inversion, and secondary excision. In primary events, 5 of 16 translational reading frames were in-phase. Thus, V kappa gene rearrangement may not be inhibited by the presence of a productively rearranged allele. An unusually large trinucleotide (P) insertion forming a palindrome of 12 nucleotides was also observed in one of the coding joints. PMID:1988542

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

    PubMed

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

    2011-06-01

    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

  4. 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

  5. Chlamydial gene encoding a 70-kilodalton antigen in Escherichia coli: analysis of expression signals and identification of the gene product.

    PubMed Central

    Sardinia, L M; Engel, J N; Ganem, D

    1989-01-01

    In an attempt to identify chlamydial genes whose native promoters allow them to be expressed in Escherichia coli, we isolated and characterized a chlamydial gene identified by screening a library of chlamydial DNA with antichlamydial antibodies. This gene encodes a 70-kilodalton immunoreactive polypeptide in E. coli hosts. Sequence analysis of the 5' portion of the gene identified its product as the chlamydial homolog of the E. coli ribosomal protein S1. The site of transcription initiation of the mRNA in chlamydiae was determined, and its putative promoter regions were identified. These regions apparently do not function efficiently in E. coli; in vitro transcripts generated by using E. coli RNA polymerase did not start at the authentic chlamydial initiation site. Several in vitro transcripts both larger and smaller than the authentic transcript were seen; presumably, these transcripts result from adventitious promoterlike elements in adjacent chlamydial DNA and may be responsible for the expression of the gene in E. coli. Images PMID:2644193

  6. Phylogenomic study of lipid genes involved in microalgal biofuel production-candidate gene mining and metabolic pathway analyses.

    PubMed

    Misra, Namrata; Panda, Prasanna Kumar; Parida, Bikram Kumar; Mishra, Barada Kanta

    2012-01-01

    Optimizing microalgal biofuel production using metabolic engineering tools requires an in-depth understanding of the structure-function relationship of genes involved in lipid biosynthetic pathway. In the present study, genome-wide identification and characterization of 398 putative genes involved in lipid biosynthesis in Arabidopsis thaliana Chlamydomonas reinhardtii, Volvox carteri, Ostreococcus lucimarinus, Ostreococcus tauri and Cyanidioschyzon merolae was undertaken on the basis of their conserved motif/domain organization and phylogenetic profile. The results indicated that the core lipid metabolic pathways in all the species are carried out by a comparable number of orthologous proteins. Although the fundamental gene organizations were observed to be invariantly conserved between microalgae and Arabidopsis genome, with increased order of genome complexity there seems to be an association with more number of genes involved in triacylglycerol (TAG) biosynthesis and catabolism. Further, phylogenomic analysis of the genes provided insights into the molecular evolution of lipid biosynthetic pathway in microalgae and confirm the close evolutionary proximity between the Streptophyte and Chlorophyte lineages. Together, these studies will improve our understanding of the global lipid metabolic pathway and contribute to the engineering of regulatory networks of algal strains for higher accumulation of oil. PMID:23032611

  7. DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli.

    PubMed Central

    Malakooti, J; Komeda, Y; Matsumura, P

    1989-01-01

    The Escherichia coli operon designated flaA contains seven flagellar genes; among them are two switch protein genes whose products are believed to interface with the motility and chemotaxis machinery of the cell. Complementation analysis using several plasmids carrying different portions of the flaA operon and analysis of expression of these plasmids in minicells allowed the identification of two flagellar gene products. The MotD (now called FliN) protein, a flagellar switch protein, was determined to have an apparent molecular weight of 16,000, and the FlaAI (FliL) protein, encoded by a previously unidentified gene, had an apparent molecular weight of 17,000. DNA sequence analysis of the motD gene revealed an open reading frame of 414 base pairs. There were two possible initiation codons (ATG) for motD translation, the first of which overlapped with the termination codon of the upstream gene, flaAII (fliN). The wild-type flaAI gene on the chromosome was replaced with a flaAI gene mutated in vitro. Loss of the flaAI gene product resulted in a nonmotile and nonflagellated phenotype. The subcellular location for both the MotD and FlaAI proteins was determined; the FlaAI protein partitioned exclusively in the insoluble fraction of a whole minicell sonic extract, whereas the MotD protein remained in both the soluble and insoluble fractions. In addition, we subcloned a 2.2-kilobase-pair DNA fragment capable of complementing the remaining four genes of the flaA operon (flbD [fliO], flaR [fliP], flaQ [fliQ], and flaP [fliR]). Images PMID:2651416

  8. Duplication of partial spinosyn biosynthetic gene cluster in Saccharopolyspora spinosa enhances spinosyn production.

    PubMed

    Tang, Ying; Xia, Liqiu; Ding, Xuezhi; Luo, Yushuang; Huang, Fan; Jiang, Yuanwei

    2011-12-01

    Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. Most of the S. spinosa genes involved in spinosyn biosynthesis are found in a contiguous c. 74-kb cluster. To increase the spinosyn production through overexpression of their biosynthetic genes, part of its gene cluster (c. 18 kb) participating in the conversion of the cyclized polyketide to spinosyn was obtained by direct cloning via Red/ET recombination rather than by constructing and screening the genomic library. The resultant plasmid pUCAmT-spn was introduced into S. spinosa CCTCC M206084 from Escherichia coli S17-1 by conjugal transfer. The subsequent single-crossover homologous recombination caused a duplication of the partial gene cluster. Integration of this plasmid enhanced production of spinosyns with a total of 388 (± 25.0) mg L(-1) for spinosyns A and D in the exconjugant S. spinosa trans1 compared with 100 (± 7.7) mg L(-1) in the parental strain. Quantitative real time polymerase chain reaction analysis of three selected genes (spnH, spnI, and spnK) confirmed the positive effect of the overexpression of these genes on the spinosyn production. This study provides a simple avenue for enhancing spinosyn production. The strategies could also be used to improve the yield of other secondary metabolites. PMID:22092858

  9. Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity.

    PubMed Central

    Radziwill, G; Tucker, W; Schaller, H

    1990-01-01

    To correlate the hepatitis B virus P gene with the enzymatic activities predicted to participate in hepadnavirus reverse transcription, a series of P gene mutants containing missense mutations, in-phase insertions, and in-phase deletions was constructed by site-directed mutagenesis. These mutants were tested in the context of otherwise intact hepatitis B virus genomes for the ability to produce core particles containing the virus-associated polymerase activity. The results obtained suggest that the P protein consists of three functional domains and a nonessential spacer arranged in the following order: terminal protein, spacer, reverse transcriptase/DNA polymerase, and RNase H. The first two domains are separated by a spacer region which could be deleted to a large extent without significant loss of endogenous polymerase activity. In cotransfection experiments, all P gene mutants could be complemented in trans by constructs expressing the wild-type gene product but not by a second P gene mutant. This indicates that the multifunctional P gene is expressed as a single translational unit and independent of the core gene and furthermore that the gene product is freely diffusible and not processed before core assembly. Images PMID:2153228

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

    PubMed

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

    2010-09-01

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

  11. Can meta-omics help to establish causality between contaminant biotransformations and genes or gene products?

    PubMed Central

    Johnson, David R.; Helbling, Damian E.; Men, Yujie; Fenner, Kathrin

    2016-01-01

    There is increasing interest in using meta-omics association studies to investigate contaminant biotransformations. The general strategy is to characterize the complete set of genes, transcripts, or enzymes from in situ environmental communities and use the abundances of particular genes, transcripts, or enzymes to establish associations with the communities’ potential to biotransform one or more contaminants. The associations can then be used to generate hypotheses about the underlying biological causes of particular biotransformations. While meta-omics association studies are undoubtedly powerful, they have a tendency to generate large numbers of non-causal associations, making it potentially difficult to identify the genes, transcripts, or enzymes that cause or promote a particular biotransformation. In this perspective, we describe general scenarios that could lead to pervasive non-causal associations or conceal causal associations. We next explore our own published data for evidence of pervasive non-causal associations. Finally, we evaluate whether causal associations could be identified despite the discussed limitations. Analysis of our own published data suggests that, despite their limitations, meta-omics association studies might still be useful for improving our understanding and predicting the contaminant biotransformation capacities of microbial communities.

  12. Regulatory Oversight of Gene Therapy and Cell Therapy Products in Korea.

    PubMed

    Choi, Minjoung; Han, Euiri; Lee, Sunmi; Kim, Taegyun; Shin, Won

    2015-01-01

    The Ministry of Food and Drug Safety regulates gene therapy and cell therapy products as biological products under the authority of the Pharmaceutical Affairs Act. As with other medicinal products, gene therapy and cell therapy products are subject to approval for use in clinical trials and for a subsequent marketing authorization and to post-market surveillance. Research and development of gene therapy and cell therapy products have been progressing rapidly in Korea with extensive investment, offering great potential for the treatment of various serious diseases. To facilitate development of safe and effective products and provide more opportunities to patients suffering from severe diseases, several regulatory programs, such as the use of investigational products for emergency situations, fast-track approval, prereview of application packages, and intensive regulatory consultation, can be applied to these products. The regulatory approach for these innovative products is case by case and founded on science-based review that is flexible and balances the risks and benefits. PMID:26374218

  13. The FRIABLE1 Gene Product Affects Cell Adhesion in Arabidopsis

    PubMed Central

    Neumetzler, Lutz; Humphrey, Tania; Lumba, Shelley; Snyder, Stephen; Yeats, Trevor H.; Usadel, Björn; Vasilevski, Aleksandar; Patel, Jignasha; Rose, Jocelyn K. C.; Persson, Staffan; Bonetta, Dario

    2012-01-01

    Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1), was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246). Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion. PMID:22916179

  14. Genes, language, cognition, and culture: towards productive inquiry.

    PubMed

    Fitch, W Tecumseh

    2011-04-01

    The Queen Mary conference on “Integrating Genetic and Cultural Evolutionary Approaches to Language,” and the papers in this special issue, clearly illustrate the excitement and potential of trans-disciplinary approaches to language as an evolved biological capacity (phylogeny) and an evolving cultural entity (glossogeny). Excepting the present author, the presenters/authors are mostly young rising stars in their respective fields, and include scientists with backgrounds in linguistics, animal communication, neuroscience, evolutionary biology, anthropology, and computer science. On display was a clear willingness to engage with different approaches and terminology and a commitment to shared standards of scientific rigor, empirically driven theory, and logical argument. Because the papers assembled here, together with the introduction, speak for themselves, I will focus in this “extro-duction” on some of the terminological and conceptual difficulties which threaten to block this exciting wave of scientific progress in understanding language evolution, in both senses of that term. In particular I will first argue against the regrettably widespread practice of opposing cultural and genetic explanations of human cognition as if they were dichotomous. Second, I will unpack the debate concerning “general-purpose” and “domain-specific” mechanisms, which masquerades as a debate about nativism but is nothing of the sort. I believe that framing discussions of language in these terms has generated more heat than light, and that a modern molecular understanding of genes, development, behavior, and evolution renders many of the assumptions underlying this debate invalid. PMID:21615292

  15. 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.

  16. 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

  17. Functional analysis of the Erwinia herbicola tutB gene and its product.

    PubMed

    Katayama, Takane; Suzuki, Hideyuki; Koyanagi, Takashi; Kumagai, Hidehiko

    2002-06-01

    The tutB gene, which lies just downstream of tpl, has been cloned from Erwinia herbicola, and its product was analyzed. Despite its high sequence similarity to tryptophan transporters, TutB was found to be a tyrosine-specific transporter. Tryptophan acted as a competitive inhibitor of tyrosine transport. Unlike the tryptophanase operon, the tpl and tutB genes do not constitute an operon. PMID:12003958

  18. Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions.

    PubMed Central

    Ghisotti, D; Finkel, S; Halling, C; Dehò, G; Sironi, G; Calendar, R

    1990-01-01

    We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome. The newly sequenced region encodes three nonessential genes, which are called gop, beta, and cII (in order, from left to right). The gop gene product kills Escherichia coli when the beta protein is absent; the gop and beta genes are transcribed rightward from the same promoter. The cII gene is transcribed leftward to a rho-independent terminator. Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the beta gene. Images PMID:2403440

  19. 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}.

  20. Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production

    PubMed Central

    Dzikowski, Ron; Frank, Matthias; Deitsch, Kirk

    2006-01-01

    The primary virulence determinant of Plasmodium falciparum malaria parasite–infected cells is a family of heterogeneous surface receptors collectively referred to as PfEMP1. These proteins are encoded by a large, polymorphic gene family called var. The family contains approximately 60 individual genes, which are subject to strict, mutually exclusive expression, with the single expressed var gene determining the antigenic, cytoadherent, and virulence phenotype of the infected cell. The mutually exclusive expression pattern of var genes is imperative for the parasite's ability to evade the host's immune response and is similar to the process of “allelic exclusion” described for mammalian Ig and odorant receptor genes. In mammalian systems, mutually exclusive expression is ensured by negative feedback inhibition mediated by production of a functional protein. To investigate how expression of the var gene family is regulated, we have created transgenic lines of parasites in which expression of individual var loci can be manipulated. Here we show that no such negative feedback system exists in P. falciparum and that this process is dependent solely on the transcriptional regulatory elements immediately adjacent to each gene. Transgenic parasites that are selected to express a var gene in which the PfEMP1 coding region has been replaced by a drug-selectable marker silence all other var genes in the genome, thus effectively knocking out all PfEMP1 expression and indicating that the modified gene is still recognized as a member of the var gene family. Mutually exclusive expression in P. falciparum is therefore regulated exclusively at the level of transcription, and a functional PfEMP1 protein is not necessary for viability or for proper gene regulation in cultured parasites. PMID:16518466

  1. Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

    PubMed Central

    Zhang, Qi; Doroghazi, James R.; Zhao, Xiling; Walker, Mark C.

    2015-01-01

    Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities. PMID:25888176

  2. 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

  3. 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.

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

    PubMed

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, João 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

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

    PubMed Central

    Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, João 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

  6. Split-gene system for hybrid wheat seed production

    PubMed Central

    Kempe, Katja; Rubtsova, Myroslava; Gils, Mario

    2014-01-01

    Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore “linked in repulsion.” Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner. PMID:24821800

  7. Investigation of genes involved in nisin production in Enterococcus spp. strains isolated from raw goat milk.

    PubMed

    Perin, Luana Martins; Todorov, Svetoslav Dimitrov; Nero, Luís Augusto

    2016-09-01

    Different strains of Lactococcus lactis are capable of producing the bacteriocin nisin. However, genetic transfer mechanisms allow the natural occurrence of genes involved in nisin production in members of other bacterial genera, such as Enterococcus spp. In a previous study, nisA was identified in eight enterococci capable of producing antimicrobial substances. The aim of this study was to verify the presence of genes involved in nisin production in Enterococcus spp. strains, as well as nisin expression. The nisA genes from eight Enterococcus spp. strains were sequenced and the translated amino acid sequences were compared to nisin amino-acid sequences previously described in databases. Although containing nisin structural and maturation related genes, the enterococci strains tested in the present study did not present the immunity related genes (nisFEG and nisI). The translated sequences of nisA showed some point mutations, identical to those presented by Lactococcus strains isolated from goat milk. All enterococci were inhibited by nisin, indicating the absence of immunity and thus that nisin cannot be expressed. This study demonstrated for the first time the natural occurrence of nisin structural genes in Enterococcus strains and highlights the importance of providing evidence of a link between the presence of bacteriocin genes and their expression. PMID:27255139

  8. The product of the imprinted H19 gene is an oncofetal RNA.

    PubMed Central

    Ariel, I.; Ayesh, S.; Perlman, E. J.; Pizov, G.; Tanos, V.; Schneider, T.; Erdmann, V. A.; Podeh, D.; Komitowski, D.; Quasem, A. S.; de Groot, N.; Hochberg, A.

    1997-01-01

    AIMS/BACKGROUND: The H19 gene is an imprinted, maternally expressed gene in humans. It is tightly linked and coregulated with the imprinted, paternally expressed gene of insulin-like growth factor 2. The H19 gene product is not translated into protein and functions as an RNA molecule. Although its role has been investigated for more than a decade, its biological function is still not understood fully. H19 is abundantly expressed in many tissues from early stages of embryogenesis through fetal life, and is down regulated postnatally. It is also expressed in certain childhood and adult tumours. This study was designed to screen the expression of H19 in human cancer and its relation to the expression of H19 in the fetus. METHODS: Using in situ hybridisation with a [35S] labelled probe, H19 mRNA was detected in paraffin wax sections of fetal tissues from the first and second trimesters of pregnancy and of a large array of human adult and childhood tumours arising from these tissues. RESULTS: The H19 gene is expressed in tumours arising from tissues which express this gene in fetal life. Its expression in the fetus and in cancer is closely linked with tissue differentiation. CONCLUSIONS: Based on these and previous data, H19 is neither a tumour suppressor gene nor an oncogene. Its product is an oncofetal RNA. The potential use of this RNA as a tumour marker should be evaluated. Images PMID:9208812

  9. 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

  10. Associations between polymorphisms of the gene and milk production traits in water buffaloes.

    PubMed

    Deng, T X; Pang, C Y; Lu, X R; Zhu, P; Duan, A Q; Liang, X W

    2016-03-01

    Signal transducer and activator of transcription 1 () is an important regulator of mammary gland differentiation and cell survival that has been regarded as a candidate gene affecting milk production traits in mammals. Therefore, this study was conducted to evaluate significant associations between SNP of the gene and milk production traits in buffaloes. Here, 18 SNP were identified in the buffalo gene, including 15 intronic mutations and 3 exon mutations. All the identified SNP were then genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry methods from 192 buffaloes. All the SNP were in Hardy-Weinberg equilibrium, and 2 haplotype blocks were successfully constructed based on these SNP data, which formed 5 and 3 major haplotypes in the population (>5%), respectively. The results of association analysis showed that only SNP13 located in exon 10 was significantly associated with the milk production traits in the population ( < 0.05). Single nucleotide polymorphism 2, SNP5, SNP8, and SNP9 were associated with protein percentage, and SNP4 and SNP10 were associated with 305-d milk yield ( < 0.05). Our results provide evidence that polymorphisms of the buffalo gene are associated with milk production traits and can be used as a candidate gene for marker-assisted selection in buffalo breeding. PMID:27065255

  11. Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

    PubMed Central

    Herod, Morgan R.; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C.; Verdaguer, Nuria; Rowlands, David J.

    2016-01-01

    occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen. PMID:27194768

  12. Microspore embryogenesis: assignment of genes to embryo formation and green vs. albino plant production.

    PubMed

    Muñoz-Amatriaín, M; Svensson, J T; Castillo, A M; Close, T J; Vallés, M P

    2009-08-01

    Plant microspores can be reprogrammed from their normal pollen development to an embryogenic route in a process termed microspore embryogenesis or androgenesis. Stress treatment has a critical role in this process, inducing the dedifferentiation of microspores and conditioning the following androgenic response. In this study, we have used three barley doubled haploid lines with similar genetic background but different androgenic response. The Barley1 GeneChip was used for transcriptome comparison of these lines after mannitol stress treatment, allowing the identification of 213 differentially expressed genes. Most of these genes belong to the functional categories "cell rescue, defense, and virulence"; "metabolism"; "transcription"; and "transport". These genes were grouped into clusters according to their expression profiles among lines. A principal component analysis allowed us to associate specific gene expression clusters to phenotypic variables. Genes associated with the ability of microspores to divide and form embryos were mainly involved in changes in the structure and function of membranes, efficient use of available energy sources, and cell fate. Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration. PMID:19229567

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

    PubMed Central

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

    2001-01-01

    A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627–1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC. PMID:11567009

  14. 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.

    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.

  15. 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.

  16. 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.

    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.

  17. Development of Ecogenomic Sensors for Remote Detection of Marine Microbes, Their Genes and Gene Products

    NASA Astrophysics Data System (ADS)

    Scholin, C.; Preston, C.; Harris, A.; Birch, J.; Marin, R.; Jensen, S.; Roman, B.; Everlove, C.; Makarewicz, A.; Riot, V.; Hadley, D.; Benett, W.; Dzenitis, J.

    2008-12-01

    An internet search using the phrase "ecogenomic sensor" will return numerous references that speak broadly to the idea of detecting molecular markers indicative of specific organisms, genes or other biomarkers within an environmental context. However, a strict and unified definition of "ecogenomic sensor" is lacking and the phrase may be used for laboratory-based tools and techniques as well as semi or fully autonomous systems that can be deployed outside of laboratory. We are exploring development of an ecogenomic sensor from the perspective of a field-portable device applied towards oceanographic research and water quality monitoring. The device is known as the Environmental Sample Processor, or ESP. The ESP employs wet chemistry molecular analytical techniques to autonomously assess the presence and abundance of specific organisms, their genes and/or metabolites in near real-time. Current detection chemistries rely on low- density DNA probe and protein arrays. This presentation will emphasize results from 2007-8 field trials when the ESP was moored in Monterey Bay, CA, as well as current engineering activities for improving analytical capacity of the instrument. Changes in microbial community structure at the rRNA level were observed remotely in accordance with changing chemical and physical oceanographic conditions. Current developments include incorporation of a reusable solid phase extraction column for purifying nucleic acids and a 4-channel real-time PCR module. Users can configure this system to support a variety of PCR master mixes, primer/probe combinations and control templates. An update on progress towards fielding a PCR- enabled ESP will be given along with an outline of plans for its use in coastal and oligotrophic oceanic regimes.

  18. In silico identification of gene amplification targets based on analysis of production and growth coupling.

    PubMed

    Jian, Xingxing; Zhou, Shengguo; Zhang, Cheng; Hua, Qiang

    2016-07-01

    Genome-scale metabolic models (GEMs) can be utilized to better understand the genotype-phenotype relationship in microbial metabolism. Manipulation strategies based on analysis of metabolic flux distributions using constraint-based methods have been validated to be effective for designing strains. Herein, we first investigated the coupled relationship of growth and production, and subsequently proposed an algorithm, called analysis of production and growth coupling (APGC), to identify amplification targets for improving production of the desired metabolite. The logical transformation of the genome-scale metabolic models (LTM) could enable a gene-level prediction, that is, direct gene targets would be determined through APGC. This algorithm was successfully employed to simulate heterogeneous biosynthesis of the antioxidant lycopene in Escherichia coli, and target genes for the improvement of lycopene production were identified. These identified gene targets were unambiguous and were closely related to the supply of essential precursors and cofactors for lycopene production, and most of these have been validated as effective in enhancing the yield of lycopene. PMID:27157785

  19. Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

    PubMed Central

    Inaba, Tomohiro; Tokumoto, Yuta; Miyazaki, Yusuke; Inoue, Naoyuki; Maseda, Hideaki; Nakajima-Kambe, Toshiaki; Uchiyama, Hiroo

    2013-01-01

    Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species. PMID:24038682

  20. 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

  1. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products.

    PubMed

    Uhlin, B E; Molin, S; Gustafsson, P; Nordström, K

    1979-06-01

    Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30 degrees C these miniplasmids are present in 20--50 copies per cell of Escherichia coli, whereas at temperatures above 35 degrees C the plasmids replicate without copy number control during 2--3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plamid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded beta-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles. PMID:383579

  2. Regulatory structures for gene therapy medicinal products in the European Union.

    PubMed

    Klug, Bettina; Celis, Patrick; Carr, Melanie; Reinhardt, Jens

    2012-01-01

    Taking into account the complexity and technical specificity of advanced therapy medicinal products: (gene and cell therapy medicinal products and tissue engineered products), a dedicated European regulatory framework was needed. Regulation (EC) No. 1394/2007, the "ATMP Regulation" provides tailored regulatory principles for the evaluation and authorization of these innovative medicines. The majority of gene or cell therapy product development is carried out by academia, hospitals, and small- and medium-sized enterprises (SMEs). Thus, acknowledging the particular needs of these types of sponsors, the legislation also provides incentives for product development tailored to them. The European Medicines Agency (EMA) and, in particular, its Committee for Advanced Therapies (CAT) provide a variety of opportunities for early interaction with developers of ATMPs to enable them to have early regulatory and scientific input. An important tool to promote innovation and the development of new medicinal products by micro-, small-, and medium-sized enterprises is the EMA's SME initiative launched in December 2005 to offer financial and administrative assistance to smaller companies. The European legislation also foresees the involvement of stakeholders, such as patient organizations, in the development of new medicines. Considering that gene therapy medicinal products are developed in many cases for treatment of rare diseases often of monogenic origin, the involvement of patient organizations, which focus on rare diseases and genetic and congenital disorders, is fruitful. Two such organizations are represented in the CAT. Research networks play another important role in the development of gene therapy medicinal products. The European Commission is funding such networks through the EU Sixth Framework Program. PMID:22365782

  3. Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

    PubMed

    Fischer, Konrad; Kraner-Scheiber, Simone; Petersen, Björn; Rieblinger, Beate; Buermann, Anna; Flisikowska, Tatiana; Flisikowski, Krzysztof; Christan, Susanne; Edlinger, Marlene; Baars, Wiebke; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Plotzki, Elena; Szczerbal, Izabela; Switonski, Marek; Denner, Joachim; Wolf, Eckhard; Schwinzer, Reinhard; Niemann, Heiner; Kind, Alexander; Schnieke, Angelika

    2016-01-01

    Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - 'gene stacking', and cointegration of multiple engineered large vectors - 'combineering', to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age. PMID:27353424

  4. Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources.

    PubMed

    Yuan, Bo; Wang, Shi-An; Li, Fu-Li

    2013-10-01

    To improve inulin utilization and ethanol fermentation, exoinulinase genes from the yeast Kluyveromyces marxianus and the recently identified yeast, Candida kutaonensis, were expressed in Saccharomyces cerevisiae. S. cerevisiae harboring the exoinulinase gene from C. kutaonensis gave higher ethanol yield and productivity from both inulin (0.38 vs. 0.34 g/g and 1.35 vs. 1.22 g l(-1) h(-1)) and Jerusalem artichoke tuber flour (0.47 vs. 0.46 g/g and 1.62 vs. 1.54 g l(-1) h(-1)) compared with the strain expressing the exoinulinase gene from K. marxianus. Thus, the exoinulinase gene from C. kutaonensis is advantageous for engineering S. cerevisiae to improve ethanol fermentation from inulin sources. PMID:23743955

  5. Differential activation of RNA polymerase III-transcribed genes by the polyomavirus enhancer and the adenovirus E1A gene products.

    PubMed Central

    Berger, S L; Folk, W R

    1985-01-01

    We have compared the effect of the polyomavirus cis-acting transcriptional enhancer and the adenovirus trans-acting E1A gene on expression of RNA polymerase III-transcribed genes (the adenovirus VAI gene and a bacterial tRNA gene) using DNA transfection and transient expression assays. The polyomavirus enhancer has little effect upon transcription of the VAI gene by RNA polymerase III in any cell type tested (murine, hamster, or human). In contrast, expression of the E1A gene within adenovirus infected cells stimulates transcription of RNA polymerase III-transcribed genes from co-transfected DNAs. Human 293 cells, which constitutively produce adenovirus E1A gene products, also express high levels of RNA polymerase III transcripts from transfected DNAs. Images PMID:2987823

  6. RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells.

    PubMed

    Grishchenko, O V; Kiselev, K V; Tchernoded, G K; Fedoreyev, S A; Veselova, M V; Bulgakov, V P; Zhuravlev, Y N

    2016-09-01

    Maackia amurensis Rupr. et Maxim is a valuable leguminous tree grown in the Russian Far East, in China, and in Korea. Polyphenols from the heartwood of this species (primarily stilbenes and isoflavonoids) possess strong hepatoprotective activity. Callus culture of M. amurensis produced isoflavonoids and their derivatives. In pharmacological experiments, the callus complex was at least as effective, as the plant complex. To increase the yield of isoflavonoids, calli were transformed with the rolB gene of Agrobacterium rhizogenes. Neomycin phosphotransferase (nptII) gene was used for transgenic cell selection. Three rolB transgenic callus lines with different levels of the rolB gene expression were established. Insertion of the rolB gene caused alterations in callus structure, growth, and isoflavonoid production, and stronger alterations were observed with higher expression levels. MB1, MB2, and MB4 cultures accumulated 1.4, 1.5, and 2.1 % of dry weight (DW) isoflavonoids, respectively. In contrast, the empty vector-transformed MV culture accumulated 1.22 % DW. Isoflavonoid productivity of the obtained MB1, MB2, and MB4 cultures was equal to 117, 112, and 199 mg/L of medium, respectively, comparing to 106 mg/L for the MV culture. High level of expression of the rolB gene in MB4 culture led to a 2-fold increase in the isoflavonoid content and productivity and reliably increased dry biomass accumulation. Lower expression levels of the rolB gene in MB1 and MB2 calli did not significantly enhance biomass accumulation and isoflavonoid content, although the rolB gene activated isoflavonoid biosynthesis during the early growth stages and caused the increased content of several distinct compounds. PMID:27063013

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

    PubMed

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

    2016-07-01

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

  8. 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...

  9. Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell; Alterthum, Flavio

    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.

  10. 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 ...

  11. Regulation of tabtoxin production by the lemA gene in Pseudomonas syringae.

    PubMed Central

    Barta, T M; Kinscherf, T G; Willis, D K

    1992-01-01

    Pseudomonas syringae pv. coronafaciens, a pathogen of oats, was mutagenized with Tn5 to generate mutants defective in tabtoxin production. From a screen of 3,400 kanamycin-resistant transconjugants, seven independent mutants that do not produce tabtoxin (Tox-) were isolated. Although the Tn5 insertions within these seven mutants were linked, they were not located in the previously described tabtoxin biosynthetic region of P. syringae. Instead, all of the insertions were within the P. syringae pv. coronafaciens lemA gene. The lemA gene is required by strains of P. syringae pv. syringae for pathogenicity on bean plants (Phaseolus vulgaris). In contrast to the phenotype of a P. syringae pv. syringae lemA mutant, the Tox- mutants of P. syringae pv. coronafaciens were still able to produce necrotic lesions on oat plants (Avena sativa), although without the chlorosis associated with tabtoxin production. Northern (RNA) hybridization experiments indicated that a functional lemA gene was required for the detection of a transcript produced from the tblA locus located in the tabtoxin biosynthetic region. Marker exchange mutagenesis of the tblA locus resulted in loss of tabtoxin production. Therefore, both the tblA and lemA genes are required for tabtoxin biosynthesis, and the regulation of tabtoxin production by lemA probably occurs at the transcriptional level. Images PMID:1314808

  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. 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

  14. Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety.

    PubMed

    Lee, Jong-Yeol; Beom, Hye-Rang; Altenbach, Susan B; Lim, Sun-Hyung; Kim, Yeong-Tae; Kang, Chon-Sik; Yoon, Ung-Han; Gupta, Ravi; Kim, Sun-Tae; Ahn, Sang-Nag; Kim, Young-Mi

    2016-05-01

    Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour. PMID:26882917

  15. Analysis of ldh genes in Lactobacillus casei BL23: role on lactic acid production.

    PubMed

    Rico, Juan; Yebra, María Jesús; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

    2008-06-01

    Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid. PMID:18231816

  16. Modern plant metabolomics: advanced natural product gene discoveries, improved technologies, and future prospects.

    PubMed

    Sumner, Lloyd W; Lei, Zhentian; Nikolau, Basil J; Saito, Kazuki

    2015-02-01

    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 review covers the approximate period of 2000 to 2014, and 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. PMID:25342293

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Correlation of gene expression and protein production rate - a system wide study

    PubMed Central

    2011-01-01

    Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR). PMID:22185473

  2. Comprehensive curation and analysis of fungal biosynthetic gene clusters of published natural products.

    PubMed

    Li, Yong Fuga; Tsai, Kathleen J S; Harvey, Colin J B; Li, James Jian; Ary, Beatrice E; Berlew, Erin E; Boehman, Brenna L; Findley, David M; Friant, Alexandra G; Gardner, Christopher A; Gould, Michael P; Ha, Jae H; Lilley, Brenna K; McKinstry, Emily L; Nawal, Saadia; Parry, Robert C; Rothchild, Kristina W; Silbert, Samantha D; Tentilucci, Michael D; Thurston, Alana M; Wai, Rebecca B; Yoon, Yongjin; Aiyar, Raeka S; Medema, Marnix H; Hillenmeyer, Maureen E; Charkoudian, Louise K

    2016-04-01

    Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, "activity-guided" approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent "genome mining" approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias toward the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and

  3. Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product.

    PubMed Central

    Allibert, P; Willison, J C; Vignais, P M

    1987-01-01

    In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product. Images PMID:3025172

  4. [Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

    PubMed

    Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

    2012-01-01

    Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials. PMID:23544311

  5. The Unfolded Protein Response and the Phosphorylations of Activating Transcription Factor 2 in the trans-Activation of il23a Promoter Produced by β-Glucans*

    PubMed Central

    Rodríguez, Mario; Domingo, Esther; Alonso, Sara; Frade, Javier García; Eiros, José; Crespo, Mariano Sánchez; Fernández, Nieves

    2014-01-01

    Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP), X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities. PMID:24982422

  6. Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae.

    PubMed

    O'Neill, E C; Saalbach, G; Field, R A

    2016-01-01

    Eukaryotic microalgae are an incredibly diverse group of organisms whose sole unifying feature is their ability to photosynthesize. They are known for producing a range of potent toxins, which can build up during harmful algal blooms causing damage to ecosystems and fisheries. Genome sequencing is lagging behind in these organisms because of their genetic complexity, but transcriptome sequencing is beginning to make up for this deficit. As more sequence data becomes available, it is apparent that eukaryotic microalgae possess a range of complex natural product biosynthesis capabilities. Some of the genes concerned are responsible for the biosynthesis of known toxins, but there are many more for which we do not know the products. Bioinformatic and analytical techniques have been developed for natural product discovery in bacteria and these approaches can be used to extract information about the products synthesized by algae. Recent analyses suggest that eukaryotic microalgae produce many complex natural products that remain to be discovered. PMID:27480684

  7. Improving heterologous polyketide production in Escherichia coli by overexpression of an S-adenosylmethionine synthetase gene.

    PubMed

    Wang, Yong; Boghigian, Brett A; Pfeifer, Blaine A

    2007-11-01

    An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l(-1) OD(600)(-1). In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds. PMID:17876579

  8. Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells.

    PubMed

    Prigojin, H; Brusel, M; Fuchs, O; Shomrat, R; Legum, C; Nudel, U; Yaffe, D

    1993-12-01

    We have examined the expression of several Duchenne muscular dystrophy (DMD) gene products in amniotic fluid (AF) and chorionic villus sampling (CVS) cells. Variable amounts of dystrophin could be detected in most CVS and AF samples by immunoprecipitation followed by Western blot analysis. PCR analysis demonstrated the presence of the muscle type dystrophin mRNA in all AF cell cultures. The brain type dystrophin mRNA was also detected in some of these cultures. These DMD gene transcripts are of fetal origin and are produced by most or all clonable AF cells. The results may facilitate the development of a method for prenatal diagnosis of DMD, based on the expression of the gene in AF and CVS cells. PMID:8253201

  9. PepPSy: a web server to prioritize gene products in experimental and biocuration workflows

    PubMed Central

    Sallou, Olivier; Duek, Paula D.; Darde, Thomas A.; Collin, Olivier; Lane, Lydie; Chalmel, Frédéric

    2016-01-01

    Among the 20 000 human gene products predicted from genome annotation, about 3000 still lack validation at protein level. We developed PepPSy, a user-friendly gene expression-based prioritization system, to help investigators to determine in which human tissues they should look for an unseen protein. PepPSy can also be used by biocurators to revisit the annotation of specific categories of proteins based on the ‘omics’ data housed by the system. In this study, it was used to prioritize 21 dubious protein-coding genes among the 616 annotated in neXtProt for reannotation. PepPSy is freely available at http://peppsy.genouest.org. Database URL: http://peppsy.genouest.org. PMID:27173522

  10. PepPSy: a web server to prioritize gene products in experimental and biocuration workflows.

    PubMed

    Sallou, Olivier; Duek, Paula D; Darde, Thomas A; Collin, Olivier; Lane, Lydie; Chalmel, Frédéric

    2016-01-01

    Among the 20 000 human gene products predicted from genome annotation, about 3000 still lack validation at protein level. We developed PepPSy, a user-friendly gene expression-based prioritization system, to help investigators to determine in which human tissues they should look for an unseen protein. PepPSy can also be used by biocurators to revisit the annotation of specific categories of proteins based on the 'omics' data housed by the system. In this study, it was used to prioritize 21 dubious protein-coding genes among the 616 annotated in neXtProt for reannotation. PepPSy is freely available at http://peppsy.genouest.orgDatabase URL: http://peppsy.genouest.org. PMID:27173522

  11. 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

  12. 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 30μl 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

  13. The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production.

    PubMed

    Ishida, Nobuhiro; Saitoh, Satoshi; Onishi, Toru; Tokuhiro, Kenro; Nagamori, Eiji; Kitamoto, Katsuhiko; Takahashi, Haruo

    2006-05-01

    A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported. PMID:16717415

  14. Genetic resources for methane production from biomass described with the Gene Ontology

    PubMed Central

    Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, João 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

  15. Genetic resources for methane production from biomass described with the Gene Ontology.

    PubMed

    Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, João 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

  16. 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.

  17. The rkpGHI and -J genes are involved in capsular polysaccharide production by Rhizobium meliloti.

    PubMed Central

    Kiss, E; Reuhs, B L; Kim, J S; Kereszt, A; Petrovics, G; Putnoky, P; Dusha, I; Carlson, R W; Kondorosi, A

    1997-01-01

    The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular polysaccharide that is involved in root nodule development and structurally analogous to group II K antigens found in Escherichia coli (G. Petrovics, P. Putnoky, R. Reuhs, J. Kim, T. A. Thorp, K. D. Noel, R. W. Carlson, and A. Kondorosi, Mol. Microbiol. 8:1083-1094, 1993; B. L. Reuhs, R. W. Carlson, and J. S. Kim, J. Bacteriol. 175:3570-3580, 1993). Here we present the nucleotide sequence for the other three complementation units of the fix-23 locus, revealing the presence of four additional open reading frames assigned to genes rkpGHI and -J. The putative RkpG protein shares similarity with acyltransferases, RkpH is homologous to short-chain alcohol dehydrogenases, and RkpJ shows significant sequence identity with bacterial polysaccharide transport proteins, such as KpsS of E. coli. No significant homology was found for RkpI. Biochemical and immunological analysis of Tn5 derivatives for each gene demonstrated partial or complete loss of capsular polysaccharides from the cell surface; on this basis, we suggest that all genes in the fix-23 region are required for K-antigen synthesis or transport. PMID:9079896

  18. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

    PubMed Central

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S. S.; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended. PMID:27175208

  19. EMEA and Gene Therapy Medicinal Products Development in the European Union

    PubMed Central

    2003-01-01

    The evaluation of quality, safety, and efficacy of medicinal products by the European Medicines Evaluation Agency (EMEA) via the centralized procedure is the only available regulatory procedure for obtaining marketing authorization for gene therapy (GT) medicinal products in the European Union. The responsibility for the authorization of clinical trials remains with the national competent authorities (NCA) acting in a harmonized framework from the scientific viewpoint. With the entry into force of a new directive on good clinical practice implementation in clinical trials as of 1 May 2004, procedural aspects will also be harmonized at EU level. Scientifically sound development of medicinal products is the key for the successful registration of dossiers and for contributing to the promotion and protection of public health. The objective of this paper is to introduce the EMEA regulatory processes and scientific activities relevant to GT medicinal products. PMID:12686717

  20. Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides.

    PubMed

    Csernetics, Arpád; Nagy, Gábor; Iturriaga, Enrique A; Szekeres, András; Eslava, Arturo P; Vágvölgyi, Csaba; Papp, Tamás

    2011-07-01

    The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone. PMID:21443966

  1. Induction of mitotic gene conversion by browning reaction products and its modulation by naturally occurring agents.

    PubMed

    Rosin, M P; Stich, H F; Powrie, W D; Wu, C H

    1982-05-01

    Mitotic gene conversion in the D7 strain of Saccharomyces cerevisiae was significantly enhanced by exposure to non-enzymatic browning reaction products. These products were formed during the heating of sugar (caramelization reaction) or sugar-amino acid mixtures (Maillard reaction) at temperatures normally used during the cooking of food. Several modulating factors of this convertogenic activity were identified. These factors included two main groups: (1) trace metals which are widely distributed in the environment; and (2) several cellular enzymatic systems. The convertogenic activities of a heated glucose-lysine mixture and a commercial caramel powder were completely suppresses when yeast were concurrently exposed to these products and to either FeIII or CuII. Equimolar concentrations of MnII or sodium selenite had no effect on the convertogenic activity of the products of either model system. Horse-radish peroxidase, beef liver catalase and rat liver S9 preparations each decreased the frequency of gene conversion induced by the caramel powder and the heated glucose-lysine products. This modulating activity of the enzymes was lost if they were heat-inactivated. These studies indicate the presence of a variety of protective mechanisms which can modify genotoxic components in complex food mixtures. PMID:7045641

  2. Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

    PubMed Central

    Niki, Tomoya; Nishijima, Takaaki; Nakayama, Masayoshi; Hisamatsu, Tamotsu; Oyama-Okubo, Naomi; Yamazaki, Hiroko; Hedden, Peter; Lange, Theo; Mander, Lewis N.; Koshioka, Masaji

    2001-01-01

    We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2–35S-Ω). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T2 generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T2 generation, indicating that the transgene was stable and dominant. The endogenous levels of GA1 and GA4 were reduced in the dwarfs, whereas large amounts of GA17 and GA25, which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

  3. Production of dwarf lettuce by overexpressing a pumpkin gibberellin 20-oxidase gene.

    PubMed

    Niki, T; Nishijima, T; Nakayama, M; Hisamatsu, T; Oyama-Okubo, N; Yamazaki, H; Hedden, P; Lange, T; Mander, L N; Koshioka, M

    2001-07-01

    We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

  4. 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

  5. NF45 and NF90 Bind HIV-1 RNA and Modulate HIV Gene Expression

    PubMed Central

    Li, Yan; Belshan, Michael

    2016-01-01

    A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1) replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies indicate that both NF45 and NF90 potentiate HIV infection through their RNA binding domains. PMID:26891316

  6. 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

  7. 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

  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, Åsa

    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. ZCT1 and ZCT2 transcription factors repress the activity of a gene promoter from the methyl erythritol phosphate pathway in Madagascar periwinkle cells.

    PubMed

    Chebbi, Mouadh; Ginis, Olivia; Courdavault, Vincent; Glévarec, Gaëlle; Lanoue, Arnaud; Clastre, Marc; Papon, Nicolas; Gaillard, Cécile; Atanassova, Rossitza; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Courtois, Martine; Oudin, Audrey

    2014-10-15

    In Catharanthus roseus, accumulating data highlighted the existence of a coordinated transcriptional regulation of structural genes that takes place within the secoiridoid biosynthetic branch, including the methyl erythritol phosphate (MEP) pathway and the following steps leading to secologanin. To identify transcription factors acting in these pathways, we performed a yeast one-hybrid screening using as bait a promoter region of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene involved in the responsiveness of C. roseus cells to hormonal signals inducing monoterpene indole alkaloid (MIA) production. We identified that ZCT2, one of the three members of the zinc finger Catharanthus protein (ZCT) family, can bind to a HDS promoter region involved in hormonal responsiveness. By trans-activation assays, we demonstrated that ZCT1 and ZCT2 but not ZCT3 repress the HDS promoter activity. Gene expression analyses in C. roseus cells exposed to methyljasmonate revealed a persistence of induction of ZCT2 gene expression suggesting the existence of feed-back regulatory events acting on HDS gene expression in correlation with the MIA production. PMID:25108262

  10. A gene from Renibacterium salmoninarum encoding a product which shows homology to bacterial zinc-metalloproteases.

    PubMed

    Grayson, T H; Evenden, A J; Gilpin, M L; Martin, K L; Munn, C B

    1995-06-01

    A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 degrees C and found to contain a 3.1 kb HindIII fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 5.57. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designed hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 degrees C to 37 degrees C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum

  11. Modulation of gene transcription by natural products--a viable anticancer strategy.

    PubMed

    D'Incalci, M; Brunelli, D; Marangon, E; Simone, M; Tavecchio, M; Gescher, A; Mantovani, R

    2007-01-01

    Drug design based on the structure of specific enzymes playing a role in carcinogenesis, e.g. tyrosine kinases, has been successful at identifying novel effective anticancer drugs. In contrast, no success has been achieved in drug design attempts, in which transcription factors or DNA-transcription factor complexes involved in the pathogenesis of human neoplasms were targeted. This failure is likely to be due to the fact that the mechanism of transcription regulation is probably too complex and still too inadequately understood to be a suitable target for drug design. It seems plausible that the high selectivity of some human tumors to some DNA-interactive anticancer drugs, e.g. cisplatin, is related to an effect on the transcription of genes that are crucial for those tumors. In this article we propose that some natural products have evolutionarily evolved to exert highly specialized functions, including modulation of the transcriptional regulation of specific genes. We discuss in detail the marine natural product Yondelis (Trabectedin, ET-743) that is effective against some soft tissue sarcoma, possibly because it interferes with the aberrant transcription mechanism in these tumors. In addition we highlight the existing evidence that many different natural products are effective inhibitors of NF-kB, a transcription factor that plays a crucial role in inflammation and cancer, indicating that some of these compounds might possess antitumor properties. We propose that large-scale characterization of natural products acting as potential modulators of gene transcription is a realistic and attractive approach to discover compounds therapeutically effective against neoplastic diseases characterized by specific aberrations of transcriptional regulation. PMID:17897020

  12. Rhf1 gene is involved in the fruiting body production of Cordyceps militaris fungus.

    PubMed

    Jiang, Keqing; Han, Richou

    2015-08-01

    Cordyceps militaris is an important medicinal fungus. Commercialization of this fungus needs to improve the fruiting body production by molecular engineering. An improved Agrobacterium tumefaciens-mediated transformation (ATMT) method was used to select an insertional mutant (g38) which exhibited fast stromatal differentiation and increased yield. The Rhf1 gene encoding filamentation protein was destroyed by a single T-DNA and no Rhf1 transcription was detected in mutant g38. To verify the function of the Rhf1 gene, RNA interference plasmid and overexpression vector of the Rhf1 gene were constructed and transferred to the wild-type JM4 by ATMT. Fast stromatal differentiation and larger fruiting bodies were found in the RNAi-Rhf1 mutants (JM-iRhf1). In the overexpression mutants (JM-OERhf1), neither stromata nor fruiting bodies appeared. The rescued strain (38-OERhf1) showed similar growth characteristics as JM4. These results indicated that the Rhf1 gene was involved in the stromatal differentiation and the shape formation of fruiting bodies. PMID:26047996

  13. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli.

    PubMed Central

    Bagg, A; Kenyon, C J; Walker, G C

    1981-01-01

    The product of the umuC gene is required for UV and chemical mutagenesis in Escherichia coli. By the use of the Mud(Ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuC gene. The strain containing the umuC::Mud(Ap, lac) fusion was identified on the basis of its UV nonmutability. Strains containing this putative null allele of umuC were (i) nonmutable by UV and other agents, (ii) slightly UV sensitive, and (iii) deficient in their ability to carry out Weigle reactivation of UV-irradiation bacteriophage lambda. The UV nonmutability of the strain could be suppressed by a derivative of the mutagenesis-enhancing plasmid pKM101. beta-Galactosidase synthesis in umuC::Mud(Ap, lac) fusion strains was inducible by UV and other DNA-damaging agents. Genetic analysis of the regulation of beta-galactosidase in umuC::Mud(Ap, lac) strains suggests that the lexA protein is the direct repressor of the umuC gene and that a function of the recA protein, probably its protease activity, is required for the removal of the lexA repressor at the time of umuC induction. PMID:7029544

  14. Global adaptive rank truncated product method for gene-set analysis in association studies.

    PubMed

    Vilor-Tejedor, Natalia; Calle, M Luz

    2014-08-01

    Gene set analysis (GSA) aims to assess the overall association of a set of genetic variants with a phenotype and has the potential to detect subtle effects of variants in a gene or a pathway that might be missed when assessed individually. We present a new implementation of the Adaptive Rank Truncated Product method (ARTP) for analyzing the association of a set of Single Nucleotide Polymorphisms (SNPs) in a gene or pathway. The new implementation, referred to as globalARTP, improves the original one by allowing the different SNPs in the set to have different modes of inheritance. We perform a simulation study for exploring the power of the proposed methodology in a set of scenarios with different numbers of causal SNPs with different effect sizes. Moreover, we show the advantage of using the gene set approach in the context of an Alzheimer's disease case-control study where we explore the endocytosis pathway. The new method is implemented in the R function globalARTP of the globalGSA package available at http://cran.r-project.org. PMID:25082012

  15. 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.

  16. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    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.

  17. Superoxide dismutase (SOD) genes in Streptomyces peucetius: effects of SODs on secondary metabolites production.

    PubMed

    Kanth, Bashistha Kumar; Jnawali, Hum Nath; Niraula, Narayan Prasad; Sohng, Jae Kyung

    2011-07-20

    Two superoxide dismutase (SOD) genes; sod1 and sod2, from Streptomyces peucetius ATCC 27952 show high similarity to other known SODs from Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680. These sod1 and sod2 were cloned into pIBR25 expression vector under a strong ermE* promoter to enhance secondary metabolites from Streptomyces strains. The recombinant expression plasmids; pIBR25SD1 and pIBR25SD2, were constructed to overexpress sod1 and sod2 respectively to enhance production of doxorubicin (DXR) in S. peucetius, clavulanic acid (CA) in Streptomyces clavuligerus NRRL 3585 and actinorhodin (ACT) and undecylprodigiosin (Red) in Streptomyces lividans TK24. Biomass variation, antibiotics production and transcriptional analysis of regulatory genes in recombinant strains have been studied to understand the effect of sod1 and sod2. The cell growth analysis shows that life span of all recombinant strains was found to be elevated as compared to wild type cells. In S. peucetius, overexpression of sod1 and sod2 was not effective in DXR production but in case of S. clavuligerus, CA production was increased by 2.5 and 1.5 times in sod1 and sod2 overexpression, respectively while in case of S. lividans, ACT production was increased by 1.4 and 1.6 times and Red production by 1.5 and 1.2 times upon sod1 and sod2 overexpressions, respectively as compared to the corresponding wild type strains. PMID:20888207

  18. Relationship between fumonisin production and FUM gene expression in Fusarium verticillioides under different environmental conditions.

    PubMed

    Fanelli, Francesca; Iversen, Anita; Logrieco, Antonio F; Mulè, Giuseppina

    2013-01-01

    Fusarium verticillioides is the main source of fumonisins, a group of mycotoxins that can contaminate maize-based food and feed and cause diseases in humans and animals. The study of the effect of different environmental conditions on toxin production should provide information that can be used to develop strategies to minimize the risk. This study analysed the effect of temperature (15°C-35°C), water activity (a(w): 0.999-0.93), salinity (0-125 g l(-1) NaCl) and pH (5-8) on the growth and production of fumonisins B(1) (FB1), B(2) (FB2) and B(3) (FB3) and the expression of FUM1 and FUM21 in F. verticillioides. The highest growth rate was measured at 25°C, a(w) of 0.998-0.99 and 0-25 g l(-1) of NaCl. Optimal conditions for fumonisin production were 30°C, a(w) of 0.99, 25 g l(-1) of NaCl and pH 5; nevertheless, the strain showed a good adaptability and was able to produce moderate levels of fumonisins under a wide range of conditions. Gene expression mirrored fumonisin production profile under all conditions with the exception of temperature: FUM1 and FUM21 expression was highest at 15°C, while maximum fumonisin production was at 30°C. These data indicate that a post-transcriptional regulation mechanism could account for the different optimal temperatures for FUM gene expression and fumonisin production. PMID:23167929

  19. Combining mouse mammary gland gene expression and comparative mapping for the identification of candidate genes for QTL of milk production traits in cattle

    PubMed Central

    Ron, Micha; Israeli, Galit; Seroussi, Eyal; Weller, Joel I; Gregg, Jeffrey P; Shani, Moshe; Medrano, Juan F

    2007-01-01

    Background Many studies have found segregating quantitative trait loci (QTL) for milk production traits in different dairy cattle populations. However, even for relatively large effects with a saturated marker map the confidence interval for QTL location by linkage analysis spans tens of map units, or hundreds of genes. Combining mapping and arraying has been suggested as an approach to identify candidate genes. Thus, gene expression analysis in the mammary gland of genes positioned in the confidence interval of the QTL can bridge the gap between fine mapping and quantitative trait nucleotide (QTN) determination. Results We hybridized Affymetrix microarray (MG-U74v2), containing 12,488 murine probes, with RNA derived from mammary gland of virgin, pregnant, lactating and involuting C57BL/6J mice in a total of nine biological replicates. We combined microarray data from two additional studies that used the same design in mice with a total of 75 biological replicates. The same filtering and normalization was applied to each microarray data using GeneSpring software. Analysis of variance identified 249 differentially expressed probe sets common to the three experiments along the four developmental stages of puberty, pregnancy, lactation and involution. 212 genes were assigned to their bovine map positions through comparative mapping, and thus form a list of candidate genes for previously identified QTLs for milk production traits. A total of 82 of the genes showed mammary gland-specific expression with at least 3-fold expression over the median representing all tissues tested in GeneAtlas. Conclusion This work presents a web tool for candidate genes for QTL (cgQTL) that allows navigation between the map of bovine milk production QTL, potential candidate genes and their level of expression in mammary gland arrays and in GeneAtlas. Three out of four confirmed genes that affect QTL in livestock (ABCG2, DGAT1, GDF8, IGF2) were over expressed in the target organ. Thus, cg

  20. Regulation of hexuronate system genes in Escherichia coli K-12: multiple regulation of the uxu operon by exuR and uxuR gene products.

    PubMed Central

    Robert-Baudouy, J; Portalier, R; Stoeber, F

    1981-01-01

    New regulatory mutants of Escherichia coli K-1 carrying alterations of the uxuR gene were isolated and characterized. In the presence of superrepressed or derepressed uxuR mutations, mannonic hydrolyase (uxuA) and oxidoreductase relationship analyses suggested that the uxuR gene product acted as a repressor in the control of uxuA-uxuB operon expression. uxuR mutations were localized near min 97, and the following gene order was established: (argH)-uxuR-uxuB-uxuA-(thr). Properties of exuR (point and deletion) mutants showed that both exuR and uxuR regulatory gene products were involved in the control of the uxuA uxuB operon. Analysis of exuR uxuR double-derepressed mutants suggested that exuR and uxuR repressors act cooperatively to repress the uxu operon. PMID:7007313

  1. Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

    PubMed

    Cho, Kwang-Soo; Han, Eun-Heui; Kwak, Sang-Soo; Cho, Ji-Hong; Im, Ju-Seong; Hong, Su-Young; Sohn, Hwang-Bae; Kim, Yun-Hee; Lee, Shin-Woo

    2016-01-01

    Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits. PMID:27212605

  2. Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus.

    PubMed

    Jiang, Lingjuan; Wei, Junhong; Li, Lei; Niu, Guoqing; Tan, Huarong

    2013-12-01

    Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus . It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics. PMID:24121866

  3. 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

  4. Effect of precisely identified mutations in the spoIIAC gene of Bacillus subtilis on the toxicity of the sigma-like gene product to Escherichia coli.

    PubMed

    Yudkin, M D; Harrison, D

    1987-09-01

    Yudkin (1986) has shown that the spoIIAC gene of Bacillus subtilis cannot be cloned in Escherichia coli in such an orientation that it is expressed. This toxicity of the gene product has been attributed to its close homology with the sigma subunit of the E. coli RNA polymerase. The effect of six individual mutations in spoIIAC has now been studied. All six mutant genes could be cloned in E. coli in an orientation that does not allow expression. When in the orientation that permits expression, one mutant gene could not be cloned, and a second substantially hampered growth; both mutations lie in the region that is believed to encode the DNA-binding domain of the protein. By contrast, two missense mutations in the region of the gene thought to encode the domain that binds to the core RNA polymerase rendered the protein harmless in E. coli, as did two nonsense mutations. PMID:3118147

  5. A large-scale genetic screen in Arabidopsis to identify genes involved in pollen exine production.

    PubMed

    Dobritsa, Anna A; Geanconteri, Aliza; Shrestha, Jay; Carlson, Ann; Kooyers, Nicholas; Coerper, Daniel; Urbanczyk-Wochniak, Ewa; Bench, Bennie J; Sumner, Lloyd W; Swanson, Robert; Preuss, Daphne

    2011-10-01

    Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production. PMID:21849515

  6. 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...

  7. Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

    PubMed Central

    Pelisser, Marcia Regina; Klein, Cátia Silene; Ascoli, Kelen Regina; Zotti, Thaís Regina; Arisi, Ana Carolina Maisonnave

    2009-01-01

    Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see. PMID:24031334

  8. Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

    DOEpatents

    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.

  9. Exploring DNA assembler, a synthetic biology tool for characterizing and engineering natural product gene clusters

    PubMed Central

    Shao, Zengyi; Zhao, Huimin

    2015-01-01

    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 et al., 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

  10. Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

    PubMed Central

    Westers, Helga; Braun, Peter G.; Westers, Lidia; Antelmann, Haike; Hecker, Michael; Jongbloed, Jan D. H.; Yoshikawa, Hirofumi; Tanaka, Teruo; van Dijl, Jan Maarten; Quax, Wim J.

    2005-01-01

    Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor. PMID:15812018

  11. 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.

  12. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    PubMed Central

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

    2015-01-01

    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. PMID:25807046

  13. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis.

    PubMed

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S; Qian, Pei-Yuan

    2015-01-01

    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. PMID:25807046

  14. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

    PubMed

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng

    2016-02-01

    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products. PMID:26011187

  15. Regulation of human immune gene expression as influenced by a commercial blended Echinacea product: preliminary studies.

    PubMed

    Randolph, R K; Gellenbeck, K; Stonebrook, K; Brovelli, E; Qian, Y; Bankaitis-Davis, D; Cheronis, J

    2003-10-01

    Consumption of Echinacea at the first sign of symptoms has been clinically shown to reduce both the severity and duration of cold and flu. Quantitative polymerase chain reaction optimized for precision and reproducibility was utilized to explore in vitro and in vivo changes in the expression of immunomodulatory genes in response to Echinacea. In vitro exposure of THP-1 cells to 250 microg/ml of Echinacea species extracts induced expression (up to 10-fold) of the interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, interleukin-8, and interleukin-10 genes. This preliminary result is consistent with a general immune response and activation of the nonspecific immune response cytokines. In vivo gene expression within peripheral leukocytes was evaluated in six healthy nonsmoking subjects (18-65 years of age). Blood samples were obtained at baseline and on Days 2, 3, 5, and 12 after consuming a commercial blended Echinacea product, three tablets three times daily (1518 mg/day) for two days plus one additional dose (506 mg) on day three. Serum chemistry and hematological values were not different from baseline, suggesting that liver or bone marrow responses were not involved in acute responses to Echinacea. The overall gene expression pattern at 48 hr to 12 days after taking Echinacea was consistent with an antiinflammatory response. The expression of interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, and interleukin-8 was modestly decreased up through Day 5, returning to baseline by day 12. The expression of interferon-alpha steadily rose through Day 12, consistent with an antiviral response. These preliminary data present a gene expression response pattern that is consistent with Echinacea's reported ability to reduce both the duration and intensity of cold and flu symptoms. PMID:14530514

  16. The multidrug resistance (mdr1) gene product functions as an ATP channel.

    PubMed Central

    Abraham, E H; Prat, A G; Gerweck, L; Seneveratne, T; Arceci, R J; Kramer, R; Guidotti, G; Cantiello, H F

    1993-01-01

    The multidrug resistance (mdr1) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein. PMID:7678345

  17. 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 H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. 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

  18. A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production.

    PubMed

    Lo, Tat-Ming; Chng, Si Hui; Teo, Wei Suong; Cho, Han-Saem; Chang, Matthew Wook

    2016-08-01

    We present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable. PMID:27559924

  19. Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product.

    PubMed Central

    Nakata, A; Amemura, M; Shinagawa, H

    1984-01-01

    The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed. Images PMID:6090402

  20. Abundance and distribution of Macrolide-Lincosamide-Streptogramin resistance genes in an anaerobic-aerobic system treating spiramycin production wastewater.

    PubMed

    Liu, Miaomiao; Ding, Ran; Zhang, Yu; Gao, Yingxin; Tian, Zhe; Zhang, Tong; Yang, Min

    2014-10-15

    The behaviors of the Macrolide-Lincosamide-Streptogramin (MLS) resistance genes were investigated in an anaerobic-aerobic pilot-scale system treating spiramycin (SPM) production wastewater. After screening fifteen typical MLS resistance genes with different mechanisms using conventional PCR, eight detected genes were determined by quantitative PCR, together with three mobile elements. Aerobic sludge in the pilot system exhibited a total relative abundance of MLS resistance genes (per 16S rRNA gene) 2.5 logs higher than those in control samples collected from sewage and inosine wastewater treatment systems (P < 0.05), implying the presence of SPM could induce the production of MLS resistance genes. However, the total relative gene abundance in anaerobic sludge (4.3 × 10(-1)) was lower than that in aerobic sludge (3.7 × 10(0)) despite of the higher SPM level in anaerobic reactor, showing the advantage of anaerobic treatment in reducing the production of MLS resistance genes. The rRNA methylase genes (erm(B), erm(F), erm(X)) were the most abundant in the aerobic sludge (5.3 × 10(-1)-1.7 × 10(0)), followed by esterase gene ere(A) (1.3 × 10(-1)) and phosphorylase gene mph(B) (5.7 × 10(-2)). In anaerobic sludge, erm(B), erm(F), ere(A), and msr(D) were the major ones (1.2 × 10(-2)-3.2 × 10(-1)). These MLS resistance genes (except for msr(D)) were positively correlated with Class 1 integron (r(2) = 0.74-0.93, P < 0.05), implying the significance of horizontal transfer in their proliferation. PMID:24973730

  1. Integrating an algal β-carotene hydroxylase gene into a designed carotenoid-biosynthesis pathway increases carotenoid production in yeast.

    PubMed

    Chang, Jui-Jen; Thia, Caroline; Lin, Hao-Yeh; Liu, Hsien-Lin; Ho, Feng-Ju; Wu, Jiunn-Tzong; Shih, Ming-Che; Li, Wen-Hsiung; Huang, Chieh-Chen

    2015-05-01

    The algal β-carotene hydroxylase gene Crchyb from Chlamydomonas reinhardtii, Czchyb from Chlorella zofingiensis, or Hpchyb from Haematococcus pluvialis and six other carotenoid-synthesis pathway genes were co-integrated into the genome of a yeast host. Each of these three algal genes showed a higher efficiency to convert β-carotene to downstream carotenoids than the fungal genes from Phaffia rhodozyma. Furthermore, the strain with Hpchyb displayed a higher carotenoid productivity than the strains integrated with Crchyb or Czchyb, indicating that Hpchyb is more efficient than Crchyb and Czchyb. These results suggest that β-carotene hydroxylase plays a crucial role in the biosynthesis of carotenoids. PMID:25537137

  2. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

    PubMed

    Li, Huan-Jun; He, Yi-Long; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Li, Na; Xu, Jun-Wei

    2016-06-10

    The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs. PMID:27080449

  3. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

    PubMed

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-05-01

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production. PMID:23610393

  4. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

    PubMed Central

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L.; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-01-01

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production. PMID:23610393

  5. Association of VIPR-1 gene polymorphisms and haplotypes with egg production in laying quails*

    PubMed Central

    Pu, Yue-jin; Wu, Yan; Xu, Xiao-juan; Du, Jin-ping; Gong, Yan-zhang

    2016-01-01

    The laying quail is a worldwide breed which exhibits high economic value. In our current study, the vasoactive intestinal peptide receptor-1 (VIPR-1) was selected as the candidate gene for identifying traits of egg production. A single nucleotide polymorphism (SNP) detection was performed in 443 individual quails, including 196 quails from the H line, 202 quails from the L line, and 45 wild quails. The SNPs were genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Two mutations (G373T, A313G) were detected in all the tested quail populations. The associated analysis showed that the SNP genotypes of the VIPR-1 gene were significantly linked with the egg weight of G373T and A313G in 398 quails. The quails with the genotype GG always exhibited the largest egg weight for the two mutations in the H and L lines. Linkage disequilibrium (LD) analysis indicated that G373T and A313G loci showed the weakest LD. Seven main diplotypes from the four main reconstructed haplotypes were observed, indicating a significant association of diplotypes with egg weight. Quails with the h1h2 (GGGT) diplotype always exhibited the smallest egg weight and largest egg number at 20 weeks of age. The overall results suggest that the alterations in quails may be linked with potential major loci or genes affecting reproductive traits. PMID:27487804

  6. Genetic Evidence for Transcriptional Activation by the Yeast Ime1 Gene Product

    PubMed Central

    Smith, H. E.; Driscoll, S. E.; Sia, RAL.; Yuan, H. E.; Mitchell, A. P.

    1993-01-01

    IME1 is required in yeast for meiosis and for expression of IME2 and other early meiotic genes. IME1 is a 360-amino acid polypeptide with central and C-terminal tyrosine-rich regions. We report here that a fusion protein composed of the lexA DNA-binding domain and IME1 activates transcription in vivo of a reporter gene containing upstream lexA binding sites. Activation by the fusion protein shares several features with natural IME1 activity: both are dependent on the RIM11 gene product; both are impaired by the same ime1 missense mutations; both are restored by intragenic suppressors. The central tyrosine-rich region is sufficient to activate transcription when fused to lexA. Deletion of this putative activation domain results in a defective IME1 derivative. Function of the deletion derivative is restored by fusion to the acidic Herpesvirus VP16 activation domain. The C-terminal tyrosine-rich region is dispensable for transcriptional activation; rather it renders activation dependent upon starvation and RIM11. Immunofluorescence studies indicate that an IME1-lacZ fusion protein is concentrated in the nucleus. These observations are consistent with a model in which IME1 normally stimulates IME2 expression by providing a transcriptional activation domain at the IME2 5' regulatory region. PMID:8462841

  7. 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

  8. 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.

  9. Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

    PubMed Central

    Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

    1998-01-01

    A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

  10. Re-examination of regulatory opinions in Europe: possible contribution for the approval of the first gene therapy product Glybera

    PubMed Central

    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

  11. 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 αTpm), Tpm2.2St(a,b,b,a) (βTpm) and Tpm3.12St(a,b,b,a) (γTpm) 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 (δTpm) 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

  12. Nucleotide and protein sequences for dog masticatory tropomyosin identify a novel Tpm4 gene product

    PubMed Central

    Reiser, Peter J.

    2016-01-01

    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 (also a jaw-closing muscle), tibialis (with 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 αTpm), Tpm2.2St(a,b,b,a) (βTpm) and Tpm3.12St(a,b,b,a) (cTpm) 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 (δTpm) 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  15. Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid.

    PubMed

    Stevenson, G; Andrianopoulos, K; Hobbs, M; Reeves, P R

    1996-08-01

    Colanic acid (CA) is an extracellular polysaccharide produced by most Escherichia coli strains as well as by other species of the family Enterobacteriaceae. We have determined the sequence of a 23-kb segment of the E. coli K-12 chromosome which includes the cluster of genes necessary for production of CA. The CA cluster comprises 19 genes. Two other sequenced genes (orf1.3 and galF), which are situated between the CA cluster and the O-antigen cluster, were shown to be unnecessary for CA production. The CA cluster includes genes for synthesis of GDP-L-fucose, one of the precursors of CA, and the gene for one of the enzymes in this pathway (GDP-D-mannose 4,6-dehydratase) was identified by biochemical assay. Six of the inferred proteins show sequence similarity to glycosyl transferases, and two others have sequence similarity to acetyl transferases. Another gene (wzx) is predicted to encode a protein with multiple transmembrane segments and may function in export of the CA repeat unit from the cytoplasm into the periplasm in a process analogous to O-unit export. The first three genes of the cluster are predicted to encode an outer membrane lipoprotein, a phosphatase, and an inner membrane protein with an ATP-binding domain. Since homologs of these genes are found in other extracellular polysaccharide gene clusters, they may have a common function, such as export of polysaccharide from the cell. PMID:8759852

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

    SciTech Connect

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

    2005-01-01

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

  17. 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

  18. Expression and Function of Enamel-related Gene Products in Calvarial Development

    PubMed Central

    Atsawasuwan, P.; Lu, X.; Ito, Y.; Chen, Y.; Gopinathan, G.; Evans, C.A.; Kulkarni, A.B.; Gibson, C.W.; Luan, X.; Diekwisch, T.G.H.

    2013-01-01

    Enamel-related gene products (ERPs) are detected in non-enamel tissues such as bone. We hypothesized that, if functional, ERP expression corresponds with distinct events during osteoblast differentiation and affects bone development and mineralization. In mouse calvariae and MC3T3 cells, expression profiles of enamel-related gene products (ERPs) correlated with key events in post-natal calvarial development and MC3T3 cell mineralization. Developing skulls from both Amel- and Ambn-deficient animals were approximately 15% shorter when compared with those of wild-type controls, and their sutures remained patent for a longer period of time. Analysis of Amel- and Ambn-deficient calvariae and calvarial osteoblast cultures revealed a dramatic reduction in mineralized nodules, a significant reduction in Runx2, Sp7, Ibsp, and Msx2 expression, and a reduction in Alx4 in Amel-deficient calvariae vs. an increase in Alx4 in Ambn-deficient calvariae. Analysis of these data indicates that ERP expression follows defined developmental profiles and affects osteoblast differentiation, mineralization, and calvarial bone development. We propose that, in parallel to their role in the developing enamel matrix, ERPs have retained an evolutionary conserved function related to the biomineralization of bones. PMID:23625374

  19. 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

  20. Trans-activation of the JC virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells

    SciTech Connect

    Tada, Hiroomi; Lashgari, M.; Amini, S.; Khalili, K. ); Rappaport, J.; Wong-Staal, F. )

    1990-05-01

    Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC virus (JCV), a human papovavirus. PML is a relatively rare disease seen predominantly in immunocompromised individuals and is a frequent complication observed in AIDS patients. The significantly higher incidence of PML in AIDS patients than in other immunosuppressive disorders has suggested that the presence of human immunodeficiency virus type 1 (HIV-1) in the brain may directly or indirectly contribute to the pathogenesis of this disease. In the present study the authors have examined the expression of the JCV genome in both glial and non-glial cells in the presence of HIV-1 regulatory proteins. They find that the HIV-1-encoded trans-regulatory protein tat increases the basal activity of the JCV late promoter, JCV{sub L}, in glial cells. They conclude that the presence of the HIV-1-encoded tat protein may positively affect the JCV lytic cycle in glial cells by stimulating JCV gene expression. The results suggest a mechanism for the relatively high incidence of PML in AIDS patients than in other immunosuppressive disorders. Furthermore, the findings indicate that the HIV-1 regulatory protein tat may stimulate other viral and perhaps cellular promoters, in addition to its own.

  1. Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL

    PubMed Central

    Zustiak, Matthew P.; Jose, Lisa; Xie, Yueqing; Zhu, Jianwei; Betenbaugh, Micheal J.

    2014-01-01

    Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells. Although significant improvements have been made, in terms of expression, more improvements are needed to compete with the yields achievable in stable gene expression. Much progress has come from optimization of transfection media and parameters, as well as altering culturing conditions to enhance productivity. Recent studies have included using cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL. In this study we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of bcl-xL and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing Bcl-xL using polyethylenimine. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta in CHO cells, or through the co-transfection with bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a prior stable cell line and subsequent screening which are both time and resource consuming. PMID:24604826

  2. Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

    PubMed Central

    Reynolds, A E; Murray, A W; Szostak, J W

    1987-01-01

    We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division. Images PMID:3316982

  3. Characterization and expression of the human rhoH12 gene product

    SciTech Connect

    Avraham, H.; Weinberg, R.A.

    1989-05-01

    The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in the place of the glycine and leucine in place of the gutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via contransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho proteins was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

  4. Quantification of Tri5 gene, expression, and deoxynivalenol production during the malting of barley.

    PubMed

    Vegi, Anuradha; Schwarz, Paul; Wolf-Hall, Charlene E

    2011-11-01

    Fusarium can survive, grow, and produce mycotoxins during malting. We evaluated the percentage of barley kernels infected with Fusarium (FI) and deoxynivalenol (DON) concentration in three barley treatments (high-quality, naturally infected, and Fusarium graminearum inoculated barley) during various stages of malting. We also applied real-time polymerase chain reaction (real-time PCR) and real-time reverse transcriptase PCR (real-time RT-PCR) methods to quantify trichothecene-producing (Tri5) DNA concentration and expression, respectively. We observed that FI significantly (P<0.05) increased during the germination stage of malting in all barley treatments. Temperatures of 49°C and higher during kilning reduced the FI in high-quality barley treatments, but for inoculated treatments temperatures in excess of 60°C were needed to reduce FI. The Tri5 DNA concentration ranged from non-detectable to 3.9 ng/50mg, 0.1 to 109.8 ng/50mg and 3.4 to 397.5 ng/50 mg in malted high-quality, inoculated and naturally infected barley treatments respectively. Strong gene expression (Tri5) in naturally infected barley treatments was found during the third day of germination, when compared to high-quality and inoculated barley treatments during malting. Deoxynivalenol was present even at high kilning temperatures, as DON is heat stable. The average DON concentration ranged from non-detectable to 0.1 μg/g, non-detectable to 1.1 μg/g, and 1.5 to 45.9 μg/g during various stages of malting in high-quality, inoculated and infected barley and malt samples respectively. Overall, the last 2 days of germination and initial stages of kilning were peak stages for FI, Tri5 gene production, Tri5 gene expression and DON production. PMID:21871683

  5. The murine Sim-2 gene product inhibits transcription by active repression and functional interference.

    PubMed

    Moffett, P; Reece, M; Pelletier, J

    1997-09-01

    The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription. PMID:9271372

  6. Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease.

    PubMed

    Haidinger, W; Mayr, U B; Szostak, M P; Resch, S; Lubitz, W

    2003-10-01

    The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression. PMID:14532068

  7. 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...

  8. Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites.

    PubMed

    Spiering, Martin J; Urban, Lori A; Nuss, Donald L; Gopalan, Vivek; Stoltzfus, Arlin; Eisenstein, Edward

    2011-04-01

    Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant. PMID:21188383

  9. 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

  10. Regulation of albumin gene expression in hepatoma cells of fetal phenotype: dominant inhibition of HNF1 function and role of ubiquitous transcription factors.

    PubMed Central

    Rollier, A; DiPersio, C M; Cereghini, S; Stevens, K; Tronche, F; Zaret, K; Weiss, M C

    1993-01-01

    Two widely used hepatoma cell lines, mouse BW1J and human HepG2, express gene products characteristic of fetal hepatocytes, including serum albumin, whereas reporter genes driven by the albumin promoter are expressed at very low levels compared with highly differentiated hepatoma cells. We have investigated the low albumin promoter activity in BW1J cells to understand differences in liver gene regulation between fetal and adult cells. Addition of the albumin upstream enhancer, or any other fragment of the albumin gene, failed to modify expression of the transfected promoter in BW1J cells. Analysis of cis elements of the albumin promoter showed that, in contrast to highly differentiated H4II cells, in BW1J cells the activity largely depends on ubiquitous transcription factors. Both BW1J and HepG2 cells produce the liver-enriched transcription factor HNF1; dimerization and DNA binding properties are identical to those of liver HNF1, yet the protein fails to show the anticipated transcriptional stimulatory activity. A transfected HNF1 expression vector strongly trans-activates the albumin promoter in HepG2 but only weakly in BW1J cells, and in hybrids (BW1J x Fao), inefficient HNF1 function is dominant. We conclude that hepatoma cells of the fetal phenotype are deficient in the use of HNF1 to drive transcription of the albumin gene and that they harbor a dominant modulator of HNF1 function. Images PMID:8443410

  11. Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance.

    PubMed Central

    Kaasen, I; Evensen, G; Seeberg, E

    1986-01-01

    We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II. The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes. High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA. Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair. Images PMID:3536857

  12. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  13. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum.

    PubMed

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport-NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885-were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  14. 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...

  15. Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

    PubMed

    Gilbert, R; Nalbantoglu, J; Howell, J M; Davies, L; Fletcher, S; Amalfitano, A; Petrof, B J; Kamen, A; Massie, B; Karpati, G

    2001-09-20

    Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer. PMID:11560768

  16. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  17. Association of adiponectin and adiponectin receptor genes with sow productivity estimated breeding values.

    PubMed

    Jafarikia, Moshen; Méthot, Steve; Maignel, Laurence; Fortin, Frédéric; Wyss, Stefanie; Sullivan, Brian; Palin, Marie-France

    2015-09-01

    Our objectives were to estimate frequencies of previously identified single nucleotide polymorphisms (SNPs) in adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) in a population of Duroc, Landrace and Yorkshire pigs and evaluate the effect of these alleles on sow productivity estimated breeding values (EBVs). Eight SNPs were genotyped on 446 pigs in the ADIPOQ (c.178G>A, c.*300A>G, c.*1094_1095insC and c.*1779A>C), ADIPOR1 (c.*129A>C) and ADIPOR2 (c.*112G>A, c.*295G>C and c.*1455G>A) genes. Association analyses were performed with sow productivity EBVs based on litter records collected in Canadian breeding farms. There were significant associations between ADIPOQ c.178G>A and c.*1094_1095insC SNPs and studied traits. However, none of these associations remained significant after applying a Bonferroni correction. The ADIPOR2 c.*112G>A SNP was associated with the total number of piglets born (TNB, P < 0.001) and litter weight at weaning (LWW, P < 0.001) EBVs. Associations were also observed between the ADIPOR2 [A;C;G] haplotype and TNB and LWW (P < 0.001). Our results demonstrate that a selection in favor of the c.*112G allele or against the [A;C;G] haplotype may have the potential to increase LWW EBVs. However, the c.*112G allele is also associated with lower TNB EBVs. Some of the alleles of the genes studied showed substantial variability and in general, the results corroborated previously reported findings for an independent sow population. However, careful cost-benefits analyses should be performed before using these markers in selection program as an improvement in TNB may translate into lighter LWW, with its associated negative impact on production traits such as growth performances. PMID:26210991

  18. A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

    PubMed Central

    Protasevich, Irina I.; Brouillette, Christie G.; Harrell, Patina M.; Hildebrandt, Ellen; Gasser, Brigitte; Mattanovich, Diethard; Ward, Andrew; Chang, Geoffrey; Urbatsch, Ina L.

    2011-01-01

    Background Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers. Methodology/Principal Findings Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from Tm ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids. Conclusion The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins. PMID:21826197

  19. Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

    PubMed Central

    Zivkovic, Milica; Miljkovic, Marija; Ruas-Madiedo, Patricia; Strahinic, Ivana; Tolinacki, Maja; Golic, Natasa

    2014-01-01

    Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11. PMID:25527533

  20. Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts.

    PubMed

    Harsch, Michael J; Gardner, Richard C

    2013-01-01

    Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice. PMID:22684328

  1. Trade-off between constitutive and inducible resistance against herbivores is only partially explained by gene expression and glucosinolate production

    PubMed Central

    Rasmann, Sergio; Chassin, Estelle; Bilat, Julia; Glauser, Gaétan; Reymond, Philippe

    2015-01-01

    The hypothesis that constitutive and inducible plant resistance against herbivores should trade-off because they use the same resources and impose costs to plant fitness has been postulated for a long time. Negative correlations between modes of deployment of resistance and defences have been observed across and within species in common garden experiments. It was therefore tested whether that pattern of resistance across genotypes follows a similar variation in patterns of gene expression and chemical defence production. Using the genetically tractable model Arabidopsis thaliana and different modes of induction, including the generalist herbivore Spodoptera littoralis, the specialist herbivore Pieris brassicae, and jasmonate application, constitutive and inducibility of resistance was measured across seven A. thaliana accessions that were previously selected based on constitutive levels of defence gene expression. According to theory, it was found that modes of resistance traded-off among accessions, particularly against S. littoralis, in which accessions investing in high constitutive resistance did not increase it substantially after attack and vice-versa. Accordingly, the average expression of eight genes involved in glucosinolate production negatively predicted larval growth across the seven accessions. Glucosinolate production and genes related to defence induction on healthy and herbivore-damaged plants were measured next. Surprisingly, only a partial correlation between glucosinolate production, gene expression, and the herbivore resistance results was found. These results suggest that the defence outcome of plants against herbivores goes beyond individual molecules or genes but stands on a complex network of interactions. PMID:25716695

  2. Trade-off between constitutive and inducible resistance against herbivores is only partially explained by gene expression and glucosinolate production.

    PubMed

    Rasmann, Sergio; Chassin, Estelle; Bilat, Julia; Glauser, Gaétan; Reymond, Philippe

    2015-05-01

    The hypothesis that constitutive and inducible plant resistance against herbivores should trade-off because they use the same resources and impose costs to plant fitness has been postulated for a long time. Negative correlations between modes of deployment of resistance and defences have been observed across and within species in common garden experiments. It was therefore tested whether that pattern of resistance across genotypes follows a similar variation in patterns of gene expression and chemical defence production. Using the genetically tractable model Arabidopsis thaliana and different modes of induction, including the generalist herbivore Spodoptera littoralis, the specialist herbivore Pieris brassicae, and jasmonate application, constitutive and inducibility of resistance was measured across seven A. thaliana accessions that were previously selected based on constitutive levels of defence gene expression. According to theory, it was found that modes of resistance traded-off among accessions, particularly against S. littoralis, in which accessions investing in high constitutive resistance did not increase it substantially after attack and vice-versa. Accordingly, the average expression of eight genes involved in glucosinolate production negatively predicted larval growth across the seven accessions. Glucosinolate production and genes related to defence induction on healthy and herbivore-damaged plants were measured next. Surprisingly, only a partial correlation between glucosinolate production, gene expression, and the herbivore resistance results was found. These results suggest that the defence outcome of plants against herbivores goes beyond individual molecules or genes but stands on a complex network of interactions. PMID:25716695

  3. Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

    PubMed

    Eakle, K A; Bernstein, M; Emr, S D

    1988-10-01

    SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind

  4. Lactobacillus reuteri-specific immunoregulatory gene rsiR modulates histamine production and immunomodulation by Lactobacillus reuteri.

    PubMed

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

    2013-12-01

    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

  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. Methyl jasmonate and miconazole differently affect arteminisin production and gene expression in Artemisia annua suspension cultures.

    PubMed

    Caretto, S; Quarta, A; Durante, M; Nisi, R; De Paolis, A; Blando, F; Mita, G

    2011-01-01

    Artemisia annua L. is a herb traditionally used for treatment of fevers. The glandular trichomes of this plant accumulate, although at low levels, artemisinin, which is highly effective against malaria. Due to the great importance of this compound, many efforts have been made to improve knowledge on artemisinin production both in plants and in cell cultures. In this study, A. annua suspension cultures were established in order to investigate the effects of methyl jasmonate (MeJA) and miconazole on artemisinin biosynthesis. Twenty-two micro molar MeJA induced a three-fold increase of artemisinin production in around 30 min; while 200 μm miconazole induced a 2.5-fold increase of artemisinin production after 24 h, but had severe effects on cell viability. The influence of these treatments on expression of biosynthetic genes was also investigated. MeJA induced up-regulation of CYP71AV1, while miconazole induced up-regulation of CPR and DBR2. PMID:21143725

  7. Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

    PubMed

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

    2015-07-01

    Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol. PMID:26002633

  8. 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

  9. Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product.

    PubMed

    Liu, Wenshuai; Zhang, Qinling; Guo, Jia; Chen, Zhi; Li, Jilun; Wen, Ying

    2015-08-01

    Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5-O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM. PMID:26002902

  10. Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product

    PubMed Central

    Liu, Wenshuai; Zhang, Qinling; Guo, Jia; Chen, Zhi; Li, Jilun

    2015-01-01

    Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5–O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM. PMID:26002902

  11. 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

  12. Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

    SciTech Connect

    Puddu, A.; Storace, D.; Odetti, P.; Viviani, G.L.

    2010-04-23

    Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic {beta}-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

  13. Gene Expression Profiles in a Rabbit Model of Systemic Lupus Erythematosus Autoantibody Production1

    PubMed Central

    Rai, Geeta; Ray, Satyajit; Milton, Jacqueline; Yang, Jun; Ren, Ping; Lempicki, Richard; Mage, Rose G.

    2010-01-01

    We previously reported the establishment of a rabbit (Oryctolagus cuniculus) model in which peptide immunization led to production of lupus-like autoantibodies including anti-Sm, -RNP, -SS-A, -SS-B and –dsDNA characteristic of those produced in Systemic Lupus Erythematosus (SLE) patients. Some neurological symptoms in form of seizures and nystagmus were observed. The animals used in the previous and in the present study were from a National Institute of Allergy and Infectious Diseases colony of rabbits that were pedigreed, immunoglobulin allotype-defined but not inbred. Their genetic heterogeneity may correspond to that found among patients of a given ethnicity. We extended the information about this rabbit model by microarray based expression profiling. We first demonstrated that human expression arrays could be used with rabbit RNA to yield information on molecular pathways. We then designed a study evaluating gene expression profiles in 8 groups of control and treated rabbits (47 rabbits in total). Genes significantly upregulated in treated rabbits were associated with NK cytotoxicity, antigen presentation, leukocyte migration, cytokine activity, protein kinases, RNA spliceosomal ribonucleoproteins, intracellular signaling cascades, and glutamate receptor activity. These results link increased immune activation with up-regulation of components associated with neurological and anti-RNP responses, demonstrating the utility of the rabbit model to uncover biological pathways related to SLE-induced clinical symptoms, including Neuropsychiatric Lupus. Our finding of distinct gene expression patterns in rabbits that made anti-dsDNA compared to those that only made other anti-nuclear antibodies should be further investigated in subsets of SLE patients with different autoantibody profiles. PMID:20817871

  14. Temperature influences β-carotene production in recombinant Saccharomyces cerevisiae expressing carotenogenic genes from Phaffia rhodozyma.

    PubMed

    Shi, Feng; Zhan, Wubing; Li, Yongfu; Wang, Xiaoyuan

    2014-01-01

    Red yeast Phaffia rhodozyma is a prominent microorganism able to synthesize carotenoid. Here, three carotenogenic cDNAs of P. rhodozyma CGMCC 2.1557, crtE, crtYB and crtI, were cloned and introduced into Saccharomyces cerevisiae INVSc1. The recombinant Sc-EYBI cells could synthesize 258.8 ± 43.8 μg g(-1) dry cell weight (DCW) of β-carotene when growing at 20 °C, about 59-fold higher than in those growing at 30 °C. Additional expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from S. cerevisiae (Sc-EYBIH) increased the β-carotene level to 528.8 ± 13.3 μg g(-1) DCW as cells growing at 20 °C, 27-fold higher than cells growing at 30 °C, although cells grew faster at 30 °C than at 20 °C. Consistent with the much higher β-carotene level in cells growing at 20 °C, transcription level of three crt genes and cHMG1 gene in cells growing at 20 °C was a little higher than in those growing at 30 °C. Meanwhile, expression of three carotenogenic genes and accumulation of β-carotene promoted cell growth. These results reveal the influence of temperature on β-carotene biosynthesis and may be helpful for improving β-carotene production in recombinant S. cerevisiae. PMID:23861041

  15. Genetic characterization of the homeodomain-independent activity of the Drosophila fushi tarazu gene product

    SciTech Connect

    Hyduk, D.; Percival-Smith, A.

    1996-02-01

    The gene products of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results. 42 refs., 8 figs., 5 tabs.

  16. The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo.

    PubMed Central

    Kalman, L V; Cheng, Y L; Kaiser, D

    1994-01-01

    The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. Anti-E. coli IF3 antibodies cross-react with the Dsg protein. Tn5 insertion mutations in dsg are lethal. When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells. Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further. The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis. The amount of IF3 protein in E. coli is known to be autoregulated at the translational level. This autoregulation is lost in an E. coli infC362 missense mutant. The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain. Dsg is distinguished from IF3 of E. coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids. Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability. Images PMID:8113185

  17. SOR1, a gene associated with bioherbicide production in sorghum root hairs.

    PubMed

    Yang, Xiaohan; Scheffler, Brian E; Weston, Leslie A

    2004-10-01

    Sorghum [Sorghum bicolor (L.) Moench] roots exude a potent bioherbicide known as sorgoleone, which is produced in living root hairs and is phytotoxic to broadleaf and grass weeds at concentrations as low as 10 microM. Differential gene expression was studied in sorghum (S. bicolorxS. sudanense) cv. SX17 between roots with abundant root hairs and those without root hairs using a modified differential display approach. A differentially expressed gene, named SOR1, was cloned by using Rapid Amplification of the 5' ends of cDNA (5'-RACE). Real-time PCR analysis of multiple tissues of sorghum SX17 revealed that the SOR1 transcript level in root hairs was more than 1000 times higher than that of other tissues evaluated, including immature leaf, mature leaf, mature stem, panicle, and roots with hairs removed. Semi-quantitative RT-PCR revealed that SOR1 was expressed in the sorgoleone-producing roots of sorghum SX17, shattercane [S. bicolor (L.) Moench], and johnsongrass [S. halepense (L.) Pers.], but not in the shoots of sorghum or in the roots of sweet corn (Zea mays L.) 'Summer Flavor 64Y', in which sorgoleone production was not detected by HPLC analysis. Similarity searches indicated that SOR1 probably encodes a novel desaturase, which might be involved in the formation of a unique and specific double bonding pattern within the long hydrocarbon tail of sorgoleone. PMID:15361534

  18. The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products.

    PubMed Central

    Becker, A; Rüberg, S; Küster, H; Roxlau, A A; Keller, M; Ivashina, T; Cheng, H P; Walker, G C; Pühler, A

    1997-01-01

    Proteins directing the biosynthesis of galactoglucan (exopolysaccharide II) in Rhizobium meliloti Rm2011 are encoded by the exp genes. Sequence analysis of a 32-kb DNA fragment of megaplasmid 2 containing the exp gene cluster identified previously (J. Glazebrook and G. C. Walker, Cell 56:661-672, 1989) revealed the presence of 25 open reading frames. Homologies of the deduced exp gene products to proteins of known function suggested that the exp genes encoded four proteins involved in the biosynthesis of dTDP-glucose and dTDP-rhamnose, six glycosyltransferases, an ABC transporter complex homologous to the subfamily of peptide and protein export complexes, and a protein homologous to Rhizobium NodO proteins. In addition, homologies of three Exp proteins to transcriptional regulators, methyltransferases, and periplasmic binding proteins were found. The positions of 26 Tn5 insertions in the exp gene cluster were determined, thus allowing the previously described genetic map to be correlated with the sequence. Operon analysis revealed that the exp gene cluster consists of five complementation groups. In comparison to the wild-type background, all exp complementation groups were transcribed at a substantially elevated level in the regulatory mucR mutant. PMID:9023225

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

    PubMed Central

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

    2005-01-01

    Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-­rays to at least 2.1 Å resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan. PMID:16508104

  20. Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli.

    PubMed Central

    Gibello, A; Ferrer, E; Sanz, J; Martin, M

    1995-01-01

    The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin. PMID:8534083

  1. The rolC gene increases caffeoylquinic acid production in transformed artichoke cells.

    PubMed

    Vereshchagina, Y V; Bulgakov, V P; Grigorchuk, V P; Rybin, V G; Veremeichik, G N; Tchernoded, G K; Gorpenchenko, T Y; Koren, O G; Phan, N H T; Minh, N T; Chau, L T; Zhuravlev, Y N

    2014-09-01

    Caffeoylquinic acids are found in artichokes, and they are currently considered important therapeutic or preventive agents for treating Alzheimer's disease and diabetes. We transformed artichoke [the cultivated cardoon or Cynara cardunculus var. altilis DC (Asteraceae)] with the rolC gene, which is a known inducer of secondary metabolism. High-performance liquid chromatography with UV and high-resolution mass spectrometry (HPLC-UV-HRMS) revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and chlorogenic acid. The rolC-transformed calli contained 1.5% caffeoylquinic acids by dry weight. The overall production of these metabolites was three times higher than that of the corresponding control calli. The enhancing effect of rolC remained stable over long-term cultivation. PMID:24938208

  2. Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli.

    PubMed

    Nielsen, Morten T; Madsen, Karina M; Seppälä, Susanna; Christensen, Ulla; Riisberg, Lone; Harrison, Scott J; Møller, Birger Lindberg; Nørholm, Morten H H

    2015-03-20

    Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering. PMID:24905856

  3. Characterization of the Escherichia coli F factor traY gene product and its binding sites.

    PubMed Central

    Nelson, W C; Morton, B S; Lahue, E E; Matson, S W

    1993-01-01

    The traY gene product (TraYp) from the Escherichia coli F factor has previously been purified and shown to bind a DNA fragment containing the F plasmid oriT region (E. E. Lahue and S. W. Matson, J. Bacteriol. 172:1385-1391, 1990). To determine the precise nucleotide sequence bound by TraYp, DNase I footprinting was performed. The TraYp-binding site is near, but not coincident with, the site that is nicked to initiate conjugative DNA transfer. In addition, a second TraYp binding site, which is coincident with the mRNA start site at the traYI promoter, is described. The Kd for each binding site was determined by a gel mobility shift assay. TraYp exhibits a fivefold higher affinity for the oriT binding site compared with the traYI promoter binding site. Hydrodynamic studies were performed to show that TraYp is a monomer in solution under the conditions used in DNA binding assays. Early genetic experiments implicated the traY gene product in the site- and strand-specific endonuclease activity that nicks at oriT (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980; S. McIntire and N. Willetts, Mol. Gen. Genet. 178:165-172, 1980). As this activity has recently been ascribed to helicase I, it was of interest to see whether TraYp had any effect on this reaction. Addition of TraYp to nicking reactions catalyzed by helicase I showed no effect on the rate or efficiency of oriT nicking. Roles for TraYp in conjugative DNA transfer and a possible mode of binding to DNA are discussed. Images PMID:8468282

  4. Ghrelin gene products and the regulation of food intake and gut motility.

    PubMed

    Chen, Chih-Yen; Asakawa, Akihiro; Fujimiya, Mineko; Lee, Shou-Dong; Inui, Akio

    2009-12-01

    A breakthrough using "reverse pharmacology" identified and characterized acyl ghrelin from the stomach as the endogenous cognate ligand for the growth hormone (GH) secretagogue receptor (GHS-R) 1a. The unique post-translational modification of O-n-octanoylation at serine 3 is the first in peptide discovery history and is essential for GH-releasing ability. Des-acyl ghrelin, lacking O-n-octanoylation at serine 3, is also produced in the stomach and remains the major molecular form secreted into the circulation. The third ghrelin gene product, obestatin, a novel 23-amino acid peptide identified from rat stomach, was found by comparative genomic analysis. Three ghrelin gene products actively participate in modulating appetite, adipogenesis, gut motility, glucose metabolism, cell proliferation, immune, sleep, memory, anxiety, cognition, and stress. Knockdown or knockout of acyl ghrelin and/or GHS-R1a, and overexpression of des-acyl ghrelin show benefits in the therapy of obesity and metabolic syndrome. By contrast, agonism of acyl ghrelin and/or GHS-R1a could combat human anorexia-cachexia, including anorexia nervosa, chronic heart failure, chronic obstructive pulmonary disease, liver cirrhosis, chronic kidney disease, burn, and postsurgery recovery, as well as restore gut dysmotility, such as diabetic or neurogenic gastroparesis, and postoperative ileus. The ghrelin acyl-modifying enzyme, ghrelin O-Acyltransferase (GOAT), which attaches octanoate to serine-3 of ghrelin, has been identified and characterized also from the stomach. To date, ghrelin is the only protein to be octanylated, and inhibition of GOAT may have effects only on the stomach and is unlikely to affect the synthesis of other proteins. GOAT may provide a critical molecular target in developing novel therapeutics for obesity and type 2 diabetes. PMID:20038570

  5. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression. PMID:27309759

  6. Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells

    PubMed Central

    Huang, Jing; Stohl, Lori L.; Zhou, Xi; Ding, Wanhong; Granstein, Richard D.

    2011-01-01

    This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP8-37 and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced IκBα degradation and NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-κB activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-κB pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation. PMID:21334428

  7. CONVECTION-ENHANCED DELIVERY AND SYSTEMIC MANNITOL INCREASE GENE PRODUCT DISTRIBUTION OF AAV VECTORS 5, 8, AND 9 AND INCREASE GENE PRODUCT IN THE ADULT MOUSE BRAIN

    PubMed Central

    Carty, Nikisha; Lee, Daniel; Dickey, Chad; Ceballos-Diaz, Carolina; Jansen-West, Karen; Golde, Todd E.; Gordon, Marcia N.; Morgan, Dave; Nash, Kevin

    2010-01-01

    The use of recombinant adeno-associated viral (rAAV) vectors as a means of gene delivery to the central nervous system has emerged as a potentially viable method for the treatment of several types of degenerative brain diseases. However, a limitation of typical intracranial injections into the adult brain parenchyma is the relatively restricted distribution of the delivered gene to large brain regions such as the cortex, presumably due to confined dispersion of the injected particles. Optimizing the administration techniques to maximize gene distribution and gene expression is an important step in developing gene therapy studies. Here, we have found additive increases in distribution when 3 methods to increase brain distribution of rAAV were combined. The convection enhanced delivery (CED) method with the step-design cannula was used to deliver rAAV vector serotypes 5, 8 and 9 encoding GFP into the hippocampus of the mouse brain. While the CED method improved distribution of all 3 serotypes, the combination of rAAV9 and CED was particularly effective. Systemic mannitol administration, which reduces intracranial pressure, also further expanded distribution of GFP expression, in particular, increased expression on the contralateral hippocampi. These data suggest that combining advanced injection techniques with newer rAAV serotypes greatly improves viral vector distribution, which could have significant benefits for implementation of gene therapy strategies. PMID:20951738

  8. Laccase Production and Differential Transcription of Laccase Genes in Cerrena sp. in Response to Metal Ions, Aromatic Compounds, and Nutrients.

    PubMed

    Yang, Jie; Wang, Guozeng; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

    2015-01-01

    Laccases can oxidize a wide range of aromatic compounds and are industrially valuable. Laccases often exist in gene families and may differ from each other in expression and function. Quantitative real-time polymerase chain reaction (qPCR) was used for transcription profiling of eight laccase genes in Cerrena sp. strain HYB07 with validated reference genes. A high laccase activity of 280.0 U/mL was obtained after submerged fermentation for 5 days. Laccase production and laccase gene transcription at different fermentation stages and in response to various environmental cues were revealed. HYB07 laccase activity correlated with transcription levels of its predominantly expressed laccase gene, Lac7. Cu(2+) ions were indispensable for efficient laccase production by HYB07, mainly through Lac7 transcription induction, and no aromatic compounds were needed. HYB07 laccase synthesis and biomass accumulation were highest with non-limiting carbon and nitrogen. Glycerol and inorganic nitrogen sources adversely impacted Lac7 transcription, laccase yields, and fungal growth. The present study would further our understanding of transcription regulation of laccase genes, which may in turn facilitate laccase production as well as elucidation of their physiological roles. PMID:26793186

  9. Laccase Production and Differential Transcription of Laccase Genes in Cerrena sp. in Response to Metal Ions, Aromatic Compounds, and Nutrients

    PubMed Central

    Yang, Jie; Wang, Guozeng; Ng, Tzi Bun; Lin, Juan; Ye, Xiuyun

    2016-01-01

    Laccases can oxidize a wide range of aromatic compounds and are industrially valuable. Laccases often exist in gene families and may differ from each other in expression and function. Quantitative real-time polymerase chain reaction (qPCR) was used for transcription profiling of eight laccase genes in Cerrena sp. strain HYB07 with validated reference genes. A high laccase activity of 280.0 U/mL was obtained after submerged fermentation for 5 days. Laccase production and laccase gene transcription at different fermentation stages and in response to various environmental cues were revealed. HYB07 laccase activity correlated with transcription levels of its predominantly expressed laccase gene, Lac7. Cu2+ ions were indispensable for efficient laccase production by HYB07, mainly through Lac7 transcription induction, and no aromatic compounds were needed. HYB07 laccase synthesis and biomass accumulation were highest with non-limiting carbon and nitrogen. Glycerol and inorganic nitrogen sources adversely impacted Lac7 transcription, laccase yields, and fungal growth. The present study would further our understanding of transcription regulation of laccase genes, which may in turn facilitate laccase production as well as elucidation of their physiological roles. PMID:26793186

  10. Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei

    PubMed Central

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

  11. Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

    PubMed

    Liu, Xiang-Lei; Lin, Jun; Hu, Hai-Feng; Zhou, Bin; Zhu, Bao-Quan

    2016-04-01

    Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3). PMID:27114316

  12. Co-ordinate regulation of herpes simplex virus gene expression is mediated by the functional interaction of two immediate early gene products.

    PubMed

    Gelman, I H; Silverstein, S

    1986-10-01

    At early times after infection with herpes simplex virus, transcription from beta-promoters is initiated only in the presence of a functional 174,000 Mr phosphoprotein (ICP4), encoded by an immediate early (alpha) gene (IE4). A transient expression assay was used to analyze the requirement for two (ICP4 and ICP0) of the five alpha-gene products in the transcriptional regulation of model alpha and beta-gene promoters. These studies reveal that cells cotransfected with plasmids containing the alpha-gene sequences for infected cell proteins (ICPs) 4 and 0 and a thymidine kinase (TK, a beta-gene) gene or the thymidine kinase promoter fused to a chloramphenicol acetyltransferase (CAT) cassette accumulate 10 to 20-fold more RNA or exhibit 10 to 20-fold more CAT activity than cells cotransfected with a plasmid encoding either alpha-gene protein and a thymidine kinase indicator gene. Functional ICP4 is required for enhanced transcriptional activation in the transient expression assay system. It is also required for the uniform dispersal of ICP0 throughout the nucleus as shown by immunofluorescence staining analysis of transfected cells. Two alpha-promoter-CAT fusions were used as targets to study what effects ICP4, ICP0 and Vmw65 (the virion-associated alpha-gene transactivator) have on expression from alpha-promoters that contain all of the sequences that confer alpha-gene regulation, or only the core sequence governing basal level expression. We conclude that ICP4 can activate alpha-gene expression from the core sequence and, depending on its abundance, activate or repress expression from a promoter containing the sequences required for alpha-gene regulation. Independent of these alpha-regulatory sequences cotransfection with low levels of sequences encoding both ICP0 and ICP4 activate expression. At higher ratios of effector (both ICP4 and ICP0) the target accumulation of CAT activity decreases. Although a ts allele of IE4 (cloned from the mutant virus tsK) does not

  13. Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume.

    PubMed

    Li, Penghui; Dong, Qiang; Ge, Shujun; He, Xianzhi; Verdier, Jerome; Li, Dongqin; Zhao, Jian

    2016-07-01

    MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying its promotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR promotes PA production by a direct repression of biosynthesis of isoflavones, the major flavonoids in legume, and by redirecting immediate precursors, such as anthocyanidins, flux into PA pathway. Ectopic expression of MtPAR repressed isoflavonoid production by directly binding and suppressing isoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up-regulated PA-specific genes and decreased the anthocyanin levels without altering the expression of anthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. MtPAR complemented PA-deficient phenotype of Arabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showed the direct interactions between MtPAR, MtTT8 and MtWD40-1 proteins from Medicago truncatula and Glycine max, to form a ternary complex to trans-activate PA-specific ANR gene. Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and whole plants only promoted the production of small amount of PAs, which was significantly enhanced by co-expression of MtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect between MtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requires more anthocyanidin precursors. This study provides new insights into the role and mechanism of MtPAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathways for a specific promotion of PA production. Based on this, a strategy by combining MtPAR and MtLAP1 co-expression to effectively improve metabolic engineering performance of PA production in legume forage was developed. PMID:26806316

  14. Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.

    PubMed Central

    Himmelfarb, H J; Maicas, E; Friesen, J D

    1985-01-01

    The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known. Images PMID:3887137

  15. Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

    PubMed Central

    2011-01-01

    Background The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions. Results Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of Bud31 and Hpr1 was found to lead to the increase of both ethanol yield and fermentation rate, while Pho85, Vrp1 and Ygl024w expression is required for maximal ethanol production in VHG fermentations. Five genes, Erg2, Prs3, Rav1, Rpb4 and Vma8, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate. Conclusions The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers. PMID:22152034

  16. Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: A new family of genes responsible for autoinducer production

    PubMed Central

    Surette, Michael G.; Miller, Melissa B.; Bassler, Bonnie L.

    1999-01-01

    In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor–autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5α does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxSV.h., luxSS.t., and luxSE.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5α can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5α luxSE.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxSE.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes. PMID:9990077

  17. Effect of inhibin gene immunization on antibody production and reproductive performance in Partridge Shank hens.

    PubMed

    Mao, Dagan; Bai, Wujiao; Hui, Fengming; Yang, Liguo; Cao, Shaoxian; Xu, Yinxue

    2016-04-01

    To investigate the effect of inhibin gene immunization on antibody production and reproductive performance in broiler breeder females, Partridge Shank hens aged 380 days were immunized with inhibin recombinant plasmid pcISI. One hundred and twenty hens were randomly assigned to four groups and treated intramuscularly with 25, 75, or 125 μg/300-μL inhibin recombinant plasmid pcISI (T1∼T3) or 300-μL saline as control (C), respectively. Booster immunization was given with the same dosage 20 days later. Blood and egg samples were collected to detect the antibody against inhibin by enzyme-linked immunosorbent assay and to evaluate egg performance. The ovaries were collected to classify the follicles and detect the FSH receptor (FSHR) messenger RNA (mRNA) expression by reverse transcription-PCR. The results showed that immunization against pcISI could elicit antibody against inhibin in both plasma and egg yolk compared with the control (P < 0.05), whereas booster immunization did not increase the antibody level in plasma. Vaccination promoted egg lay during the first 30 days after primary vaccination (P < 0.05) with no effect on egg quality and hatching rate. Immunization increased the amounts of dominant, small yellow and large white follicles in the ovary (P < 0.05). Reverse transcription-PCR results showed that immunization increased the FSHR mRNA in the large white follicles, whereas decreased the FSHR mRNA in the small yellow follicles (P < 0.05). In conclusion, inhibin vaccine pcISI can stimulate the production of antibody against inhibin as well as the follicle development and egg laying performance in Partridge Shank hens, which provides a good foundation for the application of inhibin DNA vaccine in avian production. PMID:26739531

  18. 1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon

    SciTech Connect

    I-Teh Tong; Hans H. Liao; Cameron, D.C. )

    1991-12-01

    The dha regulon in Klebsiella pneumoniae enables the organism to grown anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydoxyacetone and was screened for the production of 1, 3-PD. The cosmid pTC1 (42.5 kn total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycersol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1, 3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.

  19. DNA sequencing of the gene encoding a bacterial superantigen, Yersinia pseudotuberculosis-derived mitogen (YPM), and characterization of the gene product, cloned YPM

    SciTech Connect

    Miyoshi-Akiyama, Tohru; Kato, Hidehito; Uchiyama, Takehiko

    1995-05-15

    Previously, we found a novel bacterial superantigen from Yersinia pseudotuberculosis, designated Y. pseudotuberculosis-derived mitogen (YPM). In the present study, we analyzed the DNA sequence of the gene encoding YPM. The YPM gene was cloned into a plasmid vector pMW119 and expressed in Escherichia coli DH10B. Like the native YPM, the cloned YPM required the expression of MHC class II molecules on accessory cells in the induction of IL-2 production by human T cells. TCR-V{beta} repertoire of human T cells reactive with the cloned YPM was V{beta}3, V{beta}9, V{beta}13.1, and V{beta}13.2. This repertoire is the same as that of T cells reactive with the native YPM. These results indicate that the cloned YPM expressed in E. coli is identical to the native YPM. Sequencing of the YPM gene revealed that the gene contained an open reading frame of 456 base pairs encoding a precursor form of 151 amino acid residues with m.w. 16,679 that is processed into a mature form of 131 amino acid residues with m.w. 14,529. Homology analysis revealed that the homology of amino acid sequence is quite low among YPM and other well known bacterial superantigens. We designated the gene encoding YPM as ypm. 30 refs., 5 figs., 2 tabs.

  20. The Regulatory Pathway for Advanced Cell Therapy and Gene Therapy Products in Brazil: A Road to Be Built.

    PubMed

    de Freitas, Daniel Roberto Coradi

    2015-01-01

    The regulation of cell therapy and gene therapy products is a major challenge for the Brazilian state. From a legal point of view, the legislative apparatus, including constitutional, prohibits the marketing and patent of human substances. From the point of view of the organization of the state bureaucracy, the responsibilities for the regulation of research and application of these technologies in humans may involve up to four different institutions. The National Agency for Health Surveillance (ANVISA) has been the protagonist in structuring the regulation of cell therapy and gene therapy in Brazil, and steps have been taken to ensure quality of these products. However, obstacles such as the commercialization of these therapies and the need to determine whether these products will be regulated following the assumptions adopted in Brazil for drugs and biological products or for human blood and tissues still remain. PMID:26374221

  1. Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus

    PubMed Central

    Zheng, Zhuang-li; Qiu, Xue-hong

    2015-01-01

    A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris. PMID:25892913

  2. Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence.

    PubMed

    Kumar, Santosh; Mittal, Ekansh; Deore, Sapna; Kumar, Anil; Rahman, Aejazur; Krishnasastry, Musti V

    2015-01-01

    The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms. PMID:26347855

  3. Genistein production in rice seed via transformation with soybean IFS genes.

    PubMed

    Sohn, Soo-In; Kim, Yul-Ho; Kim, Sun-Lim; Lee, Jang-Yong; Oh, Young-Ju; Chung, Joo-Hee; Lee, Kyeong-Ryeol

    2014-03-01

    To produce genistein in rice, the isoflavone synthase (IFS) genes, SpdIFS1 and SpdIFS2 were cloned from the Korean soybean cultivar, Sinpaldalkong II as it has a higher genistein content than other soybean varieties. SpdIFS1 and SpdIFS2 show a 99.6% and 98.2% identity at the nucleotide level and 99.4% and 97.9% identity at the amino acid level, respectively, with IFS1 and IFS2 from soybean (GenBank accession Nos. AF195798 and AF195819). Plant expression vectors were constructed harboring SpdIFS1 or SpdIFS2 under the control of a rice globulin promoter that directs seed specific expression, and used to transform two rice varieties, Heugnam, a black rice, and Nakdong, a normal rice cultivar without anthocyanin pigment. Because naringenin, the substrate of SpdIFS1 and SpdIFS2, is on the anthocyanin biosynthesis pathway, the relative production rate of genistein was compared between SpdIFS-expressing transgenic Heugnam and Nakdong. Southern blot analysis of eight of the resulting transgenic rice plants revealed that the T0 plants had one to three copies of the SpdIFS1 or SpdIFS2 gene. The highest level of genistein content found in rice seeds was 103 μg/g. These levels were about 30-fold higher in our transgenic rice lines than the genistein aglycon content of a non-leguminous IFS-expressing transgenic tobacco petal, equaling about 12% of total genistein content of Sinpaldalkong II. There were no significant differences found between the genistein content in Heugnam and Nakdong transgenic rice plants. PMID:24467893

  4. [De novotranscriptomic analysis of Chlorella sorokiniana: Pathway description and gene discovery for lipid production ].

    PubMed

    Li, Lin; Wang, Qinhong; Yang, Hailin; Wang, Wu

    2014-09-01

    [ OBJECTIVE] The paucity of genomic information limits the metabolic engineering of non-model microalgae Chlorella sorokiniana. Our study aimed to elucidate the fatty acid, triacylglycerol and starch biosynthetic pathways in the microalgae C. sorokiniana based on de novo transcriptomic analysis. [METHODS] We cultured C. sorokiniana with different nitrogen concentrations (KNO3: 8g/L and 2g/L) , then sequenced the transcriptomeusing Illumina Hiseq2000 platform. We used Trinity to de novo assemble the reads so as to obtain transcripts, aligned all the transcripts with Nr database, UniProtKB/Swiss-Prot database and COG database to annotate the function and classify using BLASTx algorithm, and assigned the transcript with metabolic pathway by aligning with KEGG database. Then we used RSEM to calculate FPKM value, and used it for preliminary analysis of different gene expression in the related pathways. [RESULTS] Over 49M high quality raw reads were produced with the length of 100bp, We used Trinity to assembled these reads into 49885 transcripts with an N50 of 1941bp, ranging from 300bp to 14100bp. 26479 transcripts were annotated through BLASTx similarity search, 2357 transcripts were assigned with EC number, and 207 metabolic pathways were assigned in total. Based on these analyses, we reconstructed the fatty acids, triacylglycerol and starch biosynthetic pathways in C. sorokiniana. We also identified preliminarily different geneexpression in the pathways. [CONCLUSION] Using RNA-seq technology, we reconstructed the metabolic pathways involving in the fatty acid, triacylglycerol and starch biosynthesis in non-model microalgae C. sorokiniana without genomic data, which is consistent with those in model microalgae Chlamydomonas reinhardtii, and compared the gene expression level under different conditions. These information is very useful for the metabolic engineering of C. sorokiniana and other microalgae to enhance the production of lipids. PMID:25522590

  5. A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis.

    PubMed

    Krokida, Afrodite; Delis, Costas; Geisler, Katrin; Garagounis, Constantine; Tsikou, Daniela; Peña-Rodríguez, Luis M; Katsarou, Dimitra; Field, Ben; Osbourn, Anne E; Papadopoulou, Kalliope K

    2013-11-01

    Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development. PMID:23909862

  6. Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells

    PubMed Central

    Cavaletto, Noemi; Luganini, Anna

    2015-01-01

    ABSTRACT The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells. IMPORTANCE Human cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells, which represent

  7. The Natural Product Domain Seeker NaPDoS: A Phylogeny Based Bioinformatic Tool to Classify Secondary Metabolite Gene Diversity

    PubMed Central

    Ziemert, Nadine; Podell, Sheila; Penn, Kevin; Badger, Jonathan H.; Allen, Eric; Jensen, Paul R.

    2012-01-01

    New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry. PMID:22479523

  8. Wounding tomato fruit elicits ripening-stage specific changes in gene expression and production of volatile compounds

    PubMed Central

    Baldassarre, Valentina; Cabassi, Giovanni; Spadafora, Natasha D.; Aprile, Alessio; Müller, Carsten T.; Rogers, Hilary J.; Ferrante, Antonio

    2015-01-01

    Fleshy fruits develop from an unripe organ that needs to be protected from damage to a ripe organ that attracts frugivores for seed dispersal through production of volatile organic compounds (VOCs). Thus, different responses to wounding damage are predicted. The aim of this study was to discover whether wound-induced changes in the transcriptome and VOC production alter as tomato transitions from unripe to ripe. Transcript changes were analysed 3h post-wounding using microarray analysis in two commercial salad-tomato (Solanum lycopersicum L.) cultivars: Luna Rossa and AVG, chosen for their high aroma production. This was followed by quantitative PCR on Luna Rossa genes involved in VOC biosynthesis and defence responses. VOCs elicited by wounding at different ripening stages were analysed by solid phase micro extraction and gas chromatography–mass spectrometry. Approximately 4000 differentially expressed genes were identified in the cultivar AVG and 2500 in Luna Rossa. In both cultivars the majority of genes were up-regulated and the most affected pathways were metabolism of terpenes, carotenoids, and lipids. Defence-related genes were mostly up-regulated in immature stages of development, whereas expression of genes related to VOCs changed at riper stages. More than 40 VOCs were detected and profiles changed with ripening stage. Thus, both transcriptome and VOC profiles elicited by wounding depend on stage of ripening, indicating a shift from defence to attraction. PMID:25614658

  9. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    SciTech Connect

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  10. Dynamic Linkages between Denitrification Functional Genes/Enzymes and Biogeochemical Reaction Rates of Nitrate and Its Reduction Products

    NASA Astrophysics Data System (ADS)

    Li, M.; Shi, L.; Qian, W.; Gao, Y.; Liu, Y.; Liu, C.

    2015-12-01

    Denitrification is a respiratory process in which oxidized nitrogen compounds are used as alternative electron acceptors for energy production when oxygen is limited. Denitrification is an important process that not only accounts for the significant loss of nitrogen fertilizers from soils but also leads to NO, N2O and CO2 emissions, which are important greenhouse gas species. In this study, denitrification was investigated in Columbia River sediments, focusing on the dynamic linkages between functional genes/enzymes and biogeochemical reaction rates of nitrate and its reduction products. NO3-, NO2- and N2O were assayed in different incubation time. DNA was extracted from the sediments and functional genes were quantified as a function of time during the denitrification. Functional enzymes were extracted from the sediments and measured using a newly developed, targeted protein method. The biogeochemical, functional gene, and enzyme data were collectively used to establish the dynamic correlation of functional genes/enzymes and biogeochemical reaction rates. The results provide fundamental insights regarding the dynamic regulation of functional genes and enzymes in the processes of denitrification and greenhouse gas production, and also provide experimental data critical for the development of biogeochemical reaction models that incorporate genome-scale insights and describe macroscopic biogeochemical reaction rates in ecosystems.

  11. Study of gene expression and OTA production by Penicillium nordicum during a small-scale seasoning process of salami.

    PubMed

    Ferrara, Massimo; Magistà, Donato; Epifani, Filomena; Cervellieri, Salvatore; Lippolis, Vincenzo; Gallo, Antonia; Perrone, Giancarlo; Susca, Antonia

    2016-06-16

    Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of protein rich food with elevated NaCl. It is usually found on dry-cured meat products and is considered the main species responsible for their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN) involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30days. The expression of otapksPN gene was already detected after 4days and increased significantly after 7days of seasoning, reaching the maximum expression level after 10days (1.69×10(4)copies/100mg). Consistently with gene expression monitoring, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10days. In the late stages of the seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30days. PMID:27060649

  12. Inhibition of human immunodeficiency virus type 1 Tat-dependent activation of translation in Xenopus oocytes by the benzodiazepine Ro24-7429 requires trans-activation response element loop sequences.

    PubMed

    Braddock, M; Cannon, P; Muckenthaler, M; Kingsman, A J; Kingsman, S M

    1994-01-01

    Two benzodiazepine compounds, [7-chloro-5-(2-pyrryl)-3H-1,4 benzodiazapin-2-(H)-one] (Ro5-3335) and [7-chloro-5-(1H-pyrrol-2-yl)-3H-benzo[e] [1,4] diazepin-2-yl]- methylamine (Ro24-7429), inhibit human immunodeficiency virus type 1 (HIV-1) replication via a specific effect on the function of the transactivator protein, Tat. To gain further insight into the mechanism of action of these compounds, we have tested their effects in an alternative assay for Tat activation in Xenopus oocytes. In this system, translation of trans-activation response element (TAR)-containing RNA is activated by Tat. Both compounds specifically blocked activation of translation in a dose-dependent fashion, with Ro24-7429 showing the greater potency. In the Xenopus oocyte system, as in mammalian cells, mutation of the TAR loop sequences abolishes Tat action. However, it is possible to obtain TAR-specific, Tat-dependent activation of a target RNA with a mutation in the loop provided that this target is in large excess. This result has been interpreted as indicating that a negative factor has been titrated (M. Braddock, R. Powell, A.D. Blanchard, A.J. Kingsman, and S.M. Kingsman, FASEB J. 7:214-222, 1993). Interestingly Ro24-7429 was unable to inhibit the TAR-specific but loop sequence-independent mode of translational activation. This finding suggests that a specific loop-binding cellular factor may mediate the effects of this inhibitor of Tat action. Consistent with this notion, we could not detect any effect of Ro24-7429 on the efficiency of specific Tat binding to TAR in vitro. PMID:8254735

  13. 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

  14. Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates.

    PubMed

    Mienda, Bashir Sajo; Shamsir, Mohd Shahir; Illias, Rosli Md

    2016-04-01

    The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35gl(-1) and 1.40gl(-1) from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals. PMID:26878126

  15. A Post-GWAS Replication Study Confirming the PTK2 Gene Associated with Milk Production Traits in Chinese Holstein

    PubMed Central

    Liu, Xuan; Yang, Jie; Wei, Julong; Xu, Jingen; Zhang, Qin; Liu, Jian-Feng

    2013-01-01

    Our initial genome-wide association study (GWAS) demonstrated that two SNPs (ARS-BFGL-NGS-33248, UA-IFASA-9288) within the protein tyrosine kinase 2 (PTK2) gene were significantly associated with milk production traits in Chinese Holstein dairy cattle. To further validate if the statistical evidence provided in GWAS were true-positive findings, a replication study was performed herein through genotype-phenotype associations. The two tested SNPs were found to show significant associations with milk production traits, which confirmed the associations observed in the original study. Specifically, SNPs lying in the PTK2 gene were also detected by sequencing 14 unrelated sires in Chinese Holsteins and a total of thirty-three novel SNPs were identified. Thirteen out of these identified SNPs were genotyped and tested for association with milk production traits in an independent resource population. After Bonferroni correction for multiple testing, twelve SNPs were statistically significant for more than two milk production traits. Analyses of pairwise D’ measures of linkage disequilibrium (LD) between all SNPs were also explored. Two haplotype blocks were inferred and the association study at haplotype level revealed similar effects on milk production traits. In addition, the RNA expression analyses revealed that a non-synonymous coding SNP (g.4061098T>G) was involved in the regulation of gene expression. Thus the findings presented here provide strong evidence for associations of PTK2 variants with dairy production traits and may be applied in Chinese Holstein breeding program. PMID:24386238

  16. Pleiotropic derepression of developmentally regulated cellular and viral genes by c-myc protooncogene products in undifferentiated embryonal carcinoma cells.

    PubMed Central

    Onclercq, R; Lavenu, A; Cremisi, C

    1989-01-01

    We show here in mouse embryonal carcinoma (EC) cells that the endo A gene is negatively regulated and shares negative transacting factors with the Py and SV40 viruses. The products of the proto-oncogene c-myc derepress at the transcriptional level the appropriately initiated expression of the endo A gene and activate the Py early promoter in EC stem cells. C-myc products also activate the endo A and the Py early promoters in TDM epithelial cells, and the Py early promoter in 3T6 cells in which the two genes are already expressed or can be expressed. Furthermore we show that the myc exon 1 is essential for activation and that this activation might be mediated by AP1 family factors. Images PMID:2536923

  17. Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning.

    PubMed

    Coren, Lori V; Jain, Sumiti; Trivett, Matthew T; Ohlen, Claes; Ott, David E

    2015-03-01

    Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins. PMID:25757546

  18. Molecular Networking and Pattern-Based Genome Mining Improves discovery of biosynthetic gene clusters and their products from Salinispora species

    PubMed Central

    Duncan, Katherine R.; Crüsemann, Max; Lechner, Anna; Sarkar, Anindita; Li, Jie; Ziemert, Nadine; Wang, Mingxun; Bandeira, Nuno; Moore, Bradley S.; Dorrestein, Pieter C.; Jensen, Paul R.

    2015-01-01

    Summary Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. Here we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated the identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. These efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches. PMID:25865308

  19. Ethanol production by recombinant Escherichia coli carrying genes from Zymomonas mobilis.

    PubMed

    Lawford, H G; Rousseau, J D

    1991-01-01

    Efficient utilization of lignocellulosic feedstocks offers an opportunity to reduce the cost of producing fuel ethanol. The fermentation performance characteristics of recombinant Escherichia coli ATCC 11303 carrying the "PET plasmid" (pLOI297) with the lac operon controlling the expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes cloned from Zymomonas mobilis CP4 (Alterthum & Ingram, 1989) were assessed in batch and continuous processes with sugar mixtures designed to mimic process streams from lignocellulosic hydrolysis systems. Growth was pseudoexponential at a rate (generation time) of 1.28 h at pH 6.8 and 1.61 h at pH 6.0. The molar growth yields for glucose and xylose were 17.28 and 7.65 g DW cell/mol, respectively (at pH 6.3 and 30 degrees C), suggesting that the net yield of ATP from xylose metabolism is only 50% compared to glucose. In pH-stat batch fermentations (Luria broth with 6% sugar, pH 6.3), glucose was converted to ethanol 4-6 times faster than xylose, but the glucose conversion rate was much less than can be achieved with comparable cell densities of Zymomonas. Sugar-to-ethanol conversion efficiencies in nutrient-rich, complex LB medium were near theoretical at 98 and 88% for glucose and xylose, respectively. The yield was 10-20% less in a defined-mineral-salts medium. Acetate at a concentration of 0.1M (present in lignocellulosic hydrolysates from thermochemical processing) inhibited glucose utilization (about 50%) much more than xylose, and caused a decrease in product yield of about 30% for both sugars. With phosphate-buffered media (pH 7), glucose was a preferred substrate in mixtures with a ratio of hexose to pentose of 2.3 to 1. Xylose was consumed after glucose, and the product yield was less (0.37 g/g). Under steady-state conditions of continuous culture, the specific productivity ranged from 0.76-1.24 g EtOH/g cell/h, and the maximum volumetric productivity, 2.5 g EtOH/L/h, was achieved with a rich

  20. Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

    PubMed

    Li, Huan-Jun; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2016-01-10

    Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation. PMID:26603122

  1. Elucidation of veA Dependent Genes Associated with Aflatoxin and Sclerotial Production in Aspergillus flavus by Functional Genomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin, as well as sclerotial formation. We used microarray tech...

  2. Photorepair of ultraviolet-induced petite mutational damage in Saccharomyces cerevisiae requires the product of the PHR1 gene

    SciTech Connect

    Green, G.; MacQuillan, A.M.

    1980-11-01

    A wild-type (phr/sup +/) diploid yeast strain showed photorepair of petite mutational damage, whereas a photoreactivation-deficient (phr1/phr1) diploid strain did not, indicating that the PHR1 gene product was required for mitochondrial photorepair.

  3. Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

    PubMed

    Brueggeman, Andrew J; Bruggeman, Andrew J; Kuehler, Daniel; Weeks, Donald P

    2014-09-01

    Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides. PMID:24796724

  4. Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

    PubMed Central

    2013-01-01

    Background Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important

  5. Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

    PubMed

    Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

    2016-09-01

    Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. PMID:27156136

  6. Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle

    PubMed Central

    2013-01-01

    Background Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low-heritability traits. Semen from 550 Holstein bulls of high (≥ 1.7; n = 288) or low (≤ −2; n = 262) daughter pregnancy rate (DPR) was genotyped for 434 candidate SNPs using the Sequenom MassARRAY® system. Three types of SNPs were evaluated: SNPs previously reported to be associated with reproductive traits or physically close to genetic markers for reproduction, SNPs in genes that are well known to be involved in reproductive processes, and SNPs in genes that are differentially expressed between physiological conditions in a variety of tissues associated in reproductive function. Eleven reproduction and production traits were analyzed. Results A total of 40 SNPs were associated (P < 0.05) with DPR. Among these were genes involved in the endocrine system, cell signaling, immune function and inhibition of apoptosis. A total of 10 genes were regulated by estradiol. In addition, 22 SNPs were associated with heifer conception rate, 33 with cow conception rate, 36 with productive life, 34 with net merit, 23 with milk yield, 19 with fat yield, 13 with fat percent, 19 with protein yield, 22 with protein percent, and 13 with somatic cell score. The allele substitution effect for SNPs associated with heifer conception rate, cow conception rate, productive life and net merit were in the same direction as for DPR. Allele substitution effects for several SNPs associated with production traits were in the opposite direction as DPR. Nonetheless, there were 29 SNPs associated with DPR that were not negatively associated with production traits. Conclusion SNPs in a total of 40 genes associated with DPR were identified as well as SNPs for other traits. It might be feasible to include these SNPs into genomic tests of reproduction and other traits. The genes associated with DPR are likely to be

  7. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities.

    PubMed

    Dupont, Chris L; McCrow, John P; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L; Bai, Jing; Johnson, Zackary I; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A; Venter, J Craig; Allen, Andrew E

    2015-05-01

    Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM. PMID:25333462

  8. Tissue transglutaminase-dependent posttranslational modification of the retinoblastoma gene product in promonocytic cells undergoing apoptosis.

    PubMed Central

    Oliverio, S; Amendola, A; Di Sano, F; Farrace, M G; Fesus, L; Nemes, Z; Piredda, L; Spinedi, A; Piacentini, M

    1997-01-01

    The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program. PMID:9315663

  9. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    PubMed Central

    Dupont, Chris L; McCrow, John P; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L; Bai, Jing; Johnson, Zackary I; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A; Craig Venter, J; Allen, Andrew E

    2015-01-01

    Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM. PMID:25333462

  10. Characterization of caprine herpesvirus 1 glycoprotein D gene and its translation product.

    PubMed

    Keuser, Véronique; Detry, Bruno; Thiry, Julien; de Fays, Katalin; Schynts, Frédéric; Pastoret, Paul-Pierre; Vanderplasschen, Alain; Thiry, Etienne

    2006-02-01

    Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1. PMID:16140410

  11. Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin.

    PubMed

    Wang, L; Hilliges, M; Jernberg, T; Wiegleb-Edström, D; Johansson, O

    1990-07-01

    Sections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis. PMID:2143435

  12. Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production

    PubMed Central

    Welander, Paula V.; Summons, Roger E.

    2012-01-01

    Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group. PMID:22826256

  13. Association between ACR1 gene product expression and cardiomyopathy in children

    PubMed Central

    Wang, Yan; Niu, Ling; He, Xiuhua; Xue, Ying; Ling, Nan; Wang, Zhenzhou; An, Xinjiang

    2016-01-01

    Cardiomyopathy is a heterogeneous heart disease. Although morbidity of pediatric cardiomyopathy has been on the increase, effective treatments have not been identified. The aim of the study was to examine the expression of ACR1 gene products in association with cardiomyopathy in children. In total, 73 patients and 76 healthy subjects were enrolled in the study, from April, 2013 to April, 2015. The relative expression of ACR1 mRNA and protein were quantified in all cases, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), ELISA and western blot analysis. Immunohistochemistry was used to stain cardiac tissue samples to reveal differences between the patients and the control group. The results showed that the level of ACR1 mRNA by RT-qPCR was not different between the two study groups. However, ELISA and western blot analysis showed a significant difference, with patients expressing lower levels of ACR1. Additionally, immunohistochemistry revealed the levels of ACR1 were reduced in patients as the time course of disease increased. Thus, there is an association between the inhibition of ACR1 expression and the development of the disease. These findings are useful in the elucidation of the pathogenesis of pediatric cardiomyopathy, a severe disease with few effective treatment options available. PMID:27588091

  14. Production of L-DOPA and dopamine in recombinant bacteria bearing the Vitreoscilla hemoglobin gene.

    PubMed

    Kurt, Asli Giray; Aytan, Emel; Ozer, Ufuk; Ates, Burhan; Geckil, Hikmet

    2009-07-01

    Given the well-established beneficial effects of Vitreoscilla hemoglobin (VHb) on heterologous organisms, the potential of this protein for the production of L-DOPA and dopamine in two bacteria, Citrobacter freundii and Erwinia herbicola, was investigated. The constructed recombinants bearing the VHb gene (vgb(+)) had substantially higher levels of cytoplasmic L-DOPA (112 mg/L for C. freundii and 97 mg/L for E. herbicola) than their respective hosts (30.4 and 33.8 mg/L) and the vgb(-) control strains (35.6 and 35.8 mg/L). Further, the vgb(+) recombinants of C. freundii and E. herbicola had 20-fold and about two orders of magnitude higher dopamine levels than their hosts, repectively. The activity of tyrosine phenol-lyase, the enzyme converting L-tyrosine to L-DOPA, was well-correlated to cytoplasmic L-DOPA levels. As cultures aged, higher tyrosine phenol-lyase activity of the vgb(+) strains was more apparent. PMID:19585534

  15. Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production.

    PubMed

    Welander, Paula V; Summons, Roger E

    2012-08-01

    Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group. PMID:22826256

  16. Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production

    NASA Astrophysics Data System (ADS)

    Welander, Paula V.; Summons, Roger E.

    2012-08-01

    Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.

  17. Cardiovascular actions of DOPA mediated by the gene product of ocular albinism 1.

    PubMed

    Goshima, Yoshio; Nakamura, Fumio; Masukawa, Daiki; Chen, Sandy; Koga, Motokazu

    2014-01-01

    l-3,4-Dihydroxyphenylalanine (DOPA) is the metabolic precursor of dopamine, and the single most effective agent in the treatment of Parkinson's disease. One problem with DOPA therapy for Parkinson's disease is its cardiovascular side effects including hypotension and syncope, the underlying mechanisms of which are largely unknown. We proposed that DOPA is a neurotransmitter in the central nervous system, but specific receptors for DOPA had not been identified. Recently, the gene product of ocular albinism 1 (OA1) was shown to possess DOPA-binding activity. It was unknown, however, whether or not OA1 is responsible for the actions of DOPA itself. Immunohistochemical examination revealed that OA1 was expressed in the nucleus tractus solitarii (NTS). OA1-positive cells adjacent to tyrosine hydroxylase-positive cell bodies and nerve fibers were detected in the depressor sites of the NTS. OA1 knockdown using oa1-specific shRNA-adenovirus vectors in the NTS reduced the expression levels of OA1 in the NTS. The prior injection of the shRNA against OA1 suppressed the depressor and bradycardic responses to DOPA but not to glutamate in the NTS of anesthetized rats. Thus OA-1 is a functional receptor of DOPA in the NTS, which warrants reexamination of the mechanisms for the therapeutic and untoward actions of DOPA. PMID:25185585

  18. Effects of intronic single nucleotide polymorphisms (iSNPs) of a polysialyltransferase, ST8SIA2 gene found in psychiatric disorders on its gene products.

    PubMed

    Hane, Masaya; Kitajima, Ken; Sato, Chihiro

    2016-09-23

    Polysialic acid (polySia) is a linear homopolymer of sialic acid and mainly modifies neural cell adhesion molecule. PolySia plays important roles in synapse formation, learning and memory, social behavior and is associated with several diseases. Gene analyses of one of the biosynthetic enzymes for polySia, ST8SIA2, have revealed that several SNPs and genetic variations in the ST8SIA2 gene are associated with several psychiatric disorders; however, the mechanisms underlying these associations remain unknown. Here, we analyzed the effects of two iSNPs of ST8SIA2, rs2168351 and rs3784730, which are associated with bipolar disorder and autism spectrum disorder, respectively, on the expression of mRNA, ST8SIA2 and its final product, polySia in mouse neuroblastoma and human adenocarcinoma cell lines. We found that both iSNPs affected the expression of pre-mRNA and mRNA of ST8SIA2, and altered the cellular levels of ST8SIA2 and polySia. Taken together, these results indicate that impairment of the regulated expression of ST8SIA2 and the resulting downstream effects on gene products by these two iSNPs contribute to the development of these psychiatric disorders. PMID:27565727

  19. Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

    PubMed Central

    2010-01-01

    In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications. PMID:21054894

  20. Detection and diversity evaluation of tetracycline resistance genes in grassland-based production systems in Colombia, South america.

    PubMed

    Santamaría, Johanna; López, Liliana; Soto, Carlos Yesid

    2011-01-01

    Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals' diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE-PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment. PMID:22174707

  1. Detection and Diversity Evaluation of Tetracycline Resistance Genes in Grassland-Based Production Systems in Colombia, South America

    PubMed Central

    Santamaría, Johanna; López, Liliana; Soto, Carlos Yesid

    2011-01-01

    Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals’ diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE–PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment. PMID:22174707

  2. Photosynthetic electron transport controls nitrogen assimilation in cyanobacteria by means of posttranslational modification of the glnB gene product.

    PubMed Central

    Tsinoremas, N F; Castets, A M; Harrison, M A; Allen, J F; Tandeau de Marsac, N

    1991-01-01

    A glnB gene is identified in the cyanobacterium Synechococcus sp. PCC 7942, and its gene product is found to be covalently modified as a result of imbalance in electron transfer in photosynthesis, where photosystem II is favored over photosystem I. The gene was cloned and sequenced and found to encode a polypeptide of 112 amino acid residues, whose sequence shows a high degree of similarity to the Escherichia coli regulatory protein, PII. In E. coli, PII is involved in signal transduction in transcriptional and post-translational regulation of nitrogen assimilation. Increase in ammonium ion concentration is shown to decrease covalent modification of the Synechococcus PII protein, as in enteric bacteria. We therefore propose that the photosynthetic electron transport chain may regulate the pathway of nitrogen assimilation in cyanobacteria by means of posttranslational, covalent modification of the glnB gene product. The existence of the glnB gene in different strains of cyanobacteria is demonstrated and its implications are discussed. Images PMID:1905010

  3. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example

    PubMed Central

    Taniguchi, Hironori; Wendisch, Volker F.

    2015-01-01

    Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. PMID:26257719

  4. SxtA and sxtG Gene Expression and Toxin Production in the Mediterranean Alexandrium minutum (Dinophyceae)

    PubMed Central

    Perini, Federico; Galluzzi, Luca; Dell’Aversano, Carmela; Dello Iacovo, Emma; Tartaglione, Luciana; Ricci, Fabio; Forino, Martino; Ciminiello, Patrizia; Penna, Antonella

    2014-01-01

    The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions. PMID:25341029

  5. Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila.

    PubMed

    Hirth, F; Loop, T; Egger, B; Miller, D F; Kaufman, T C; Reichert, H

    2001-12-01

    Hox genes encode evolutionarily conserved transcription factors involved in the specification of segmental identity during embryonic development. This specification of identity is thought to be directed by differential Hox gene action, based on differential spatiotemporal expression patterns, protein sequence differences, interactions with co-factors and regulation of specific downstream genes. During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere; in the absence of labial, the cells in this brain region do not acquire a neuronal identity and major axonal pathfinding deficits result. We have used genetic rescue experiments to investigate the functional equivalence of the Drosophila Hox gene products in the specification of the tritocerebral neuromere. Using the Gal4-UAS system, we first demonstrate that the labial mutant brain phenotype can be rescued by targeted expression of the Labial protein under the control of CNS-specific labial regulatory elements. We then show that under the control of these CNS-specific regulatory elements, all other Drosophila Hox gene products, except Abdominal-B, are able to efficiently replace Labial in the specification of the tritocerebral neuromere. We also observe a correlation between the rescue efficiency of the Hox proteins and the chromosomal arrangement of their encoding loci. Our results indicate that, despite considerably diverged sequences, most Hox proteins are functionally equivalent in their ability to replace Labial in the specification of neuronal identity. This suggests that in embryonic brain development, differences in Hox gene action rely mainly on cis-acting regulatory elements and not on Hox protein specificity. PMID:11731458

  6. High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

    PubMed Central

    Catone, Mariela V.; Ruiz, Jimena A.; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I.

    2014-01-01

    Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases. PMID:24887088

  7. High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

    PubMed

    Catone, Mariela V; Ruiz, Jimena A; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I

    2014-01-01

    Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases. PMID:24887088

  8. LmTDRM database: a comprehensive database on thiol metabolic gene/gene products in Listeria monocytogenes EGDe.

    PubMed

    Srinivas, Vanishree; Gopal, Shubha

    2014-01-01

    There are a number of databases on the Listeria species and about their genome. However, these databases do not specifically address a set of network that is important in defence mechanism of the bacteria. Listeria monocytogenes EGDe is a well-established intracellular model organism to study host pathogenicity because of its versatility in the host environment. Here, we have focused on thiol disulphide redox metabolic network proteins, specifically in L. monocytogenes EGDe. The thiol redox metabolism is involved in oxidative stress mechanism and is found in all living cells. It functions to maintain the thiol disulphide balance required for protein folding by providing reducing power. Nevertheless, they are involved in the reversible oxidation of thiol groups in biomolecules by creating disulphide bonds; therefore, the term thiol disulphide redox metabolism (TDRM). TDRM network genes play an important role in oxidative stress mechanism and during host–pathogen interaction. Therefore, it is essential to have detailed information on these proteins with regard to other bacteria and its genome analysis to understand the presence of tRNA, transposons, and insertion elements for horizontal gene transfer. LmTDRM database is a new comprehensive web-based database on thiol proteins and their functions. It includes: Description, Search, TDRM analysis, and genome viewer. The quality of these data has been evaluated before they were aggregated to produce a final representation. The web interface allows for various queries to understand the protein function and their annotation with respect to their relationship with other bacteria. LmTDRM is a major step towards the development of databases on thiol disulphide redox proteins; it would definitely help researchers to understand the mechanism of these proteins and their interaction. Database URL: www.lmtdrm.com. PMID:25228549

  9. Cloning and expression analyses of interferon regulatory factor (IRF) 3 and 7 genes in European eel, Anguilla anguilla with the identification of genes involved in IFN production.

    PubMed

    Huang, Bei; Huang, Wen Shu; Nie, P

    2014-04-01

    Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel. PMID:24565894

  10. Reference genes selection and relative expression analysis from Shiraia sp. SUPER-H168 productive of hypocrellin.

    PubMed

    Deng, Huaxiang; Gao, Ruijie; Liao, Xiangru; Cai, Yujie

    2016-04-10

    Shiraia bambusicola is an essential pharmaceutical fungus due to its production of hypocrellin with antiviral, antidepressant, and antiretroviral properties. Based on suitable reference gene (RG) normalization, gene expression analysis enables the exploitation of significant genes relative to hypocrellin biosynthesis by quantitative real-time polymerase chain reaction. We selected and assessed nine candidate RGs in the presence and absence of hypocrellin biosynthesis using GeNorm and NormFinder algorithms. After stepwise exclusion of unstable genes, GeNorm analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytochrome oxidase (CyO) as the most stable expression, while NormFinder determined 18S ribosomal RNA (18S rRNA) as the most appropriate candidate gene for normalization. Tubulin (Tub) was observed to be the least stable gene and should be avoided for relative expression analysis. We further analyzed relative expression levels of essential proteins correlative with hypocrellin biosynthesis, including polyketide synthase (PKS), O-methyltransferase (Omef), FAD/FMN-dependent oxidoreductase (FAD), and monooxygenase (Mono). Compared to PKS, Mono kept a similar expression pattern and simulated PKS expression, while FAD remained constantly expressed. Omef presented lower transcript levels and had no relation to PKS expression. These relative expression analyses will pave the way for further interpretation of the hypocrellin biosynthesis pathway. PMID:26779826

  11. Overexpression of aflR Leads to Upregulation of Pathway Gene Transcription and Increased Aflatoxin Production in Aspergillus flavus

    PubMed Central

    Flaherty, J. E.; Payne, G. A.

    1997-01-01

    The aflatoxin biosynthetic pathway regulatory gene, aflR, encodes a putative 47-kDa protein containing a zinc cluster DNA binding motif. It is required for the transcription of all of the characterized aflatoxin pathway genes in both Aspergillus flavus and Aspergillus parasiticus. The objective of this study was to examine the effects of aflR overexpression on temporal gene expression, aflatoxin production, and nitrate inhibition of aflatoxin biosynthesis in A. flavus. An inducible expression construct was made by fusing the coding region of aflR to the promoter region of the A. flavus adh1 gene. This construct was transformed into A. flavus 656-2 (FGSC A1010), a strain mutated at the aflR locus. Strain 656-2 containing the adh1(p)::aflR construct had induced transcription of two early aflatoxin pathway genes, nor-1 and pksA, and produced wild-type concentrations of aflatoxin in a temporal pattern similar to that of wild-type strains of A. flavus. Strains 656-2 and 86-10 (FGSC A1009) an aflatoxigenic strain, were transformed with a construct containing the constitutive promoter gpdA driving aflR. Transformants of these strains constitutively expressed aflR, fas-1A, pksA, nor-1, and omtA but did not constitutively produce aflatoxin. Strain 86-10 containing the gpdA(p)::aflR construct produced 50 times more aflatoxin than 86-10, but the temporal pattern of aflatoxin production was the same as for 86-10, and aflatoxin production was also induced by sucrose. The addition of 10 g of nitrate per liter to sucrose low salts medium inhibited aflatoxin production by both strain 86-10 and a transformant of 86-10 containing the gpdA(p)::aflR construct, indicating that nitrate inhibition of aflatoxin biosynthesis does not occur solely at the level of aflR transcription. These studies show that constitutive overexpression of the pathway transcriptional regulatory gene aflR leads to higher transcript accumulation of pathway genes and increased aflatoxin production but that the

  12. The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product.

    PubMed

    Fabry, S; Nass, N; Huber, H; Palme, K; Jaenicke, L; Schmitt, R

    1992-09-10

    Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed. PMID:1511889

  13. Cloning of the Thermomonospora fusca Endoglucanase E2 gene in Streptomyces lividans: Affinity purification and functional domains of the cloned gene product

    SciTech Connect

    Ghangas, G.S.; Wilson, D.B. )

    1988-10-01

    Thermomonospora fusca YX grown in the presence of cellulose produces a number of {beta}-1-4-endoglucanases, some of which bind to microcrystalline cellulose. By using a multicopy plasmid, pIJ702, a gene coding for one of these enzymes (E2) was cloned into Streptomyces lividans and then mobilized into both Escherichia coli and Streptomyces albus. The gene was localized to a 1.6-kilobase PvuII-ClaI segment of the originally cloned 3.0-kilobase SstI fragment of Thermomonospora DNA. The culture supernatants of Streptomyces transformants contain a major endoglucanase that cross-reacts with antibody against Thermomonospora cellulase E2 and has the same molecular weight (43,000) as T. fusca E2. This protein binds quickly and tightly to Avicel. It also binds to filter paper but at a slower rate than to Avicel. Several large proteolytic degradation products of this enzyme generated in vivo lose the ability to bind to Avicel and have higher activity on carboxymethyl cellulose than the native enzyme. Other smaller products bind to Avicel but lack activity. A weak cellobiose-binding site not observed in the native enzyme was present in one of the degradation products. In E. coli, the cloned gene produced a cellulase that also binds tightly to Avicel but appeared to be slightly larger than T. fusca E2. The activity of intact E2 from all organisms can be inactivated by Hg{sup 2+} ions. Dithiothreitol protected against Hg{sup 2+} inactivation and reactivated both unbound and Avicel-bound Hg{sub 2+}-inhibited E2, but at different rates.

  14. De Novo Assembly, Gene Annotation, and Marker Discovery in Stored-Product Pest Liposcelis entomophila (Enderlein) Using Transcriptome Sequences

    PubMed Central

    Wei, Dan-Dan; Chen, Er-Hu; Ding, Tian-Bo; Chen, Shi-Chun; Dou, Wei; Wang, Jin-Jun

    2013-01-01

    Background As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. Methodology/Principal Findings We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. Conclusions/Significance We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance

  15. Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products

    PubMed Central

    2014-01-01

    Background The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana. Results This study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3′-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase

  16. Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds

    SciTech Connect

    Gracia, Tannia Hilscherova, Klara; Jones, Paul D.; Newsted, John L.; Higley, Eric B.; Zhang, Xiaowei; Hecker, Markus; Murphy, Margaret B.; Yu, Richard M.K.; Lam, Paul K.S.; Wu, Rudolf S.S.; Giesy, John P.

    2007-12-01

    The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3{beta}HSD2, CYP11{beta}1, CYP11{beta}2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11{beta}2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantly increased CYP11{beta}2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The {beta}-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different.

  17. Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol.

    PubMed

    Mienda, Bashir Sajo; Shamsir, Mohd Shahir; Md Illias, Rosli

    2016-08-01

    Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli. PMID:26513379

  18. Effect of incorporation of thermo-regulatory genes into exotic layers on egg production and quality under tropical environment.

    PubMed

    Hagan, Julius K; Adomako, Kwaku; Olympio, Simon Oscar

    2014-01-01

    A breed development strategy aimed at making exotic layers (Lohmann Brown) more productive under tropical environment using thermo-regulatory genes is underway at Akate Farms in Kumasi, Ghana. The present experiment was carried out to find out the effect of the genes on egg production in hot and humid environments. Three genetic groups comprising naked-neck, frizzle and their normally feathered sibs were obtained after successive generations of crossing between naked-neck and frizzle cocks and Lohmann brown hens. A total of 270 18-week-old pullets, 90 each of the 3 groups, were selected randomly and assigned to a completely randomized design experiment with 3 replicates, with 30 birds in each replicate group and kept up to a period of 72 weeks. The birds were kept in a partitioned open-sided deep-litter house constructed with sandcrete blocks with 30 pullets in each compartment. They were fed ad libitum with layer diets containing 18 % crude protein and 2,800 kcal ME/kg. Results obtained showed that the crossbred naked-neck and frizzle phenotypes produced eggs at a significantly (P < 0.05) higher rates than their normally feathered sibs and also out-performed their normally feathered sibs in other egg production parameters measured, even though they all segregated from similar parents. This is an indication of the favourable effect of the genes on egg production under hot and humid environments. PMID:23955013

  19. Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols.

    PubMed

    Styger, Gustav; Jacobson, Dan; Bauer, Florian F

    2011-08-01

    During alcoholic fermentation, many volatile aroma compounds are formed by Saccharomyces cerevisiae, including esters, fatty acids, and higher alcohols. While the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the regulation of the network, and the physiological roles of individual pathways within the network. Furthermore, different yeast strains tend to produce significantly different aroma profiles. These differences are of tremendous biotechnological interest, since producers of alcoholic beverages such as wine and beer are searching for means to diversify and improve their product range. Various factors such as the redox, energy, and nutritional balance of a cell have previously been suggested to directly or indirectly affect and regulate the network. To gain a better understanding of the regulations and physiological role of this network, we screened a subset of the EUROSCARF strain deletion library for genes that, when deleted, would impact most significantly on the aroma profile produced under fermentative conditions. The 10 genes whose deletion impacted most significantly on higher alcohol production were selected and further characterized to assess their mode of action within or on this metabolic network. This is the first description of a large-scale screening approach using aroma production as the primary selection criteria, and the data suggest that many of the identified genes indeed play central and direct roles within the aroma production network of S. cerevisiae. PMID:21547456

  20. Identification of Genetic Associations and Functional Polymorphisms of SAA1 Gene Affecting Milk Production Traits in Dairy Cattle.

    PubMed

    Yang, Shaohua; Gao, Yahui; Zhang, Shengli; Zhang, Qin; Sun, Dongxiao

    2016-01-01

    Our initial RNA sequencing (RNA-seq) revealed that the Serum amyloid A1 (SAA1) gene was differentially expressed in the mammary glands of lactating Holstein cows with extremely high versus low phenotypic values of milk protein and fat percentage. To further validate the genetic effect and potential molecular mechanisms of SAA1 gene involved in regulating milk production traits in dairy cattle, we herein performed a study through genotype-phenotype associations. Six identified SNPs were significantly associated with one or more milk production traits (0.00002< P < 0.0025), providing additional evidence for the potential role of SAA1 variants in milk production traits in dairy cows. Subsequently, both luciferase assay and electrophoretic mobility shift assay (EMSA) clearly demonstrated that the allele A of g.-963C>A increased the promoter activity by binding the PARP factor while allele C did not. Bioinformatics analysis indicated that the secondary structure of SAA protein changed by the substitution A/G in the locus c. +2510A>G. Our findings were the first to reveal the significant associations of the SAA1 gene with milk production traits, providing basis for further biological function validation, and two identified SNPs, g.-963C>A and c. +2510A>G, may be considered as genetic markers for breeding in dairy cattle. PMID:27610623

  1. MAp19, the alternative splice product of the MASP2 gene.

    PubMed

    Degn, Søren E; Thiel, Steffen; Nielsen, Ole; Hansen, Annette G; Steffensen, Rudi; Jensenius, Jens C

    2011-10-28

    The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217 ng/ml, i.e., 11nM, comparable to the 7 nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent. PMID:21871896

  2. Distribution of the longevity gene product, SIRT1, in developing mouse organs.

    PubMed

    Ogawa, Tetsuo; Wakai, Chizu; Saito, Tomomi; Murayama, Aya; Mimura, Yuuichi; Youfu, Sachiko; Nakamachi, Tomoya; Kuwagata, Makiko; Satoh, Kazue; Shioda, Seiji

    2011-06-01

    A longevity gene product, Sir2 (silent information regulator 2) is a NAD-dependent histone deacetylase involved in longevity in yeasts, worms and flies. The mammalian homolog of Sir2, SIRT1(sirtuin 1), has been shown to play important roles related to anti-aging effects (regulating apoptosis, stress tolerance, insulin resistance, and fat metabolism). Recently, SIRT1 expression has been demonstrated to occur at as early as embryonic day 10.5 in mice. SIRT1 during developing period may be involved in the mechanism of developmental origins of adult diseases, such as diabetes and cardiovascular disease. To investigate the contribution of SIRT1, it is important to reveal the distribution of this protein during development. In the present study, we demonstrated the distribution of immunoreactivity of SIRT1 in mouse organs during prenatal and neonatal development by staining a wide variety of serial sections. The SIRT1 immunoreactivity was strongly observed in the neuroepithelial layer, dorsal root ganglion, trigeminal ganglion, eyes, roots of whiskers, and internal organs, including the testis, liver, heart, kidney, and lung during the fetal period. Neurons which had finished migrating still showed relatively strong immunoreactivity. The immunoreactivity was completely absorbed by the blocking peptide in an absorption test. During the postnatal period, the immunoreactivities in most of these organs, except the heart and testis weakened, with the liver most dramatically affected. As SIRT1 expression was demonstrated in a wide variety of developing organs, further study to investigate prenatal factors which affect SIRT1 expression and its activity is important. PMID:21054562

  3. ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation[S

    PubMed Central

    Stephensen, Charles B.; Armstrong, Patrice; Newman, John W.; Pedersen, Theresa L.; Legault, Jillian; Schuster, Gertrud U.; Kelley, Darshan; Vikman, Susanna; Hartiala, Jaana; Nassir, Rami; Seldin, Michael F.; Allayee, Hooman

    2011-01-01

    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 of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20–59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats (“55” genotype) than in subjects with one deletion (d) (three or four repeats) and one common (“d5” genotype) allele or with two deletion (“dd”) alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk. PMID:21296957

  4. Growth-related gene product {alpha}: A chemotactic cytokine for neutrophils in rheumatoid arthritis

    SciTech Connect

    Koch, A.E.; Pope, R.M. |; Shah, M.R.; Hosaka, S.

    1995-10-01

    Leukocyte recruitment is critical in the inflammation seen in rheumatoid arthritis (RA). To determine whether the chemokine growth-related gene product {alpha} (gro{alpha}) plays a role in this process, we examined synovial tissue (ST), synovial fluid (SF), and plasma samples from 102 patients with arthritis. RA SF contained more antigenic gro{alpha} (mean 5.3 {+-} 1.9 ng/ml) than did SFs from either osteoarthritis (OA) or other forms of arthritis (mean 0.1 ng/ml) (p < 0.05). RA plasma contained more gro{alpha} (mean 4.3 {+-} 1.8 ng/ml) than normal plasma (mean 0.1 ng/ml) (p < 0.05). RA ST fibroblasts (1.2 x 10{sup 5}/cells/ml RPMI 1640/24 h) produced antigenic gro{alpha} (mean 0.2 {+-} 0.1 ng/ml), and this production was increased significantly upon incubation with TNF-{alpha} (mean 1.3 {+-} 0.3 ng/ml) or IL-1{beta} (mean 2.3 {+-} 0.6 ng/ml) (p < 0.05). Cells from RA SF also produced gro{alpha}: neutrophils (PMNs) (10{sup 7} cells/ml/24 h) produced 3.7 {+-} 0.7 ng/ml. RA SF mononuclear cells produced gro{alpha}, particularly upon incubation with LPS or PHA. Immunoreactive ST gro{alpha} was found in greater numbers of RA compared with either OA or normal lining cells, as well as in RA compared with OA subsynovial macrophages (p < 0.05). IL-8 accounted for a mean of 36% of the RA SF chemotactic activity for PMNs, while epithelial neutrophil-activating peptide-78 accounted for 34%, and gro{alpha} for 28%, of this activity. Combined neutralization of all three chemokines in RA SFs resulted in a mean decrease of 50% of the chemotactic activity for PMNs present in the RA SFs. These results indicate that gro{alpha} plays an important role in the ingress of PMNs into the RA joint. 54 refs., 6 figs., 1 tab.

  5. FUM Gene Expression Profile and Fumonisin Production by Fusarium verticillioides Inoculated in Bt and Non-Bt Maize

    PubMed Central

    Rocha, Liliana O.; Barroso, Vinícius M.; Andrade, Ludmila J.; Pereira, Gustavo H. A.; Ferreira-Castro, Fabiane L.; Duarte, Aildson P.; Michelotto, Marcos D.; Correa, Benedito

    2016-01-01

    This study aimed to determine the levels of fumonisins produced by Fusarium verticillioides and FUM gene expression on Bt (Bacillus thuringiensis) and non-Bt maize, post harvest, during different periods of incubation. Transgenic hybrids 30F35 YG, 2B710 Hx and their isogenic (30F35 and 2B710) were collected from the field and a subset of 30 samples selected for the experiments. Maize samples were sterilized by gamma radiation at a dose of 20 kGy. Samples were then inoculated with F. verticillioides and analyzed under controlled conditions of temperature and relative humidity for fumonisin B1 and B2 (FB1 and FB2) production and FUM1, FUM3, FUM6, FUM7, FUM8, FUM13, FUM14, FUM15, and FUM19 expression. 2B710 Hx and 30F35 YG kernel samples were virtually intact when compared to the non-Bt hybrids that came from the field. Statistical analysis showed that FB1 production was significantly lower in 30F35 YG and 2B710 Hx than in the 30F35 and 2B710 hybrids (P < 0.05). However, there was no statistical difference for FB2 production (P > 0.05). The kernel injuries observed in the non-Bt samples have possibly facilitated F. verticillioides penetration and promoted FB1 production under controlled conditions. FUM genes were expressed by F. verticillioides in all of the samples. However, there was indication of lower expression of a few FUM genes in the Bt hybrids; and a weak association between FB1 production and the relative expression of some of the FUM genes were observed in the 30F35 YG hybrid. PMID:26779158

  6. FUM Gene Expression Profile and Fumonisin Production by Fusarium verticillioides Inoculated in Bt and Non-Bt Maize.

    PubMed

    Rocha, Liliana O; Barroso, Vinícius M; Andrade, Ludmila J; Pereira, Gustavo H A; Ferreira-Castro, Fabiane L; Duarte, Aildson P; Michelotto, Marcos D; Correa, Benedito

    2015-01-01

    This study aimed to determine the levels of fumonisins produced by Fusarium verticillioides and FUM gene expression on Bt (Bacillus thuringiensis) and non-Bt maize, post harvest, during different periods of incubation. Transgenic hybrids 30F35 YG, 2B710 Hx and their isogenic (30F35 and 2B710) were collected from the field and a subset of 30 samples selected for the experiments. Maize samples were sterilized by gamma radiation at a dose of 20 kGy. Samples were then inoculated with F. verticillioides and analyzed under controlled conditions of temperature and relative humidity for fumonisin B1 and B2 (FB1 and FB2) production and FUM1, FUM3, FUM6, FUM7, FUM8, FUM13, FUM14, FUM15, and FUM19 expression. 2B710 Hx and 30F35 YG kernel samples were virtually intact when compared to the non-Bt hybrids that came from the field. Statistical analysis showed that FB1 production was significantly lower in 30F35 YG and 2B710 Hx than in the 30F35 and 2B710 hybrids (P < 0.05). However, there was no statistical difference for FB2 production (P > 0.05). The kernel injuries observed in the non-Bt samples have possibly facilitated F. verticillioides penetration and promoted FB1 production under controlled conditions. FUM genes were expressed by F. verticillioides in all of the samples. However, there was indication of lower expression of a few FUM genes in the Bt hybrids; and a weak association between FB1 production and the relative expression of some of the FUM genes were observed in the 30F35 YG hybrid. PMID:26779158

  7. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

    PubMed

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  8. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

    PubMed Central

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A.; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4+ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  9. Production of Truncated Candida antarctica Lipase B Gene Using Automated PCR Gene Assembly Protocol and Expression in Yeast for use in Ethanol and Biodiesel Production.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An improved column-based process for production of biodiesel was developed using a column containing a strongly basic anion-exchange resin in sequence with a column containing a resin to which a lipase biocatalyst is bound. Currently most biodiesel is produced by transesterification of triglyceride...

  10. Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production.

    PubMed

    Dowd, Patrick F; Johnson, Eric T

    2015-05-01

    Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease resistance-associated genes in milk stage kernels from commercial popcorn fields over 3 years. Relatively lower expression of resistance gene types was noted in years with higher temperatures and lower rainfall, which was consistent with prior results for many previously identified resistance response-associated genes. The lower rates of expression occurred for genes such as chitinases, protease inhibitors, and peroxidases; enzymes involved in the synthesis of cell wall barriers and secondary metabolites; and regulatory proteins. However, expression of several specific resistance genes previously associated with mycotoxins, such as aflatoxin in dent maize, was not affected. Insect damage altered the spectrum of resistance gene expression differences compared to undamaged ears. Correlation analyses showed expression differences of some previously reported resistance genes that were highly associated with mycotoxin levels and included glucanases, protease inhibitors, peroxidases, and thionins. PMID:25512225

  11. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

    PubMed Central

    Fischer, Konrad; Kraner-Scheiber, Simone; Petersen, Björn; Rieblinger, Beate; Buermann, Anna; Flisikowska, Tatiana; Flisikowski, Krzysztof; Christan, Susanne; Edlinger, Marlene; Baars, Wiebke; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Plotzki, Elena; Szczerbal, Izabela; Switonski, Marek; Denner, Joachim; Wolf, Eckhard; Schwinzer, Reinhard; Niemann, Heiner; Kind, Alexander; Schnieke, Angelika

    2016-01-01

    Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age. PMID:27353424

  12. Unraveling antimicrobial resistance genes and phenotype patterns among Enterococcus faecalis isolated from retail chicken products in Japan.

    PubMed

    Hidano, Arata; Yamamoto, Takehisa; Hayama, Yoko; Muroga, Norihiko; Kobayashi, Sota; Nishida, Takeshi; Tsutsui, Toshiyuki

    2015-01-01

    Multidrug-resistant enterococci are considered crucial drivers for the dissemination of antimicrobial resistance determinants within and beyond a genus. These organisms may pass numerous resistance determinants to other harmful pathogens, whose multiple resistances would cause adverse consequences. Therefore, an understanding of the coexistence epidemiology of resistance genes is critical, but such information remains limited. In this study, our first objective was to determine the prevalence of principal resistance phenotypes and genes among Enterococcus faecalis isolated from retail chicken domestic products collected throughout Japan. Subsequent analysis of these data by using an additive Bayesian network (ABN) model revealed the co-appearance patterns of resistance genes and identified the associations between resistance genes and phenotypes. The common phenotypes observed among E. faecalis isolated from the domestic products were the resistances to oxytetracycline (58.4%), dihydrostreptomycin (50.4%), and erythromycin (37.2%), and the gene tet(L) was detected in 46.0% of the isolates. The ABN model identified statistically significant associations between tet(L) and erm(B), tet(L) and ant(6)-Ia, ant(6)-Ia and aph(3')-IIIa, and aph(3')-IIIa and erm(B), which indicated that a multiple-resistance profile of tetracycline, erythromycin, streptomycin, and kanamycin is systematic rather than random. Conversely, the presence of tet(O) was only negatively associated with that of erm(B) and tet(M), which suggested that in the presence of tet(O), the aforementioned multiple resistance is unlikely to be observed. Such heterogeneity in linkages among genes that confer the same phenotypic resistance highlights the importance of incorporating genetic information when investigating the risk factors for the spread of resistance. The epidemiological factors that underlie the persistence of systematic multiple-resistance patterns warrant further investigations with appropriate

  13. Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

    PubMed

    Douëllou, T; Delannoy, S; Ganet, S; Mariani-Kurkdjian, P; Fach, P; Loukiadis, E; Montel, Mc; Thevenot-Sergentet, D

    2016-09-01

    Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples. PMID:27257743

  14. Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple.

    PubMed

    Nieuwenhuizen, Niels J; Green, Sol A; Chen, Xiuyin; Bailleul, Estelle J D; Matich, Adam J; Wang, Mindy Y; Atkinson, Ross G

    2013-02-01

    Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple 'Royal Gala' expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies. PMID:23256150

  15. Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple1[W

    PubMed Central

    Nieuwenhuizen, Niels J.; Green, Sol A.; Chen, Xiuyin; Bailleul, Estelle J.D.; Matich, Adam J.; Wang, Mindy Y.; Atkinson, Ross G.

    2013-01-01

    Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple ‘Royal Gala’ expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies. PMID:23256150

  16. Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

    PubMed

    De Clercq, N; Vlaemynck, G; Van Pamel, E; Van Weyenberg, S; Herman, L; Devlieghere, F; De Meulenaer, B; Van Coillie, E

    2016-03-01

    Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, β-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in

  17. Extended region of nodulation genes in Rhizobium meliloti 1021. II. Nucleotide sequence, transcription start sites and protein products

    SciTech Connect

    Fisher, R.F.; Swanson, J.A.; Mulligan, J.T.; Long, S.R.

    1987-10-01

    The authors have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the nod box, suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site.

  18. Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii.

    PubMed

    La Russa, M; Bogen, C; Uhmeyer, A; Doebbe, A; Filippone, E; Kruse, O; Mussgnug, J H

    2012-11-30

    Photosynthetic organisms like plants and algae can use sunlight to produce lipids as important metabolic compounds. Plant-derived triacylglycerols (TAGs) are valuable for human and animal nutrition because of their high energy content and are becoming increasingly important for the production of renewable biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) have been demonstrated to play an important role in the accumulation of TAG compounds in higher plants. DGAT homologue genes have been identified in the genome of the green alga Chlamydomonas reinhardtii, however their function in vivo is still unknown. In this work, the three most promising type-2 DGAT candidate genes potentially involved in TAG lipid accumulation (CrDGAT2a, b and c) were investigated by constructing overexpression strains. For each of the genes, three strains were identified which showed enhanced mRNA levels of between 1.7 and 29.1 times that of the wild type (wt). Total lipid contents, neutral lipids and fatty acid profiles were determined and showed that an enhanced mRNA expression level of the investigated DGAT genes did not boost the intracellular TAG accumulation or resulted in alterations of the fatty acid profiles compared to wild type during standard growth condition or during nitrogen or sulfur stress conditions. We conclude that biotechnological efforts to enhance cellular TAG amount in microalgae need further insights into the complex network of lipid biosynthesis to identify potential bottlenecks of neutral lipid production. PMID:22542934

  19. Heterologous expression of an orphan NRPS gene cluster from Paenibacillus larvae in Escherichia coli revealed production of sevadicin.

    PubMed

    Tang, Ying; Frewert, Simon; Harmrolfs, Kirsten; Herrmann, Jennifer; Karmann, Lisa; Kazmaier, Uli; Xia, Liqiu; Zhang, Youming; Müller, Rolf

    2015-01-20

    The Gram-positive bacterium Paenibacillus larvae is the causative agent of the fateful honey bee disease American Foulbrood (AFB). Sequence analysis of P. larvae genomic DNA showed the presence of numerous nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) encoding gene clusters, not correlating with secondary metabolite production. As NRPS and PKS derived metabolites are known to exhibit diverse biological activities, their identification is of particular interest for infection and drug research. Here an 11.6kb orphan NRPS gene cluster was directly cloned from the genomic DNA of P. larvae and expressed in Escherichia coli resulting in the production of sevadicin. Isolation of the metabolite was followed by structural characterization, synthesis and bioactivity studies. PMID:25529345

  20. Engineered Production of Tryprostatins in E. coli through Reconstitution of a Partial ftm Biosynthetic Gene Cluster from Aspergillus sp.

    PubMed Central

    Shah, Gopitkumar R; Wesener, Shane R.; Cheng, Yi-Qiang

    2015-01-01

    Tryprostatin A and B are indole alkaloid-based fungal products that inhibit mammalian cell cycle at the G2/M phase. They are biosynthetic intermediates of fumitremorgins produced by a complex pathway involving a nonribosomal peptide synthetase (FtmA), a prenyltransferase (FtmB), a cytochrome P450 hydroxylase (FtmC), an O-methyltransferase (FtmD), and several additional enzymes. A partial fumitremorgin biosynthetic gene cluster (ftmABCD) from Aspergillus sp. was reconstituted in Escherichia coli BL21(DE3) cells, with or without the co-expression of an Sfp-type phosphopantetheinyltransferase gene (Cv_sfp) from Chromobacterium violaceum No. 968. Several recombinant E. coli strains produced tryprostatin B up to 106 mg/l or tryprostatin A up to 76 mg/l in the fermentation broth under aerobic condition, providing an effective way to prepare those pharmaceutically important natural products biologically. PMID:26640821

  1. Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities

    USGS Publications Warehouse

    Chee-Sanford, J. C.; Aminov, R.I.; Krapac, I.J.; Garrigues-Jeanjean, N.; Mackie, R.I.

    2001-01-01

    In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.

  2. Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes.

    PubMed

    Nakatsuka, Takashi; Abe, Yoshiko; Kakizaki, Yuko; Yamamura, Saburo; Nishihara, Masahiro

    2007-11-01

    Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3'-hydroxylase [F3'H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. PMID:17639403

  3. Biotransformation of dihydroisosteviol and the effects of transformed products on steroidogenic gene expressions.

    PubMed

    Chang, Shwu-Fen; Yang, Li-Ming; Huang, Tsurng-Juhn; Chen, Chin-Yang; Sheu, Shiow-Yunn; Liu, Pan-Chun; Lin, Shwu-Jiuan

    2013-11-01

    The biotransformation of dihydroisosteviol with Absidia pseudocylindrospora ATCC 24169, Streptomyces griseus ATCC 10137, Mucor recurvatus MR36, and Aspergillus niger BCRC 31130 yielded 15 metabolites, eight of which were previously unknown. Structures of metabolites were established by 2D NMR techniques and HRMS data, two of which were further corroborated by chemical means, and another via single-crystal X-ray diffraction analysis. Subsequently, two steroidogenic cell lines (Y-1 mouse adrenal tumor and MA-10 mouse Leydig tumor cells) were used in a reverse transcription-PCR analysis to assess the effects of all compounds on steroidogenic gene expressions using forskolin as a positive control. The tested gene expressions included steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) enzyme. Gene expression profiles showed that ten of the tested compounds effectively suppressed P450SCC mRNA expression in both Y-1 and MA-10 cells. Several induced SF-1 gene expression and two enhanced StAR gene expression in Y-1 cells. By contrast, in MA-10 cells, one compound effectively suppressed StAR mRNA expression, whereas for others effectively suppressed SF-1 gene expression. The results suggest that analogs of dihydroisosteviol can be potential modulators to alter steroidogenic gene expressions and subsequent enzyme activities. PMID:23948258

  4. The gene bap, involved in biofilm production, is present in Staphylococcus spp. strains from nosocomial infections.

    PubMed

    Potter, Amina; Ceotto, Hilana; Giambiagi-Demarval, Marcia; dos Santos, Kátia Regina Netto; Nes, Ingolf F; Bastos, Maria do Carmo de Freire

    2009-06-01

    This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii. PMID:19557349

  5. Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9

    PubMed Central

    Zhang, Xiya; Liang, Puping; Ding, Chenhui; Zhang, Zhen; Zhou, Jianwen; Xie, Xiaowei; Huang, Rui; Sun, Ying; Sun, Hongwei; Zhang, Jinran; Xu, Yanwen; Songyang, Zhou; Huang, Junjiu

    2016-01-01

    The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5′-NGG-3′) recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5′-NNGRRT-3′) preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models. PMID:27586692

  6. Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9.

    PubMed

    Zhang, Xiya; Liang, Puping; Ding, Chenhui; Zhang, Zhen; Zhou, Jianwen; Xie, Xiaowei; Huang, Rui; Sun, Ying; Sun, Hongwei; Zhang, Jinran; Xu, Yanwen; Songyang, Zhou; Huang, Junjiu

    2016-01-01

    The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5'-NGG-3') recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5'-NNGRRT-3') preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models. PMID:27586692

  7. Expression profiling of cell cycle genes reveals key facilitators of cell production during carpel development, fruit set, and fruit growth in apple (Malus×domestica Borkh.)

    PubMed Central

    Malladi, Anish; Johnson, Lisa Klima

    2011-01-01

    Cell production is an essential facilitator of fruit growth and development. Cell production during carpel/floral-tube growth, fruit set, and fruit growth, and its regulation by cell cycle genes were investigated in apple (Malus×domestica Borkh.). Cell production was inhibited during late carpel/floral-tube development, resulting in growth arrest before bloom. Fruit set re-activated cell production between 8 d and 11 d after full bloom (DAFB) and triggered fruit growth. The early phase of fruit growth involved rapid cell production followed by exit from cell proliferation at ∼24 DAFB. Seventy-one cell cycle genes were identified, and expression of 59 genes was investigated using quantitative RT-PCR. Changes in expression of 19 genes were consistently associated with transitions in cell production during carpel/floral-tube growth, fruit set, and fruit growth. Fourteen genes, including B-type cyclin-dependent kinases (CDKs) and A2-, B1-, and B2-type cyclins, were positively associated with cell production, suggesting that availability of G2/M phase regulators of the cell cycle is limiting for cell proliferation. Enhanced expression of five genes including that of the putative CDK inhibitors, MdKRP4 and MdKRP5, was associated with reduced cell production. Exit from cell proliferation at G0/G1 during fruit growth was facilitated by multiple mechanisms including down-regulation of putative regulators of G1/S and G2/M phase progression and up-regulation of KRP genes. Interestingly, two CDKA genes and several CDK-activating factors were up-regulated during this period, suggesting functions for these genes in mediating exit from cell proliferation at G0/G1. Together, the data indicate that cell cycle genes are important facilitators of cell production during apple fruit development. PMID:20732881

  8. Role of Nitric Oxide and Flavohemoglobin Homolog Genes in Aspergillus nidulans Sexual Development and Mycotoxin Production ▿ †

    PubMed Central

    Baidya, Sachin; Cary, Jeffrey W.; Grayburn, W. Scott; Calvo, A. M.

    2011-01-01

    Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the Aspergillus nidulans flavohemoglobin gene fhbA induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased nsdD and steA expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi. PMID:21642398

  9. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

    PubMed Central

    Ritzerfeld, Julia; Remmele, Steffen; Wang, Tao; Temmerman, Koen; Brügger, Britta; Wegehingel, Sabine; Tournaviti, Stella; Strating, Jeroen R.P.M.; Wieland, Felix T.; Neumann, Beate; Ellenberg, Jan; Lawerenz, Chris; Hesser, Jürgen; Erfle, Holger; Pepperkok, Rainer; Nickel, Walter

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes. PMID:21795383

  10. [Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production].

    PubMed

    Sawasdee, K; Rudeekulthamrong, P; Zimmermann, W; Murakami, S; Pongsawasdi, P; Kaulpiboon, J

    2014-01-01

    The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM-T easy vector to transform into Escherichia coli DH5 alpha for sequencing. The obtained gene encoding an amylomaltase consisted of 1.503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21 (DE3). The maximum expression was observed when the cloned cells were cultured at 37 degrees C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70 degrees C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the large-ring cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions. PMID:25272748

  11. [Adeno-associated virus mediated T-bet gene transfer into SGC-7901 cell to regulate IFN-gamma production].

    PubMed

    Qiu, Gufeng; Wang, Suoying; Wang, Shengjun; Shao, Qixiang; Ma, Jie; Yang, Ming; Xu, Xiaopeng; Mao, Chaoming; Su, Zhaoliang; Huang, Xinxiang; Xu, Huaxi

    2009-06-01

    In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene. PMID:19634682

  12. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments.

    PubMed

    Breitling, Rainer; Armengaud, Patrick; Amtmann, Anna; Herzyk, Pawel

    2004-08-27

    One of the main objectives in the analysis of microarray experiments is the identification of genes that are differentially expressed under two experimental conditions. This task is complicated by the noisiness of the data and the large number of genes that are examined simultaneously. Here, we present a novel technique for identifying differentially expressed genes that does not originate from a sophisticated statistical model but rather from an analysis of biological reasoning. The new technique, which is based on calculating rank products (RP) from replicate experiments, is fast and simple. At the same time, it provides a straightforward and statistically stringent way to determine the significance level for each gene and allows for the flexible control of the false-detection rate and familywise error rate in the multiple testing situation of a microarray experiment. We use the RP technique on three biological data sets and show that in each case it performs more reliably and consistently than the non-parametric t-test variant implemented in Tusher et al.'s significance analysis of microarrays (SAM). We also show that the RP results are reliable in highly noisy data. An analysis of the physiological function of the identified genes indicates that the RP approach is powerful for identifying biologically relevant expression changes. In addition, using RP can lead to a sharp reduction in the number of replicate experiments needed to obtain reproducible results. PMID:15327980

  13. Evaluation of Gene Expression and Alginate Production in Response to Oxygen Transfer in Continuous Culture of Azotobacter vinelandii

    PubMed Central

    Díaz-Barrera, Alvaro; Martínez, Fabiola; Guevara Pezoa, Felipe; Acevedo, Fernando

    2014-01-01

    Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h−1) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h−1, the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h−1 showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor

  14. Evaluation of gene expression and alginate production in response to oxygen transfer in continuous culture of Azotobacter vinelandii.

    PubMed

    Díaz-Barrera, Alvaro; Martínez, Fabiola; Pezoa, Felipe Guevara; Acevedo, Fernando

    2014-01-01

    Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h(-1)) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h(-1), the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h(-1) showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor

  15. [The exploration and practice of production of transgenic zebrafish into undergraduate student gene engineering experimental teaching].

    PubMed

    Yuan, Wu-Zhou; Deng, Yun

    2013-11-01

    The preparation of transgenic animals is one of the core technology and critical achievement of gene engineering. However, it has not been reported that the gene engineering experimental course of undergraduate students in universities of mainland China has carried out the preparation of transgenic animals. In this paper, the authors took the advantage of scientific research platform, introduced the transgenic zebrafish technology to gene engineering experimental course of undergraduate students, and explored and practiced related teaching model, which had achieved good results and had great value to popularize. PMID:24579316

  16. Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production

    PubMed Central

    Vuoristo, Kiira S.; Mars, Astrid E.; van Loon, Stijn; Orsi, Enrico; Eggink, Gerrit; Sanders, Johan P. M.; Weusthuis, Ruud A.

    2015-01-01

    Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L. PMID:26347730

  17. Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production.

    PubMed

    Vuoristo, Kiira S; Mars, Astrid E; van Loon, Stijn; Orsi, Enrico; Eggink, Gerrit; Sanders, Johan P M; Weusthuis, Ruud A

    2015-01-01

    Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L. PMID:26347730

  18. Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil.

    PubMed

    Reis, Andre L S; Montanhini, Maike T M; Bittencourt, Juliana V M; Destro, Maria T; Bersot, Luciano S

    2013-12-01

    Bacillus cereusis an ubiquitous, spore-forming bacteria that can survive pasteurization and the majority of the heating processes used in the dairy industry. Besides, it is a pathogen responsible for different types of food poisoning. One type of foodborne disease caused by B.cereusis the diarrheal syndrome, which is caused by the ingestion of vegetative cells producing toxins in the small intestine. One virulence factor for the diarrheal syndrome is the toxin hemolysin BL (HBL), a three-component protein formed by the L1, L2 and B components. In order to evaluate the presence of diarrheal strains isolated from milk and dairy products, 63 B. cereus isolates were obtained from 260 samples of UHT milk, pasteurized milk and powdered milk, sold in commercial establishments and from different brands. The isolates were subjected to the Polymerase Chain Reaction (PCR) for the detection of the encoding genes for the L1, L2 and B components and the toxin production capacity were evaluated with an immunoassay. A total of 23 [36.5%] isolates were identified carrying simultaneously the three tested genes, from which, 20 [86.9%] showed toxigenic capacity. 26 [41.3%] isolates did not carry any of genes tested and the other 14 [22.2%] were positive for one or two of them. The results showed a high toxigenic capacity among the B. cereus isolates able to produce the HBL, indicating a potential risk for consumers. PMID:24688511

  19. A mutation upstream of an ATPase gene significantly increases magnetosome production in Magnetospirillum gryphiswaldense.

    PubMed

    Liu, Jiangning; Ding, Yao; Jiang, Wei; Tian, Jiesheng; Li, Ying; Li, Jilun

    2008-12-01

    A mutant of Magnetospirillum gryphiswaldense, NPHB, was obtained from a conjugation experiment. An aberrant recombination occurred between a putative elongation factor-G gene (fus-like) of the bacterial chromosome and the chloramphenicol resistant gene (cat) of a suicide vector, pSUP202. Complementary experiments and transcription analysis of genes around the recombinant site showed that the cat promoter enhanced the expression of adenosine triphosphatase gene downstream. Adenosine triphosphate hydrolyzing activity in NPHB was 35% higher than in the wild-type strain (M. gryphiswaldense MSR-1). NPHB accumulated 71% less poly-beta-hydroxybutyrate and consumed 56% more oxygen and 40% more lactate than MSR-1. The magnetosome content of NPHB was 69% higher than MSR-1 in flask culture. NPHB cultured in a 7.5-L bioreactor gave a maximum yield of 58.4 +/- 6.4 mg magnetosomes per liter. PMID:18800186

  20. Analysis of transfer genes and gene products within the traB-traC region of the Escherichia coli fertility factor, F

    SciTech Connect

    Moore, D.; Wu, J.H.; Kathir, P.; Hamilton, C.M.; Ippen-Ihler, K.

    1987-09-01

    A series of plasmids that carry overlapping segments of F DNA encoding the genes in the traB-traC interval was constructed, and a restriction enzyme map of the region was derived. Plasmids carrying deletions that had been introduced at an HpaI sites within this interval were also isolated. The ability of these plasmids to complement transfer of F lac plasmids carrying mutations in traB, traV, traW, and traC was analyzed. The protein products of the plasmids were labeled in UV-irradiated cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These analyses showed that the product of traV is a polypeptide that migrates with an apparent molecular weight of 21,000. It was not detected when (/sup 35/S)methionine was used to label plasmid products, but was readily detected in /sup 14/C-amino acid labeling experiments. A 21,500-dalton product appeared to stem from the region assigned to traP. A 9000-dalton product was found to stem from a locus, named traR, that is located between traV and traC. No traW activity could be detected from the region of tra DNA examined. The authors data also indicated that traC is located in a more promoter-proximal position than suggested on earlier maps. The plasmids constructed are expected to be useful in studies designed to identify the specific functions of the traB, -P, -V, -R, and -C products.

  1. Female-specific gene expression in dioecious liverwort Pellia endiviifolia is developmentally regulated and connected to archegonia production

    PubMed Central

    2014-01-01

    Background In flowering plants a number of genes have been identified which control the transition from a vegetative to generative phase of life cycle. In bryophytes representing basal lineage of land plants, there is little data regarding the mechanisms that control this transition. Two species from bryophytes - moss Physcomitrella patens and liverwort Marchantia polymorpha are under advanced molecular and genetic research. The goal of our study was to identify genes connected to female gametophyte development and archegonia production in the dioecious liverwort Pellia endiviifolia species B, which is representative of the most basal lineage of the simple thalloid liverworts. Results The utility of the RDA-cDNA technique allowed us to identify three genes specifically expressed in the female individuals of P.endiviifolia: PenB_CYSP coding for cysteine protease, PenB_MT2 and PenB_MT3 coding for Mysterious Transcripts1 and 2 containing ORFs of 143 and 177 amino acid residues in length, respectively. The exon-intron structure of all three genes has been characterized and pre-mRNA processing was investigated. Interestingly, five mRNA isoforms are produced from the PenB_MT2 gene, which result from alternative splicing within the second and third exon. All observed splicing events take place within the 5′UTR and do not interfere with the coding sequence. All three genes are exclusively expressed in the female individuals, regardless of whether they were cultured in vitro or were collected from a natural habitat. Moreover we observed ten-fold increased transcripts level for all three genes in the archegonial tissue in comparison to the vegetative parts of the same female thalli grown in natural habitat suggesting their connection to archegonia development. Conclusions We have identified three genes which are specifically expressed in P.endiviifolia sp B female gametophytes. Moreover, their expression is connected to the female sex-organ differentiation and is

  2. Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells.

    PubMed

    Montano, Marco Aurélio Echart; da Cruz, Ivana Beatrice Mânica; Duarte, Marta Maria Medeiros Frescura; Krewer, Cristina da Costa; da Rocha, Maria Izabel de Ugalde Marques; Mânica-Cattani, Maria Fernanda; Soares, Felix Alexandre Antunes; Rosa, Guilherme; Maris, Angélica Francesca; Battiston, Francielle Garghetti; Trott, Alexis; Lera, Juan Pablo Barrio

    2012-10-01

    Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question. PMID:22688013

  3. Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes.

    PubMed

    Macaluso, A; Best, E A; Bender, R A

    1990-12-01

    A positive, genetic selection against the activity of the nitrogen regulatory (NTR) system was used to isolate insertion mutations affecting nitrogen regulation in Klebsiella aerogenes. Two classes of mutation were obtained: those affecting the NTR system itself and leading to the loss of almost all nitrogen regulation, and those affecting the nac locus and leading to a loss of nitrogen regulation of a family of nitrogen-regulated enzymes. The set of these nac-dependent enzymes included histidase, glutamate dehydrogenase, glutamate synthase, proline oxidase, and urease. The enzymes shown to be nac independent included glutamine synthetase, asparaginase, tryptophan permease, nitrate reductase, the product of the nifLA operon, and perhaps nitrite reductase. The expression of the nac gene was itself highly nitrogen regulated, and this regulation was mediated by the NTR system. The loss of nitrogen regulation was found in each of the four insertion mutants studied, showing that loss of nitrogen regulation resulted from the absence of nac function rather than from an altered form of the nac gene product. Thus we propose two classes of nitrogen-regulated operons: in class I, the NTR system directly activates expression of the operon; in class II, the NTR system activates nac expression and the product(s) of the nac locus activates expression of the operon. PMID:1979323

  4. Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system.

    PubMed

    Lwa, Teng Rhui; Tan, Chuan Hao; Lew, Qiao Jing; Chu, Kai Ling; Tan, Janice; Lee, Yih Yean; Chao, Sheng-Hao

    2010-04-15

    Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells. PMID:20188772

  5. Production of Xylitol from d-Xylose by a Xylitol Dehydrogenase Gene-Disrupted Mutant of Candida tropicalis

    PubMed Central

    Ko, Byoung Sam; Kim, Jinmi; Kim, Jung Hoe

    2006-01-01

    Xylitol dehydrogenase (XDH) is one of the key enzymes in d-xylose metabolism, catalyzing the oxidation of xylitol to d-xylulose. Two copies of the XYL2 gene encoding XDH in the diploid yeast Candida tropicalis were sequentially disrupted using the Ura-blasting method. The XYL2-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing d-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter−1 h−1, a specific productivity of 0.76 g g−1 h−1, and a xylitol yield of 98%. This is the first report of gene disruption of C. tropicalis for enhancing the efficiency of xylitol production. PMID:16751533

  6. 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

  7. Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

    PubMed

    Chen, Chun-Yen; Kao, Ai-Ling; Tsai, Zheng-Chia; Chow, Te-Jin; Chang, Hsin-Yueh; Zhao, Xin-Qing; Chen, Po-Ting; Su, Hsiang-Yen; Chang, Jo-Shu

    2016-03-01

    Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production. PMID:26849021

  8. Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene.

    PubMed

    Nebreda, A R; Villa, T G; Villanueva, J R; del Rey, F

    1986-01-01

    The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain (exg1-2) with highly reduced extracellular exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg- mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+ wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed Schizosaccharomyces pombe, yielding extracellular EXG activity which showed cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg+ strain. Genetic and Southern analysis of several stable Exg- transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exg1-2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related

  9. Modulation of Type III Secretion System in Pseudomonas aeruginosa: Involvement of the PA4857 Gene Product

    PubMed Central

    Zhu, Miao; Zhao, Jingru; Kang, Huaping; Kong, Weina; Zhao, Yuanyu; Wu, Min; Liang, Haihua

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes serious acute or chronic infections in humans. Acute infections typically involve the type III secretion systems (T3SSs) and bacterial motility, whereas chronic infections are often associated with biofilm formation and the type VI secretion system. To identify new genes required for pathogenesis, a transposon mutagenesis library was constructed and the gene PA4857, named tspR, was found to modulate T3SS gene expression. Deletion of P. aeruginosa tspR reduced the virulence in a mouse acute lung infection model and diminished cytotoxicity. Suppression of T3SS gene expression in the tspR mutant resulted from compromised translation of the T3SS master regulator ExsA. TspR negatively regulated two small RNAs, RsmY and RsmZ, which control RsmA. Our data demonstrated that defects in T3SS expression and biofilm formation in retS mutant could be partially restored by overexpression of tspR. Taken together, our results demonstrated that the newly identified retS-tspR pathway is coordinated with the retS-gacS system, which regulates the genes associated with acute and chronic infections and controls the lifestyle choice of P. aeruginosa. PMID:26858696

  10. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    PubMed

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes. PMID:27343449

  11. The association of very low-density lipoprotein receptor (VLDLR) haplotypes with egg production indicates VLDLR is a candidate gene for modulating egg production.

    PubMed

    Wang, ZhePeng; Meng, GuoHua; Li, Na; Yu, MingFen; Liang, XiaoWei; Min, YuNa; Liu, FuZhu; Gao, YuPeng

    2016-01-01

    The very low-density lipoprotein receptor (VLDLR) transports egg yolk precursors into oocytes. However, our knowledge of the distribution patterns of VLDLR variants among breeds and their relationship to egg production is still incomplete. In this study, eight single nucleotide polymorphisms (SNPs) that account for 87% of all VLDLR variants were genotyped in Nick Chick (NC, n=91), Lohmann Brown (LohB, n=50) and Lueyang (LY, n=381) chickens, the latter being an Chinese indigenous breed. Egg production by NC and LY chickens was recorded from 17 to 50 weeks. Only four similar haplotypes were found in NC and LohB, of which two accounted for 100% of all NC haplotypes and 92.5% of LohB haplotypes. In contrast, there was considerable haplotypic diversity in LY. Comparison of egg production in LY showed that hens with NC-like haplotypes had a significantly higher production (p < 0.05) than those without the haplotypes. However, VLDLR expression was not significantly different between the haplotypes. These findings indicate a divergence in the distribution of VLDLR haplotypes between selected and non-selected breeds and suggest that the near fixation of VLDLR variants in NC and LohB is compatible with signature of selection. These data also support VLDLR as a candidate gene for modulating egg production. PMID:27560838

  12. The association of very low-density lipoprotein receptor (VLDLR) haplotypes with egg production indicates VLDLR is a candidate gene for modulating egg production

    PubMed Central

    Wang, ZhePeng; Meng, GuoHua; Li, Na; Yu, MingFen; Liang, XiaoWei; Min, YuNa; Liu, FuZhu; Gao, YuPeng

    2016-01-01

    Abstract The very low-density lipoprotein receptor (VLDLR) transports egg yolk precursors into oocytes. However, our knowledge of the distribution patterns of VLDLR variants among breeds and their relationship to egg production is still incomplete. In this study, eight single nucleotide polymorphisms (SNPs) that account for 87% of all VLDLR variants were genotyped in Nick Chick (NC, n=91), Lohmann Brown (LohB, n=50) and Lueyang (LY, n=381) chickens, the latter being an Chinese indigenous breed. Egg production by NC and LY chickens was recorded from 17 to 50 weeks. Only four similar haplotypes were found in NC and LohB, of which two accounted for 100% of all NC haplotypes and 92.5% of LohB haplotypes. In contrast, there was considerable haplotypic diversity in LY. Comparison of egg production in LY showed that hens with NC-like haplotypes had a significantly higher production (p < 0.05) than those without the haplotypes. However, VLDLR expression was not significantly different between the haplotypes. These findings indicate a divergence in the distribution of VLDLR haplotypes between selected and non-selected breeds and suggest that the near fixation of VLDLR variants in NC and LohB is compatible with signature of selection. These data also support VLDLR as a candidate gene for modulating egg production. PMID:27560838

  13. Genes Involved in the Production of Antimetabolite Toxins by Pseudomonas syringae Pathovars

    PubMed Central

    Arrebola, Eva; Cazorla, Francisco M; Pérez-García, Alejandro; de Vicente, Antonio

    2011-01-01

    Pseudomonas syringae is pathogenic in a wide variety of plants, causing diseases with economic impacts. Pseudomonas syringae pathovars produce several toxins that can function as virulence factors and contribute to disease symptoms. These virulence factors include antimetabolite toxins, such as tabtoxin, phaseolotoxin and mangotoxin, which target enzymes in the pathways of amino acid metabolism. The antimetabolite toxins are generally located in gene clusters present in the flexible genomes of specific strains. These gene clusters are typically present in blocks of genes that appear to be integrated into specific sites in the P. syringae core genome. A general overview of the genetic organization and biosynthetic and regulatory functions of these genetic traits of the antimetabolite toxins will be given in the present work. PMID:24710214

  14. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

    PubMed

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-05-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  15. Tissue-specific Ctr1 Gene Expression and in silico Analysis of Its Putative Protein Product

    NASA Astrophysics Data System (ADS)

    Samsonov, Sergey A.; Nordlund, Eija; Platonova, Natalia A.; Skvortsov, Alexey N.; Tsymbalenko, Nadezhda V.; Puchkova, Ludmila V.

    2006-08-01

    Investigations of the links between Ctr1 gene activity and copper status in rat organs (liver, cerebellum, choroid plexus and mammary gland) with distinct types of copper metabolism as well as theoretical analysis of CTR1 domains structure were carried out in the research. The results suggest that (i) activity of mammalian Ctr1 gene is tissue-specific regulated at least by two different mechanisms: the gene activity is repressed by high intracellular Cu content and is activated/inactivated dependently on the cuproenzymes synthesis level required by physiological conditions. (ii) Multimerized conservative transmembrane domains 2 and 3 form the channel with copper binding amino acid side chains groups oriented inside this channel. These groups can transfer copper to the cytosolic domain, where Cu binds to CTR1 cytosolic HCH-motifs and can be further transferred to CXXC-motif of any known Cu(I)-chaperon.

  16. Associations of Leptin and Pituitary-Specific Transcription Factor Genes' Polymorphisms with Reproduction and Production Traits in Dairy Buffalo.

    PubMed

    Nasr, Maf; Awad, A; El Araby, I E

    2016-08-01

    This study aimed to detect the genetic variability in Leptin and Pit-1 genes using polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing also to explore their possible associations with reproductive and productive traits of Egyptian buffaloes. Regarding Leptin gene, three genotypes (AA, AG and GG) were identified with frequency of 0.54, 0.40 and 0.06, respectively, and the genotypes were distributed according to the Hardy-Weinberg equilibrium. Allele A was comparatively higher than G with frequency of 0.74 and 0.26, respectively. For Pit-1 gene, the association could not be performed due to the monomorphism (BB). The results showed that AA genotypes were found to be superior in most of production and reproduction traits. AA genotypes yielded more milk (2332.34 kg, p = 0.04) with higher fat% (6.10, p = 0.004) and fat yield (155.75 kg, p = 0.06), reach peak milk production at 42.19 days and required 2.19 services for conception in comparison with GG genotypes. Birthweight of animals with AA genotype was lesser than with GG genotype (39.35 and 43.67 kg, p = 0.02, respectively). The days open is numerically better in AA genotype animals (99.35 days), but the difference between the three genotypes was non-significant. The distinct significant associations reported in this study suggested that Leptin is reputable candidate genetic marker, which might be used to enhance animals' genetic potential for milk production in conjunction with reproduction. PMID:27334051

  17. Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.

    PubMed

    Ma, Zheng; Luo, Shuai; Xu, Xianhao; Bechthold, Andreas; Yu, Xiaoping

    2016-04-01

    Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase β-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic. PMID:26790416

  18. Production of Candida antarctica lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in an ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production.

    PubMed

    Hughes, Stephen R; Moser, Bryan R; Harmsen, Amanda J; Bischoff, Kenneth M; Jones, Marjorie A; Pinkelman, Rebecca; Bang, Sookie S; Tasaki, Ken; Doll, Kenneth M; Qureshi, Nasib; Saha, Badal C; Liu, Siqing; Jackson, John S; Robinson, Samantha; Cotta, Michael C; Rich, Joseph O; Caimi, Paolo

    2011-02-01

    A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. Saccharomyces cerevisiae strains expressing CALB protein or CALB Lyt-1 fusion protein were first grown on 2% (w/v) glucose, producing 9.3 g/L ethanol during fermentation. The carbon source was switched to galactose for GAL1-driven expression, and the CALB and CALB Lyt-1 enzymes expressed were tested for fatty acid ethyl ester (biodiesel) production. The synthetic enzymes catalyzed the formation of fatty acid ethyl esters from ethanol and either corn or soybean oil. It was further demonstrated that a one-step-charging resin, specifically selected for binding to lipase, was capable of covalent attachment of the CALB Lyt-1 enzyme, and that the resin-bound enzyme catalyzed the production of biodiesel. High-level expression of lipase in an ethanologenic yeast strain has the potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel. PMID:21609683

  19. Cloning heterologous genes into E. Coli for enzyme production and crystal growth: Problems of expression and microheterogeneity

    NASA Astrophysics Data System (ADS)

    Carter, Charles W.

    1988-07-01

    Protein crystal growth is heavily dependant on provision of large amounts of very pure protein. For this reason, molecular cloning will be used increasingly to permit the study of proteins which cannot otherwise be prepared in sufficient amounts, or purity, or both. We have obtained a stable clone of the tryptophanyl-tRNA synthetase from Bacillus stearothermophilus that is active in enzyme production. This result entailed two unusual aspects of interest to those using molecular cloning for enzyme production and crystal growth: (1) The cloning steps required stringent selection procedures that may have selected an unspecified mutational event 5' to the structural gene, because an as yet unknown flanking element of the B. stearothermophilus DNA produces a marked instability in plasmids containing the native DNA. (2) The homologous Escherichia coli trpS enzyme apparently interferes with crystallization of B. stearothermophilus tryptophanyl-tRNA synthetase purified from an E. coli strain. We have therefore deleted the E. coli chromosomal trpS gene by site-specific recombination of a recombinant lambda phage containing a marked deletion of the E. coli trpS gene. Enzyme prepared from this deletion strain crystallizes in a normal fashion, suitable for high-resolution X-ray crystallography studies. Crystallographic data sets from isomorphous crystals grown with native and cloned protein are identical to 3Åresolution to within normal scaling statistics.

  20. Lessons in détente or know thy host: the immunomodulatory gene products of myxoma virus.

    PubMed

    Zúñiga, Martha C

    2003-04-01

    The poxvirus, myxoma virus, encodes within its genome at least eleven different proteins that compromise, skew, or disable the innate and adaptive responses of its hosts. In the laboratory rabbit, Oryctolagus cuniculus, these effects result in myxomatosis, a fatal condition characterized by skin lesions and systemic immunosuppression. Interestingly, while myxoma infection also causes skin lesions in its natural host and in natural populations of O. cuniculus in Australia where this novel host and the virus have co-evolved, the condition of myxomatosis does not ensue and infection is not fatal. In this review I discuss the biochemical properties of the characterized immunomodulatory proteins of myxoma virus, and their pathogenic effects in laboratory rabbits. Disruption of any one myxoma immunomodulatory gene diminishes the severity of the infection without compromising infectivity. Thus, the characterized immunomodulatory genes appear not to be required for a productive infection in vivo. The differences in the severity of their effects in laboratory-bred versus wild O. cuniculus suggest that the outcome of myxoma infection is a consequence of the interplay between the viral immunomodulatory gene products and the cells and molecules of the host immune system. PMID:12734406

  1. Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production

    PubMed Central

    2010-01-01

    Background Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials. Results To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb) was introduced into nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIK)was introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L-1, 4 -fold nikkomycin X and 210 mg L-1, 1.8-fold nikkomycin Z) in the resulting exconjugants comparing with the parent strain (220 mg L-1 nikkomycin X and 120 mg L-1 nikkomycin Z). The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. Conclusion A high nikkomycins producing strain (1100 mg L-1 nikkomycins) was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites. PMID:20096125

  2. Analysis of 6-pyruvyl tetrahydropterin synthase, a target gene product of su(s) suppressor in Drosophila

    SciTech Connect

    Yim, J.J.; Park, Y.S.; Kim, J.H.; Jacobson, K.B. . Dept. of Microbiology; Oak Ridge National Lab., TN )

    1989-01-01

    Of the 20 suppressor mutants listed in Drosophila, su(s){sup 2} has been studied for the biochemical characterization of the suppression. In su(s){sup 2} suppression, some mutants at vermilion (v, 1-33.0), sable (s, 1-43.0), speck (sp, 2-107.0), and purple (pr, 2-54.5) are suppressible by mutants at the suppressor of sable (su(s)) locus. There have been many reports of suppression mechanism studies in purple led to purple loci. The biochemical studies in purple led to in vitro demonstration of a su(s){sup +} gene product as a negative effector against suppressed purple PPH{sub 4} synthase, which has been also demonstrated in the gene product of vermilion more recently. Both studies led to a proposal for a posttranslational mechanism in which the su(s){sup +} gene produces a substance that interferes with the function of the target enzymes from the purple and vermilion mutant. In contrast to the transcriptional regulation of the su(s) locus the posttranslational mechanism is based on qualitative differences between wild and suppressed enzyme. Therefore this investigation was to determine if any quantitative or qualitative differences exist between the mutant and wild type target enzymes. 7 refs., 4 figs.

  3. The bldB Gene Encodes a Small Protein Required for Morphogenesis, Antibiotic Production, and Catabolite Control in Streptomyces coelicolor

    PubMed Central

    Pope, Margaret K.; Green, Brian; Westpheling, Janet

    1998-01-01

    Mutants blocked at the earliest stage of morphological development in Streptomyces species are called bld mutants. These mutants are pleiotropically defective in the initiation of development, the ability to produce antibiotics, the ability to regulate carbon utilization, and the ability to send and/or respond to extracellular signals. Here we report the identification and partial characterization of a 99-amino-acid open reading frame (ORF99) that is capable of restoring morphogenesis, antibiotic production, and catabolite control to all of the bldB mutants. Of the existing bld mutants, bldB is of special interest because the phenotype of this mutant is the most pleiotropic. DNA sequence analysis of ORF99 from each of the existing bldB mutants identified base changes either within the coding region of the predicted protein or in the regulatory region of the gene. Primer extension analysis identified an apparent transcription start site. A promoter fusion to the xylE reporter gene showed that expression of bldB is apparently temporally regulated and that the bldB gene product is involved in the regulation of its own expression. PMID:9515926

  4. Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol.

    PubMed Central

    Tarczynski, M C; Jensen, R G; Bohnert, H J

    1992-01-01

    A bacterial gene encoding mannitol-1-phosphate dehydrogenase, mtlD, was engineered for expression in higher plants. Gene constructions were stably incorporated into tobacco plants. The mtlD gene was expressed and translated into a functional enzyme in tobacco, resulting in the synthesis and accumulation of mannitol, which was identified by NMR and mass spectroscopy. Mannitol concentrations exceeded 6 mumol/g (fresh weight) in the leaves and in the roots of some transformants, whereas this sugar alcohol was not detected in these organs of wild-type tobacco plants or of untransformed tobacco plants that underwent the same regeneration scheme. These experiments demonstrate that branch-points in plant carbohydrate metabolism can be generated by which novel gene products can utilize endogenous substrates to divert metabolic energy into novel compounds. Additionally, the system described here allows for physiological studies in which the responses of wild-type and transgenic tobacco to various environmental stimuli can be compared directly. Such studies will facilitate our understanding of the roles of sugar alcohols (e.g., in stress tolerance) in higher plants. Images PMID:1557364

  5. Volatile Gas Production by Methyl Halide Transferase: An In Situ Reporter Of Microbial Gene Expression In Soil.

    PubMed

    Cheng, Hsiao-Ying; Masiello, Caroline A; Bennett, George N; Silberg, Jonathan J

    2016-08-16

    Traditional visual reporters of gene expression have only very limited use in soils because their outputs are challenging to detect through the soil matrix. This severely restricts our ability to study time-dependent microbial gene expression in one of the Earth's largest, most complex habitats. Here we describe an approach to report on dynamic gene expression within a microbial population in a soil under natural water levels (at and below water holding capacity) via production of methyl halides using a methyl halide transferase. As a proof-of-concept application, we couple the expression of this gas reporter to the conjugative transfer of a bacterial plasmid in a soil matrix and show that gas released from the matrix displays a strong correlation with the number of transconjugant bacteria that formed. Gas reporting of gene expression will make possible dynamic studies of natural and engineered microbes within many hard-to-image environmental matrices (soils, sediments, sludge, and biomass) at sample scales exceeding those used for traditional visual reporting. PMID:27415416

  6. Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones

    PubMed Central

    Plevova, Karla; Francova, Hana Skuhrova; Burckova, Katerina; Brychtova, Yvona; Doubek, Michael; Pavlova, Sarka; Malcikova, Jitka; Mayer, Jiri; Tichy, Boris; Pospisilova, Sarka

    2014-01-01

    In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. PMID:24038023

  7. Prenatal exposure to dexamethasone disturbs sex-determining gene expression and fetal testosterone production in male embryos.

    PubMed

    Yun, Hyo Jung; Lee, Ji-Yeon; Kim, Myoung Hee

    2016-02-26

    Prenatal stress is known to cause intrauterine fetal growth retardation, and is also associated with various long-term effects in the form of metabolic and neurodevelopmental diseases in adults. Many of the diseases associated with prenatal stress exhibit a sex bias. Perturbations and vulnerability to prenatal stress are often more profound in males, but the mechanisms responsible for this relationship are not clear. We have previously shown that administration of the synthetic glucocorticoid, dexamethasone (Dex), at embryonic days 7.5, 8.5, and 9.5, induces embryonic growth restriction in a sex-dependent manner in a mouse model. Here we examined the effect of prenatal exposure to Dex on gonadal development. During male gonadal development, sex-determining genes, such as Sry, Sox9, and other downstream genes, were found to be dysregulated in response to prenatal Dex, whereas the genes for the ovarian pathway were affected to a lesser degree in females. In addition, fetal testosterone concentrations were decreased by prenatal exposure to Dex, in parallel with reduced numbers of 3β-hydroxysteroid dehydrogenase (3β-HSD)-positive cells in the embryonic testis. These results show that prenatal exposure to Dex differentially influences male versus female on the gene expression and hormone production during sex determination. We believe these studies provide valuable insights into possible mechanisms responsible for sex-specific responses to prenatal stress. PMID:26827828

  8. A Sulfonylurea Herbicide Resistance Gene from Arabidopsis thaliana as a New Selectable Marker for Production of Fertile Transgenic Rice Plants.

    PubMed

    Li, Z; Hayashimoto, A; Murai, N

    1992-10-01

    A mutant acetolactate synthase (ALS) gene, csr1-1, isolated from sulfonylurea herbicide-resistant Arabidopsis thaliana, was placed under control of a cauliflower mosaic virus 35S promoter (35S). Rice protoplasts were transformed with the 35S/ALS chimeric gene and regenerated into fertile transgenic rice (Oryza sativa) plants. The 35S/ALS gene was expressed effectively as demonstrated by northern blot hybridization analysis, and conferred to transformed calli at least 200-fold greater chlorsulfuron resistance than nontransformed control calli. Effective selection of 35S/ALS-transformed protoplasts was achieved at extremely low chlorsulfuron concentrations of 10 nm. The results demonstrated that the 35S/ALS gene is an alternative selectable marker for rice protoplast transformation and fertile transgenic rice production. The results also suggest that the mutant form of Arabidopsis ALS enzyme operates normally in rice cells. Thus, the mechanism of protein transport to chloroplast and ALS inhibition by chlorsulfuron is apparently conserved among plant species as diverse as Arabidopsis (dicotyledon) and rice (monocotyledon). PMID:16653044

  9. Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product.

    PubMed Central

    Brooks, A D; He, S Y; Gold, S; Keen, N T; Collmer, A; Hutcheson, S W

    1990-01-01

    The ability of Erwinia chrysanthemi to cause soft-rot diseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase E. chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone (pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase (ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6. Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL- ExoPL- mutant of EC16 was constructed that exhibited reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005. Images PMID:2254266

  10. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.

    PubMed Central

    Cyert, M S; Kunisawa, R; Kaim, D; Thorner, J

    1991-01-01

    Calcineurin, or phosphoprotein phosphatase type 2B (PP2B), is a calmodulin-regulated phosphoprotein phosphatase. We isolated a gene encoding a yeast PP2B homolog (CNA1) by screening a yeast genomic DNA library in the expression vector lambda gt11, first with 125I-labeled yeast calmodulin and then with a human cDNA encoding the catalytic (or A) subunit of calcineurin. The predicted CNA1 gene product is 54% identical to its mammalian counterpart. Using the polymerase chain reaction (PCR) with oligonucleotide primers based on sequences conserved between CNA1 and mammalian PP2B genes, we isolated a second gene, CNA2. CNA2 is identical to PP2Bw, a partial cDNA clone previously described by others as originating from rabbit brain tissue. Our findings demonstrate that a unicellular eukaryote contains phosphoprotein phosphatases of the 2B class. Haploid cells containing a single cna1 or cna2 null mutation, or both mutations, were viable. MATa cna1 cna2 double mutants were more sensitive than wild-type cells or either single mutant to growth arrest induced by the mating pheromone alpha factor and failed to resume growth during continuous exposure to alpha factor. Thus, calcineurin action antagonizes the mating-pheromone response pathway. Images PMID:1651503

  11. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  12. 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

  13. Significant enhancement of methionol production by co-expression of the aminotransferase gene ARO8 and the decarboxylase gene ARO10 in Saccharomyces cerevisiae.

    PubMed

    Yin, Sheng; Lang, Tiandan; Xiao, Xiao; Liu, Li; Sun, Baoguo; Wang, Chengtao

    2015-03-01

    Methionol is an important volatile sulfur flavor compound, which can be produced via the Ehrlich pathway in Saccharomyces cerevisiae. Aminotransferase and decarboxylase are essential enzymes catalyzing methionol biosynthesis. In this work, two aminotransferase genes ARO8 and ARO9 and one decarboxylase gene ARO10 were introduced into S. cerevisiae S288c, respectively, via an expression vector. Over-expression of ARO8 resulted in higher aminotransferase activity than that of ARO9. And the cellular decarboxylase activity was remarkably increased by over-expression of ARO10. A co-expression vector carrying both ARO8 and ARO10 was further constructed to generate the recombinant strain S810. Shaking flask experiments showed that the methionol yield from S810 reached 1.27 g L(-1), which was increased by 51.8 and 68.8% compared to that from the wild-type strain and the control strain harboring the empty vector. The fed-batch fermentation by strain S810 produced 3.24 g L(-1) of methionol after 72 h of cultivation in a bioreactor. These results demonstrated that co-expression of ARO8 and ARO10 significantly boosted the methionol production. It is the first time that more than 3.0 g L(-1) of methionol produced by genetically engineered yeast strain was reported by co-expression of the aminotransferase and decarboxylase via the Ehrlich pathway. PMID:25743068

  14. Evaluation of bovine chemerin (RARRES2) gene variation on beef cattle production traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous genome-wide association study in cattle based on >48,000 single nucleotide polymorphism (SNP) markers identified markers on chromosome 4 near the chemerin gene associated with average daily feed intake (ADFI) in steers (P<0.008). Chemerin is an adipokine that has been associated with obe...

  15. Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions o...

  16. Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease res...

  17. Controlled Striatal DOPA Production From a Gene Delivery System in a Rodent Model of Parkinson's Disease

    PubMed Central

    Cederfjäll, Erik; Broom, Lauren; Kirik, Deniz

    2015-01-01

    Conventional symptomatic treatment for Parkinson's disease (PD) with long-term L-3,4-dihydroxyphenylalanine (DOPA) is complicated with development of drug-induced side effects. In vivo viral vector-mediated gene expression encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) provides a drug delivery strategy of DOPA with distinct advantages over pharmacotherapy. Since the brain alterations made with current gene transfer techniques are irreversible, the therapeutic approaches taken to the clinic should preferably be controllable to match the needs of each individual during the course of their disease. We used a recently described tunable gene expression system based on the use of destabilized dihydrofolate reductase (DD) and generated a N-terminally coupled GCH1 enzyme (DD-GCH1) while the TH enzyme was constitutively expressed, packaged in adeno-associated viral (AAV) vectors. Expression of DD-GCH1 was regulated by the activating ligand trimethoprim (TMP) that crosses the blood–brain barrier. We show that the resulting intervention provides a TMP-dose-dependent regulation of DOPA synthesis that is closely linked to the magnitude of functional effects. Our data constitutes the first proof of principle for controlled reconstitution of dopamine capacity in the brain and suggests that such next-generation gene therapy strategies are now mature for preclinical development toward use in patients with PD. PMID:25592335

  18. BIOFILTRATION INCORPORATING GENE SILENCING TECHNOLOGY FOR THE PRODUCTION OF METHANOL FROM METHANE CONTAINING WASTE GASES

    EPA Science Inventory

    I expect the proposed and revised approach will work, as there are multiple examples of plasmid-based gene silencing systems in nature (HOK/SOK is a perfect example). The challenge will be in developing a strong plasmid for use in methanotrophs.

    Potential to ...

  19. Pollen and seed mediated gene flow in commercial alfalfa seed production fields

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potential for gene flow has been widely recognized since alfalfa is pollinated by bees. The Western US is a major exporter of alfalfa seed and hay and the organic dairy industry is one of the fastest growing agricultural sectors. Because of this, many alfalfa producers are impacted by market sen...

  20. Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 segrega...

  1. Nasopharyngeal carcinomas frequently lack the p16/MTS1 tumor suppressor protein but consistently express the retinoblastoma gene product.

    PubMed Central

    Gulley, M. L.; Nicholls, J. M.; Schneider, B. G.; Amin, M. B.; Ro, J. Y.; Geradts, J.

    1998-01-01

    The p16/MTS1 gene is altered by deletion, mutation, or hypermethylation in a wide variety of human cancers. As a result of deficient p16 protein, these cancers lack a critical mechanism for halting G1/S cell cycle progression. In the current study, 59 cases of nasopharyngeal carcinoma were evaluated for expression of the p16 tumor suppressor protein by immunohistochemical analysis of paraffin-embedded tissue. There was no detectable p16 in 38/59 cases (64%), which implies a very high rate of p16 inactivation in this type of cancer. On the other hand, the retinoblastoma gene product, which also regulates the G1 to S phase transition of the cell cycle, was consistently expressed in nasopharyngeal carcinomas by immunohistochemical analysis. These results implicate p16 inactivation but not Rb alteration in the stepwise progression of nasopharyngeal carcinogenesis. Images Figure 1 Figure 2 PMID:9546345

  2. Structural analysis of the RH-like blood group gene products in nonhuman primates

    SciTech Connect

    Salvignol, I.; Calvas, P.; Blancher, A.; Socha, W.W.; Colin, Y.; Le Van Kim, C.; Bailly, P.; Cartron, J.P.; Ruffie, J.; Blancher, A.

    1995-03-01

    Rh-related transcripts present in bone marrow samples from several species of nonhuman primates (chimpanzee, gorilla, gibbon, crab-eating macaque) have been amplified by RT-polymerase chain reaction using primers deduced from the sequence of human RH genes. Nucleotide sequence analysis of the nonhuman transcripts revealed a high degree of similarity to human blood group Rh sequences, suggesting a great conservation of the RH genes throughout evolution. Full-length transcripts, potentially encoding 417 amino acid long proteins homologous to Rh polypeptides, were characterized, as well as mRNA isoforms which harbored nucleotide deletions or insertions and potentially encode truncated proteins. Proteins of 30-40,000 M{sub r}, immunologically related to human Rh proteins, were detected by western blot analysis with antipeptide antibodies, indicating that Rh-like transcripts are translated into membrane proteins. Comparison of human and nonhuman protein sequences was pivotal in clarifying the molecular basis of the blood group C/c polymorphism, showing that only the Pro103Ser substitution was correlated with C/c polymorphism. In addition, it was shown that a proline residue at position 102 was critical in the expression of C and c epitopes, most likely by providing an appropriate conformation of Rh polypeptides. From these data a phylogenetic reconstruction of the RH locus evolution has been calculated from which an unrooted phylogenetic tree could be proposed, indicating that African ape Rh-like genes would be closer to the human RhD gene than to the human RhCE gene. 55 refs., 4 figs., 1 tab.

  3. Evaluation of Bovine chemerin (RARRES2) Gene Variation on Beef Cattle Production Traits1

    PubMed Central

    Lindholm-Perry, Amanda K.; Kuehn, Larry A.; Rempel, Lea A.; Smith, Timothy P. L.; Cushman, Robert A.; McDaneld, Tara G.; Wheeler, Tommy L.; Shackelford, Steven D.; King, David A.; Freetly, Harvey C.

    2012-01-01

    A previous study in cattle based on >48,000 markers identified markers on chromosome 4 near the chemerin gene associated with average daily feed intake (ADFI) in steers (P < 0.008). Chemerin is an adipokine associated with obesity and metabolic syndrome in humans, representing a strong candidate gene potentially underlying the observed association. To evaluate whether the bovine chemerin gene is involved in feed intake, 16 markers within and around the gene were tested for association in the same resource population. Eleven were nominally significant for ADFI (P < 0.05) and two were significant after Bonferroni correction. Two and five SNP in this region were nominally significant for the related traits of average daily gain (ADG) and residual feed intake (RFI), respectively. All markers were evaluated for effects on meat quality and carcass phenotypes. Many of the markers associated with ADFI were associated with hot carcass weight (HCW), adjusted fat thickness (AFT), and marbling (P < 0.05). Marker alleles that were associated with lower ADFI were also associated with lower HCW, AFT, and marbling. Markers associated with ADFI were genotyped in a validation population of steers representing 14 breeds to determine predictive merit across populations. No consistent relationships for ADFI were detected. To determine whether cattle feed intake or growth phenotypes might be related to chemerin transcript abundance, the expression of chemerin was evaluated in adipose of 114 heifers that were siblings of the steers in the discovery population. Relative chemerin transcript abundance was not correlated with ADFI, ADG, or RFI, but associations with body condition score and yearling weight were observed. We conclude that variation in the chemerin gene may underlie observed association in the resource population, but that additional research is required to determine if this variation is widespread among breeds and to develop robust markers with predictive merit

  4. The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

    PubMed

    Sato, Masahiro; Maeda, Kosuke; Koriyama, Miyu; Inada, Emi; Saitoh, Issei; Miura, Hiromi; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi; Miyoshi, Kazuchika

    2016-01-01

    The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particul