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Sample records for reca gene products

  1. Cloning of the Helicobacter pylori recA gene and functional characterization of its product.

    PubMed

    Schmitt, W; Odenbreit, S; Heuermann, D; Haas, R

    1995-09-20

    The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned a recA homolog from Helicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to the Campylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus sigma 70 promoter sequence was found upstream of recA. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream of recA. Compared to the wild-type strains, isogenic H. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinant H. pylori RecA protein produced in Escherichia coli strain GC6 (recA-) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein in H. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of the H. pylori mutant using the cloned recA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA in H. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced in E. coli, giving rise to a smaller but inactive protein. PMID:7476856

  2. Cloning and sequencing the recA+ genes of Acetobacter polyoxogenes and Acetobacter aceti: construction of recA- mutants of by transformation-mediated gene replacement.

    PubMed

    Tayama, K; Fukaya, M; Takemura, H; Okumura, H; Kawamura, Y; Horinouchi, S; Beppu, T

    1993-05-15

    The recA+ gene of Acetobacter polyoxogenes was cloned as a gene that conferred methyl methanesulfonate resistance (MMSR) on the RecA- Escherichia coli HB101. The cloned recA+ gene also conferred (i) resistance to UV irradiation, (ii) enhanced intrachromosomal recombination, and (iii) permitted prophage phi 80 induction in E. coli recA- lysogens. Nucleotide sequence determination revealed that the recA product consists of 348 amino acids (aa) corresponding to 38 kDa, and shows significant similarity to RecA proteins from other Gram- bacteria. Next, a portion of recA from Acetobacter aceti was cloned by using polymerase chain reaction with oligodeoxyribonucleotide primers design based on the A. polyoxogenes recA sequence. Due to availability of efficient host-vector and transformation systems in A. aceti, recA mutants of A. aceti were obtained by transformation-mediated gene replacement with the cloned A. aceti recA gene which was inactivated by insertion of the kanamycin-resistance-encoding gene from pACYC177. The recA mutants obtained in this way showed similar phenotypes to those of E. coli recA strains, such as increased sensitivity to MMS and to UV irradiation, and decreased homologous recombination.

  3. Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA.

    PubMed

    García-Vázquez, Francisco A; Ruiz, Salvador; Matás, Carmen; Izquierdo-Rico, M José; Grullón, Luis A; De Ondiz, Aitor; Vieira, Luis; Avilés-López, Karen; Gutiérrez-Adán, Alfonso; Gadea, Joaquín

    2010-08-01

    Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 microg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

  4. Isolation of the Helicobacter pylori recA gene and involvement of the recA region in resistance to low pH.

    PubMed

    Thompson, S A; Blaser, M J

    1995-06-01

    To understand the potential roles of the important DNA repair protein RecA in Helicobacter pylori pathogenesis, we cloned the recA gene from H. pylori 84-183. Degenerate PCR primers based on conserved RecA protein regions were used to amplify a portion of H. pylori recA, which was used as a probe to isolate the full-length recA gene from H. pylori genomic libraries. The H. pylori recA gene encoded a protein of 347 amino acids with a molecular mass of 37.6 kDa. As expected, H. pylori RecA was highly similar to other RecA proteins and most closely resembled that of Campylobacter jejuni (75% identity). Immediately downstream of recA was an open reading frame whose predicted product showed 58% identity to the Bacillus subtilis enolase protein. recA and eno were disrupted in H. pylori 84-183 by insertion of antibiotic resistance genes. Reverse transcription-PCR demonstrated that recA and eno were cotranscribed and that insertion of the kanamycin resistance gene into recA had polar effects on expression of the downstream eno. The H. pylori recA mutants were severely impaired in their ability to survive treatment with UV light and methyl methanesulfonate and with the antimicrobial agents ciprofloxacin and metronidazole. The eno mutant had sensitivities to UV light and metronidazole intermediate to those of wild-type and recA strains, suggesting that truncation of the recA-eno transcript resulted in lowered recA expression. For survival at low pH, a recA mutant was approximately 10-fold more sensitive than strain 84-183, while the eno mutant demonstrated intermediate susceptibility. This difference occurred in the presence or absence of urea, implying the involvement of a gene in the recA region in an acid resistance mechanism distinct from that mediated by urease. PMID:7768597

  5. Isolation and characterization of the recA gene of Rhizobium meliloti.

    PubMed Central

    Better, M; Helinski, D R

    1983-01-01

    Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation. Images PMID:6305915

  6. Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida.

    PubMed Central

    Berg, J M; Mdluli, K E; Nano, F E

    1992-01-01

    A gene locus that is functionally analogous to the recA gene of Escherichia coli was molecularly cloned from Francisella novicida. The cloned gene was found to suppress the sensitivity of an E. coli strain to DNA-damaging agents and to support genetic recombination in E. coli. After transposon mutagenesis, the recA-like gene locus was returned to F. novicida and a UV-sensitive F. novicida strain was isolated. In contrast to the wild-type strain, this UV-sensitive strain could not be transformed with chromosomal DNA. Images PMID:1309722

  7. Direct evidence of recombination in the recA gene of Aeromonas bestiarum.

    PubMed

    Sanglas, Ariadna; Albarral, Vicenta; Farfán, Maribel; Lorén, J Gaspar; Fusté, M Carmen

    2016-03-01

    Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248 bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures.

  8. Recombinogenic activity of chimeric recA genes (Pseudomonas aeruginosa/Escherichia coli): a search for RecA protein regions responsible for this activity.

    PubMed Central

    Bakhlanova, I V; Ogawa, T; Lanzov, V A

    2001-01-01

    In the background of weak, if any, constitutive SOS function, RecA from Pseudomonas aeruginosa (RecAPa) shows a higher frequency of recombination exchange (FRE) per DNA unit length as compared to RecA from Escherichia coli (RecAEc). To understand the molecular basis for this observation and to determine which regions of the RecAPa polypeptide are responsible for this unusual activity, we analyzed recAX chimeras between the recAEc and recAPa genes. We chose 31 previously described recombination- and repair-proficient recAX hybrids and determined their FRE calculated from linkage frequency data and constitutive SOS function expression as measured by using the lacZ gene under control of an SOS-regulated promoter. Relative to recAEc, the FRE of recAPa was 6.5 times greater; the relative alterations of FRE for recAX genes varied from approximately 0.6 to 9.0. No quantitative correlation between the FRE increase and constitutive SOS function was observed. Single ([L29M] or [I102D]), double ([G136N, V142I]), and multiple substitutions in related pairs of chimeric RecAX proteins significantly altered their relative FRE values. The residue content of three separate regions within the N-terminal and central but not the C-terminal protein domains within the RecA molecule also influenced the FRE values. Critical amino acids in these regions were located close to previously identified sequences that comprise the two surfaces for subunit interactions in the RecA polymer. We suggest that the intensity of the interactions between the subunits is a key factor in determining the FRE promoted by RecA in vivo. PMID:11560883

  9. Characterization of the Pseudomonas aeruginosa recA gene: the Les- phenotype

    SciTech Connect

    Kokjohn, T.A.; Miller, R.V.

    1988-02-01

    The Les- phenotype (lysogeny establishment deficient) is a pleiotropic effect of the lesB908 mutation of Pseudomonas aeruginosa PAO. lesB908-containing strains are also (i) deficient in general recombination, (ii) sensitive to UV irradiation, and (iii) deficient in UV-stimulated induction of prophages. The P. aeruginosa recA-containing plasmid pKML3001 complemented each of these pleiotropic characteristics of the lesB908 mutation, supporting the hypothesis that lesB908 is an allele of the P. aeruginosa recA gene. The phenotypic effects of the lesB908 mutation may be best explained by the hypothesis that the lesB908 gene product is altered in such a way that it has lost synaptase activity but possesses intrinsic protease activity in the absence of DNA damage. The Les- phenotype is a result of the rapid destruction of newly synthesized phage repressor, resulting in lytic growth of the infecting virus. This hypothesis is consistent with the observations that increasing the number of copies of the phage repressor gene by increasing the multiplicity of infection (i.e., average number of phage genomes per cell) or by introducing the cloned phage repressor gene into a lesB908 mutant will also suppress the Les- phenotype in a phage-specific fashion.

  10. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    PubMed

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

  11. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    PubMed

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products. PMID:26898909

  12. Comparative evolution of the recA gene of surface and deep subsurface microorganisms (an evolutionary clock of intermediate rate). Final report

    SciTech Connect

    Miller, R.V.

    1998-04-01

    Because of the ability of the recA protein product to maintain both DNA integrity and increase genetic diversity, this gene may be essential to the survival of microorganisms following the damaging effects of numerous environmental stresses such as exposure to solar UV radiation, exposure to gamma radiation, starvation, and changing environments. While the various activities and amino-acid sequence of recA have been highly conserved among the eubacteria and archaea, little is known as to whether a strict structure-function relationship has been conserved. In other words, are the same regions of this highly plastic, functionally heterogeneous protein involved in the same catalytic capacities throughout the bacterial kingdom? While it is reasonable to assume that this type of conservation has also occurred, we felt it necessary to test the assumption by demonstrating that mutations in different genera of bacteria which eliminate similar functions (i.e., lead to similar phenotypes) are caused by changes in the amino-acid sequence in the same regions of their recA proteins. Therefore, we located the changes in nucleotide sequence in two recA mutants of P. aeruginosa which displayed mutant phenotypes in recombination and UV resistance. Our assumption was that if structure-function relationships held, these mutations would be found in areas already identified as essential for the function of the E. coli recA protein.

  13. Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

    PubMed Central

    Duwat, P; Ehrlich, S D; Gruss, A

    1992-01-01

    Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair. PMID:1514816

  14. Nucleotide fluctuation of RecA repair gene in Siberian permafrost Psychrobacter cryohalolentis K5

    NASA Astrophysics Data System (ADS)

    Tremberger, George, Jr.; Holden, T.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Marchese, P.; Lieberman, D.; Cheung, T.

    2008-08-01

    A nucleotide sequence can be expressed as a numerical sequence when each nucleotide is assigned its proton number. A resulting gene numerical sequence can be investigated for its fractal dimension in terms of evolution and chemical properties for comparative studies. We have investigated such nucleotide fluctuation in the RecA repair gene of Psychrobacter cryohalolentis K5, Psychrobacter arcticus 273-4, and Psychrobacter sp. PRwf-1. The fractal dimension was found to correlate with the gene's operating temperature with the highest fractal dimension associated with P. cryohalolentis K5 living at the low temperatures found in Siberian permafrost. The CpG dinucleotide content was found to be about 5% for the three species of Psychrobacters, which is substantially lower than that of Deinococcus radiodurans at about 12%. The average nucleotide pair-wise free energy was found to be lowest for Psychrobacter sp. PRwf-1, the species with the lowest fractal dimension of the three, consistent with the recent finding that Psychrobacter sp. PRw-f1 has a temperature growth maximum of 15-20°C higher than P. arcticus 273-4 and P. cryohaloentis K5. The results suggest that microbial vitality in extreme environments is associated with fractal dimension as well as high CpG dinucleotide content, while the average nucleotide pair-wise free energy is related to the operating environment. Evidence that extreme temperature operation would impose constraints measurable by Shannon entropy is also discussed. A quantitative estimate of an entropy-based measure having the characteristics of a mechanical pressure shows that the Psychrobacter RecA sequence experiences lower pressure than that of the human HAR1 sequence.

  15. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

    PubMed

    Yassien, M A M; Elfaky, M A

    2015-11-01

    A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α. PMID:26375447

  16. Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda

    SciTech Connect

    Calsou, P.; Villaverde, A.; Defais, M.

    1987-10-01

    The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts.

  17. Regulation of bacterial RecA protein function.

    PubMed

    Cox, Michael M

    2007-01-01

    The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes. PMID:17364684

  18. Directed molecular screening for RecA ATPase inhibitors.

    PubMed

    Wigle, Tim J; Singleton, Scott F

    2007-06-15

    The roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make it an attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z' factor of 0.83 in 96-well microplates, to assess whether 18 select compounds could inhibit ATP hydrolysis by RecA. The compounds represented five sets of related inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotide analogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not active against RecA, we found that three suramin-like agents substantially inhibited RecA's ATPase activity. PMID:17499507

  19. The RecA protein of Helicobacter pylori requires a posttranslational modification for full activity.

    PubMed

    Fischer, Wolfgang; Haas, Rainer

    2004-02-01

    The RecA protein is a central component of the homologous recombination machinery and of the SOS system in most bacteria. In performing these functions, it is involved in DNA repair processes and plays an important role in natural transformation competence. This may be especially important in Helicobacter pylori, where an unusually high degree of microdiversity among strains is generated by homologous recombination. We have suggested previously that the H. pylori RecA protein is subject to posttranslational modifications that result in a slight shift in its electrophoretic mobility. Here we show that at least two genes downstream of recA are involved in this modification and that this process is dependent on genes involved in glycosylation and lipopolysaccharide biosynthesis. Site-directed mutagenesis of a putative glycosylation site results in production of an unmodified RecA protein. This posttranslational modification is not involved in membrane targeting or cell division functions but is necessary for the full function of RecA in DNA repair. Thus, it might be an adaptation to the specific requirements of H. pylori in its natural environment. PMID:14729704

  20. Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.

    PubMed

    Stefanska, Aleksandra; Kaczorowska, Anna-Karina; Plotka, Magdalena; Fridjonsson, Olafur H; Hreggvidsson, Gudmundur O; Hjorleifsdottir, Sigridur; Kristjansson, Jakob K; Dabrowski, Slawomir; Kaczorowski, Tadeusz

    2014-07-20

    The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.

  1. Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants.

    PubMed Central

    Tessman, E S; Peterson, P

    1985-01-01

    As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized lambda recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The lambda recA phages were recognized by their altered plaque colors, and the RecA protease activity of the lambda recA mutant lysogens was measured by expression of beta-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prtc; in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prtc mutants were recombinase negative and were designated Prtc Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prtc Rec+ mutant (recA441), the new Prtc Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prtc Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prtc mutants as the strongest among 150. These five strongest Prtc mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prtc mutants. Strong Prtc Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described. PMID:3160686

  2. Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

    PubMed Central

    Palejwala, V A; Wang, G E; Murphy, H S; Humayun, M Z

    1995-01-01

    The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene. PMID:7592365

  3. Phylogeny of Vibrio cholerae Based on recA Sequence

    PubMed Central

    Stine, O. Colin; Sozhamannan, Shanmuga; Gou, Qing; Zheng, Siqen; Morris, J. Glenn; Johnson, Judith A.

    2000-01-01

    We sequenced a 705-bp fragment of the recA gene from 113 Vibrio cholerae strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly, Vibrio parahaemolyticus and Vibrio vulnificus strains formed out-groups; we also identified isolates which resembled V. cholerae biochemically but which did not cluster with V. cholerae. In many instances, V. cholerae serogroup designations did not correlate with phylogeny, as reflected by recA sequence divergence. This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among V. cholerae strains. PMID:11083852

  4. Modulating Cellular Recombination Potential through Alterations in RecA Structure and Regulation

    PubMed Central

    Bakhlanova, Irina V.; Dudkina, Alexandra V.; Baitin, Dima M.; Knight, Kendall L.; Cox, Michael M.; Lanzov, Vladislav A.

    2010-01-01

    The wild type E. coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to 6 fold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to 4 fold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50 fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of Escherichia coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function – filament formation and the inherent DNA pairing activity of the formed filaments. PMID:21143322

  5. Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

    PubMed Central

    Kim, Taejin; Chitteni-Pattu, Sindhu; Cox, Benjamin L.; Wood, Elizabeth A.; Sandler, Steven J.; Cox, Michael M.

    2015-01-01

    The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism. PMID:26047498

  6. recA and catalase in H sub 2 O sub 2 -mediated toxicity in Neisseria gonorrhoeae

    SciTech Connect

    Hassett, D.J.; Charniga, L.; Cohen, M.S. )

    1990-12-01

    Neisseria gonorrhoeae cells defective in the biosynthesis of the recA gene product are no more sensitive to hydrogen peroxide than wild-type cells. Although gonococci possess nearly 100-fold-greater catalase levels than Escherichia coli, they are more susceptible to hydrogen peroxide than this organism. The natural niche of gonococci undoubtedly results in exposure to oxidant stress; however, they do not demonstrate particularly efficient antioxidant defense systems.

  7. RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens.

    PubMed

    Odahara, Masaki; Inouye, Takayuki; Nishimura, Yoshiki; Sekine, Yasuhiko

    2015-11-01

    Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear-encoded chloroplast-localized homolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P. patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13-63 bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.

  8. Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation.

    PubMed

    Vial, Ludovic; Pothier, Joël F; Normand, Philippe; Moënne-Loccoz, Yvan; Bally, René; Wisniewski-Dyé, Florence

    2004-07-15

    The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4VI at frequencies of 10(-4) to 10(-3) per cell per generation. Variant 4VI displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A. lipoferum 4B was cloned and sequenced and a recA mutant (termed 4BrecA) was constructed by allelic exchange. Strain 4BrecA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity. The ability to generate variants in vitro was not altered; the variants from 4BrecA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4BrecA was increased by up to 10-fold. So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA. PMID:15251210

  9. Characterization of dinY, a new Escherichia coli DNA repair gene whose products are damage inducible even in a lexA(Def) background.

    PubMed Central

    Petit, C; Cayrol, C; Lesca, C; Kaiser, P; Thompson, C; Defais, M

    1993-01-01

    Bacteriophage Mu dX(Ap lac) was used to isolate a mutation in an Escherichia coli lexA(Def) strain representing a previously undescribed gene (dinY) which does not seem to be under the direct control of LexA. The insertion created a dinY::lacZ fusion in which beta-galactosidase expression required a DNA-damaging treatment (UV irradiation or mitomycin) and activable RecA protein. This strain showed a decreased Weigle reactivation of bacteriophage lambda. However, it was fully inducible for UV mutagenesis. Two-dimensional gel electrophoresis analysis identified two spots absent in the mutant which were both UV inducible only in the presence of activated RecA protein (RecA*). This finding suggests that the dinY::lacZ fusion lies in a gene either that is under the direct control of activated RecA or whose product undergoes RecA*-dependent posttranscriptional/posttranslational modification(s). The dinY gene may also control the expression of some other gene(s) and/or lie in an operon. The fusion was mapped at a position between 41 and 41.5 min on the E. coli chromosome, in the vicinity of the ruv operon. Images PMID:8423139

  10. A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage.

    PubMed

    Rapa, Rita A; Islam, Atiqul; Monahan, Leigh G; Mutreja, Ankur; Thomson, Nicholas; Charles, Ian G; Stokes, Harold W; Labbate, Maurizio

    2015-04-01

    Lateral gene transfer (LGT) has been crucial in the evolution of the cholera pathogen, Vibrio cholerae. The two major virulence factors are present on two different mobile genetic elements, a bacteriophage containing the cholera toxin genes and a genomic island (GI) containing the intestinal adhesin genes. Non-toxigenic V. cholerae in the aquatic environment are a major source of novel DNA that allows the pathogen to morph via LGT. In this study, we report a novel GI from a non-toxigenic V. cholerae strain containing multiple genes involved in DNA repair including the recombination repair gene recA that is 23% divergent from the indigenous recA and genes involved in the translesion synthesis pathway. This is the first report of a GI containing the critical gene recA and the first report of a GI that targets insertion into a specific site within recA. We show that possession of the island in Escherichia coli is protective against DNA damage induced by UV-irradiation and DNA targeting antibiotics. This study highlights the importance of genetic elements such as GIs in the evolution of V. cholerae and emphasizes the importance of environmental strains as a source of novel DNA that can influence the pathogenicity of toxigenic strains.

  11. Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect*

    PubMed Central

    Cox, Julia M.; Li, Hao; Wood, Elizabeth A.; Chitteni-Pattu, Sindhu; Inman, Ross B.; Cox, Michael M.

    2008-01-01

    The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture. PMID:18603529

  12. Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence ▿

    PubMed Central

    Aranda, Jesús; Bardina, Carlota; Beceiro, Alejandro; Rumbo, Soraya; Cabral, Maria P.; Barbé, Jordi; Bou, Germán

    2011-01-01

    RecA is the major enzyme involved in homologous recombination and plays a central role in SOS mutagenesis. In Acinetobacter spp., including Acinetobacter baumannii , a multidrug-resistant bacterium responsible for nosocomial infections worldwide, DNA repair responses differ in many ways from those of other bacterial species. In this work, the function of A. baumannii RecA was examined by constructing a recA mutant. Alteration of this single gene had a pleiotropic effect, showing the involvement of RecA in DNA damage repair and consequently in cellular protection against stresses induced by DNA damaging agents, several classes of antibiotics, and oxidative agents. In addition, the absence of RecA decreased survival in response to both heat shock and desiccation. Virulence assays in vitro (with macrophages) and in vivo (using a mouse model) similarly implicated RecA in the pathogenicity of A. baumannii . Thus, the data strongly suggest a protective role for RecA in the bacterium and indicate that inactivation of the protein can contribute to a combined therapeutic approach to controlling A. baumannii infections. PMID:21642465

  13. Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

    PubMed Central

    Piechura, Joseph R.; Tseng, Tzu-Ling; Hsu, Hsin-Fang; Byrne, Rose T.; Windgassen, Tricia A.; Chitteni-Pattu, Sindhu; Battista, John R.; Li, Hung-Wen; Cox, Michael M.

    2015-01-01

    Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism. PMID:25559557

  14. Image analysis reveals that Escherichia coli RecA protein consists of two domains.

    PubMed Central

    Yu, X; Egelman, E H

    1990-01-01

    The Escherichia coli RecA protein catalyzes homologous genetic recombination by forming helical polymers around DNA molecules. These polymers have an ATPase activity, which is essential for the movement of strands between two DNA molecules. One obstacle to structural studies of the RecA filament has been that the ATPase results in a dynamical polymer containing a mixture of states with respect to the bound ATP and its hydrolytic products. We have formed filaments which are trapped in the ADP-Pi state by substituting AIF4- for the Pi, and have used these stable filaments to generate a three-dimensional reconstruction from electron micrographs. The resolution of the reconstruction is sufficient to resolve the 38-k RecA subunit into two nearly equal domains. This reconstruction provides the most detailed view yet of the RecA protein, and serves as a framework within which existing biochemical data on RecA can be understood. Images FIGURE 1 FIGURE 8 FIGURE 12 PMID:2137715

  15. RecA Expression in Response to Solar UVR in the Marine Bacterium Vibrio natriegens.

    PubMed

    Booth, M.G.; Jeffrey, W.H.; Miller, R.V.

    2001-12-01

    Solar ultraviolet radiation may produce daily stress on marine and estuarine communities as cells are damaged and repair that damage. Reduction in the earth's stratospheric ozone layer has increased awareness of the potential effects that ultraviolet radiation may have in the environment, including how marine bacteria respond to changes in solar radiation. We examined the use of the bacterial RecA protein as an indicator of the potential of bacteria to repair DNA damage caused by solar UV irradiation using the marine bacterium Vibrio natriegens as a model. RecA is universally present in bacteria and is a regulator protein for the so-called Dark Repair Systems, which include excision repair, postreplication recombinational repair, and mutagenic or SOS repair. Solar UVB and UVA both reduced V. natriegens viability in seawater microcosms. After exposure to unfiltered solar radiation or radiation in which UVB was blocked, survival dropped below 1%, whereas visible light from which UVA and UVB had been filtered had no effect on survival. Using a RecA-specific antibody for detection, RecA protein was induced by solar radiation in a diel pattern in marine microcosms conducted in the Gulf of Mexico. Peak induction was observed at dusk each day. Although RecA expression was correlated with the formation of UVB-induced cyclobutyl pyrimidine dimers, longer wavelength UVA radiation also induced recA gene expression. Our results demonstrate that RecA-regulated, light-independent repair is an important component in the ability of marine bacteria to survive exposure to solar ultraviolet radiation and that RecA expression is a useful monitor of bacterial repair after exposure to solar UVR.

  16. RecX Facilitates Homologous Recombination by Modulating RecA Activities

    PubMed Central

    Cárdenas, Paula P.; Carrasco, Begoña; Defeu Soufo, Clarisse; César, Carolina E.; Herr, Katharina; Kaufenstein, Miriam; Graumann, Peter L.; Alonso, Juan C.

    2012-01-01

    The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the “length or packing” of a RecA filament, facilitates the initiation of recombination and increases recombination across species. PMID:23284295

  17. Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

    PubMed Central

    Million-Weaver, Samuel; Samadpour, Ariana Nakta

    2015-01-01

    ABSTRACT Efficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts. IMPORTANCE Head-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene

  18. Conservation of an ATP-binding domain among recA proteins from Proteus vulgaris, erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r

    SciTech Connect

    Knight, K.L.; Hess, R.M.; McEntee, K.

    1988-06-01

    The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.

  19. Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA*

    PubMed Central

    Manfredi, Candela; Carrasco, Begoña; Ayora, Silvia; Alonso, Juan C.

    2008-01-01

    Subsaturating amounts of Bacillus subtilis SsbA, independently of the order of addition, partially inhibit the single-stranded DNA-dependent dATPase activity of RecA. This negative effect is fully overcome when a substoichiometric amount of RecO is added. SsbA added prior to RecA does not stimulate the dATP-dependent DNA strand exchange activity; however, added after RecA it enhances the extent of strand exchange. The addition of RecO stimulates RecA-mediated joint molecule formation, although it limits the accumulation of final recombination products. Thus we suggest that RecO has a dual activity: RecO acts as a RecA mediator enabling RecA to utilize SsbA-coated single-stranded DNA as a polymerization substrate and controls RecA-mediated DNA strand exchange by limiting its extent. We herein discuss the possible mechanisms of RecO involvement in the regulation of double strand break repair and genetic transformation. PMID:18599486

  20. Bacillus subtilis RecO nucleates RecA onto SsbA-coated single-stranded DNA.

    PubMed

    Manfredi, Candela; Carrasco, Begoña; Ayora, Silvia; Alonso, Juan C

    2008-09-01

    Subsaturating amounts of Bacillus subtilis SsbA, independently of the order of addition, partially inhibit the single-stranded DNA-dependent dATPase activity of RecA. This negative effect is fully overcome when a substoichiometric amount of RecO is added. SsbA added prior to RecA does not stimulate the dATP-dependent DNA strand exchange activity; however, added after RecA it enhances the extent of strand exchange. The addition of RecO stimulates RecA-mediated joint molecule formation, although it limits the accumulation of final recombination products. Thus we suggest that RecO has a dual activity: RecO acts as a RecA mediator enabling RecA to utilize SsbA-coated single-stranded DNA as a polymerization substrate and controls RecA-mediated DNA strand exchange by limiting its extent. We herein discuss the possible mechanisms of RecO involvement in the regulation of double strand break repair and genetic transformation. PMID:18599486

  1. Conjugational hyperrecombination achieved by derepressing the LexA regulon, altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12.

    PubMed Central

    Lanzov, Vladislav A; Bakhlanova, Irina V; Clark, Alvin J

    2003-01-01

    The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination. PMID:12702672

  2. Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

    PubMed

    Grinholc, Mariusz; Rodziewicz, Aleksandra; Forys, Katarzyna; Rapacka-Zdonczyk, Aleksandra; Kawiak, Anna; Domachowska, Anna; Golunski, Grzegorz; Wolz, Christiane; Mesak, Lili; Becker, Karsten; Bielawski, Krzysztof P

    2015-11-01

    Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

  3. Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

    PubMed

    Grinholc, Mariusz; Rodziewicz, Aleksandra; Forys, Katarzyna; Rapacka-Zdonczyk, Aleksandra; Kawiak, Anna; Domachowska, Anna; Golunski, Grzegorz; Wolz, Christiane; Mesak, Lili; Becker, Karsten; Bielawski, Krzysztof P

    2015-11-01

    Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations. PMID:26252968

  4. UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli.

    PubMed

    Veaute, Xavier; Delmas, Stéphane; Selva, Marjorie; Jeusset, Josette; Le Cam, Eric; Matic, Ivan; Fabre, Francis; Petit, Marie-Agnès

    2005-01-12

    The roles of UvrD and Rep DNA helicases of Escherichia coli are not yet fully understood. In particular, the reason for rep uvrD double mutant lethality remains obscure. We reported earlier that mutations in recF, recO or recR genes suppress the lethality of uvrD rep, and proposed that an essential activity common to UvrD and Rep is either to participate in the removal of toxic recombination intermediates or to favour the proper progression of replication. Here, we show that UvrD, but not Rep, directly prevents homologous recombination in vivo. In addition to RecFOR, we provide evidence that RecA contributes to toxicity in the rep uvrD mutant. In vitro, UvrD dismantles the RecA nucleoprotein filament, while Rep has only a marginal activity. We conclude that UvrD and Rep do not share a common activity that is essential in vivo: while Rep appears to act at the replication stage, UvrD plays a role of RecA nucleoprotein filament remover. This activity of UvrD is similar to that of the yeast Srs2 helicase. PMID:15565170

  5. The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames.

    PubMed Central

    Daugelat, S.; Jacobs, W. R.

    1999-01-01

    The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening. PMID:10091667

  6. The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli.

    PubMed

    Šimatović, Ana; Mitrikeski, Petar T; Vlašić, Ignacija; Sopta, Mary; Brčić-Kostić, Krunoslav

    2016-01-01

    In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730.

  7. A RecA Protein Surface Required for Activation of DNA Polymerase V

    PubMed Central

    Gruber, Angela J.; Erdem, Aysen L.; Sabat, Grzegorz; Karata, Kiyonobu; Jaszczur, Malgorzata M.; Vo, Dan D.; Olsen, Tayla M.; Woodgate, Roger; Goodman, Myron F.; Cox, Michael M.

    2015-01-01

    DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. PMID:25811184

  8. Active displacement of RecA filaments by UvrD translocase activity

    PubMed Central

    Petrova, Vessela; Chen, Stefanie H.; Molzberger, Eileen T.; Tomko, Eric; Chitteni-Pattu, Sindhu; Jia, Haifeng; Ordabayev, Yerdos; Lohman, Timothy M.; Cox, Michael M.

    2015-01-01

    The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode. PMID:25824953

  9. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    PubMed Central

    Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B. R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M.

    2016-01-01

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament. PMID:27447485

  10. Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

    PubMed

    Leite, Wellington C; Galvão, Carolina W; Saab, Sérgio C; Iulek, Jorge; Etto, Rafael M; Steffens, Maria B R; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L; Cox, Michael M

    2016-01-01

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament. PMID:27447485

  11. 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.

  12. Molecular genetic and biochemical analyses of a DNA repair gene from Serratia marcescens

    SciTech Connect

    Murphy, K.E.

    1989-01-01

    In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity.

  13. Dissecting the dissociation process of RecA monomer from a nucleoprotein filament

    NASA Astrophysics Data System (ADS)

    Kim, Doseok; Kim, Sung Hyun; Joo, Chirlmin; Park, Jeehae; Ragunathan, Kaushik; Ha, Taekjip

    2010-03-01

    RecA protein is a DNA-dependent ATPase and plays a key role in DNA repair mechanisms. RecA proteins form a helical filament on a single-strand DNA mediating homologous recombination. Understanding the molecular mechanisms of RecA-DNA interaction is crucial for further investigation on its biochemical properties. Using a single-molecule fluorescence technique, we dissected the dissociation process with single-monomer resolution. We could resolve the existence of an intermediate state after ATP hydrolysis in the dissociation process. In the nucleotide cofactor free environment, RecA did not dissociate indicating that the bound ADP is required for the monomer dissociation. Based on our observation, we suggest a model for the RecA dissociation process coupled with ATP hydrolysis cycle.

  14. RecA: Regulation and Mechanism of a Molecular Search Engine.

    PubMed

    Bell, Jason C; Kowalczykowski, Stephen C

    2016-06-01

    Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.

  15. Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.

    PubMed Central

    Ohta, K; Beall, D S; Mejia, J P; Shanmugam, K T; Ingram, L O

    1991-01-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA). PMID:2059047

  16. sbmC, a stationary-phase induced SOS Escherichia coli gene, whose product protects cells from the DNA replication inhibitor microcin B17.

    PubMed

    Baquero, M R; Bouzon, M; Varea, J; Moreno, F

    1995-10-01

    Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double-strand breaking of DNA in a DNA gyrase-dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC, that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids (M(r), 18,095) that has been visualized in SDS-polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA-damaging agents and, also, by the entry of cells into the stationary growth phase. A G-->T transversion at the fifth nucleotide of the quasicanonical LexA-box preceding the gene makes recA cells 16-fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way.

  17. Specificity in suppression of SOS expression by recA4162 and uvrD303.

    PubMed

    Massoni, Shawn C; Sandler, Steven J

    2013-12-01

    Detection and repair of DNA damage is essential in all organisms and depends on the ability of proteins recognizing and processing specific DNA substrates. In E. coli, the RecA protein forms a filament on single-stranded DNA (ssDNA) produced by DNA damage and induces the SOS response. Previous work has shown that one type of recA mutation (e.g., recA4162 (I298V)) and one type of uvrD mutation (e.g., uvrD303 (D403A, D404A)) can differentially decrease SOS expression depending on the type of inducing treatments (UV damage versus RecA mutants that constitutively express SOS). Here it is tested using other SOS inducing conditions if there is a general feature of ssDNA generated during these treatments that allows recA4162 and uvrD303 to decrease SOS expression. The SOS inducing conditions tested include growing cells containing temperature-sensitive DNA replication mutations (dnaE486, dnaG2903, dnaN159, dnaZ2016 (at 37°C)), a del(polA)501 mutation and induction of Double-Strand Breaks (DSBs). uvrD303 could decrease SOS expression under all conditions, while recA4162 could decrease SOS expression under all conditions except in the polA strain or when DSBs occur. It is hypothesized that recA4162 suppresses SOS expression best when the ssDNA occurs at a gap and that uvrD303 is able to decrease SOS expression when the ssDNA is either at a gap or when it is generated at a DSB (but does so better at a gap).

  18. Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis

    SciTech Connect

    Owttrim, G.W.; Coleman, J.R.

    1987-05-01

    A recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system.

  19. Gene products of corticosteroid action in hippocampus.

    PubMed

    Nichols, N R; Finch, C E

    1994-11-30

    We used two methods to examine altered patterns of gene expression in rat hippocampus in response to administered glucocorticoids: analysis of RNA in vitro translation products on 2-d gels and cloning of cDNAs from a rat hippocampal library by differential hybridization (+/- CORT). We determined that two of the CORT-responsive cDNA clones encoded the 35- and 50-kd RNA translation products and identified them as GPDH and GFAP, respectively, by sequence analysis. Cloned mRNAs that increased and decreased in response to CORT were determined to be under positive and negative regulation by glucocorticoids in intact rats. Despite their similarities in glucocorticoid response characteristics, we found three subsets of hippocampal mRNA responses to CORT and shaking stress which differ in temporal and level-dependent aspects of CORT regulation. In addition, GPDH gene expression represents a glucocorticoid-dependent stress response which is rapidly increased in a dose- and stressor-dependent manner. It is a candidate for a sensitive indicator of stress responsiveness in the brain as a function of neuroendocrine activity. Mechanisms of adaptation to stress in the brain are likely to involve responses that are both mediated by glucocorticoids and opposed by them. GFAP and TGF-beta 1 mRNA responses may be examples of the latter, since they are decreased in response to glucocorticoids, are under negative regulation by glucocorticoids in intact rats, and are increased in response to brain injury and disease and during aging. If these astrocytic and microglial responses are involved in cellular defense mechanisms in the brain, then their regulation by glucocorticoids would be important in maintaining and restoring cellular homeostasis in physiological and pathophysiological states. Future studies using these sensitive probes for glucocorticoid-regulated gene expression may identify new mechanisms by which the brain coordinates acute and chronic responses to stress and disease.

  20. Conformational flexibility of RecA protein filament: transitions between compressed and stretched states.

    PubMed

    Petukhov, Michael; Lebedev, Dmitry; Shalguev, Valery; Islamov, Akhmed; Kuklin, Aleksandr; Lanzov, Vladislav; Isaev-Ivanov, Vladimir

    2006-11-01

    RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations. PMID:16909421

  1. An integrative approach to the study of filamentous oligomeric assemblies, with application to RecA.

    PubMed

    Boyer, Benjamin; Ezelin, Johann; Poulain, Pierre; Saladin, Adrien; Zacharias, Martin; Robert, Charles H; Prévost, Chantal

    2015-01-01

    Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called "extended" and "compressed" forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.

  2. An Integrative Approach to the Study of Filamentous Oligomeric Assemblies, with Application to RecA

    PubMed Central

    Boyer, Benjamin; Ezelin, Johann; Poulain, Pierre; Saladin, Adrien; Zacharias, Martin; Robert, Charles H.; Prévost, Chantal

    2015-01-01

    Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called “extended” and “compressed” forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general. PMID:25785454

  3. Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization.

    PubMed

    Amundsen, Susan K; Fero, Jutta; Hansen, Lori M; Cromie, Gareth A; Solnick, Jay V; Smith, Gerald R; Salama, Nina R

    2008-08-01

    Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP-dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double-strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection. PMID:18573180

  4. Optimal conditions for decorating outer surface of single-walled carbon nanotubes with RecA proteins

    NASA Astrophysics Data System (ADS)

    Oura, Shusuke; Umemura, Kazuo

    2016-03-01

    In this study, we estimated the optimal reaction conditions for decorating the outer surface of single-walled carbon nanotubes (SWNTs) with RecA proteins by comparison with hybrids of RecA and single-stranded DNA (ssDNA). To react SWNTs with RecA proteins, we first prepared ssDNA-SWNT hybrids. The heights of the ssDNA-SWNT hybrids increased as the amount of RecA used in the reaction increased, as determined from atomic force microscopy images. We further confirmed the increasing adsorption of RecA proteins onto ssDNA on SWNT surfaces by agarose gel electrophoresis. These results suggest that the combination of RecA proteins and ssDNA-SWNT hybrids forms RecA-ssDNA-SWNT hybrids. We also successfully controlled the amount of RecA adsorbed on the ssDNA-SWNT hybrids. Our results thus indicate the optimized reaction conditions for decorating the outer surface of SWNTs with RecA proteins, which is the key to the development of novel biosensors and nanomaterial-based bioelectronics.

  5. Effects of DNA sequence and structure on binding of RecA to single-stranded DNA.

    PubMed

    Bar-Ziv, R; Libchaber, A

    2001-07-31

    Fluorescence anisotropy is used to follow the binding of RecA to short single-stranded DNA (ssDNA) sequences (39 bases) at low DNA and RecA concentration where the initial phase of polymerization occurs. We observe that RecA condensation is extremely sensitive to minute changes in DNA sequences. RecA binds strongly to sequences that are rich in pyrimidines and that lack significant secondary structure and base stacking. We find a correlation between the DNA folding free energy and the onset concentration for RecA binding. These results suggest that the folding of ssDNA and base stacking represent a barrier for RecA binding. The link between secondary structure and binding affinity is further analyzed with two examples: discrimination between two naturally occurring polymorphisms differing by one base and RecA binding on a molecular beacon. A self-assembly model is introduced to explain these observations. We propose that RecA may be used to sense ssDNA sequence and structure.

  6. Biochemical and cellular characterization of Helicobacter pylori RecA, a protein with high-level constitutive expression.

    PubMed

    Orillard, Emilie; Radicella, J Pablo; Marsin, Stéphanie

    2011-12-01

    Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonization. The biochemical characterization of the purified recombinase reveals activities similar to those of Escherichia coli RecA (EcRecA). We show that in H. pylori, HpRecA is present in about 80,000 copies per cell during exponential growth and decreases to about 50,000 copies in stationary phase. The amount of HpRecA remains unchanged after induction of DNA lesions, suggesting that HpRecA is always expressed at a high level in order to repair DNA damage or facilitate recombination. We performed HpRecA localization analysis by adding a Flag tag to the protein, revealing two different patterns of localization. During exponential growth, RecA-Flag presents a diffuse pattern, overlapping with the DAPI (4',6-diamidino-2-phenylindole) staining of DNA, whereas during stationary phase, the protein is present in more defined areas devoid of DAPI staining. These localizations are not affected by inactivation of competence or DNA recombination genes. Neither UV irradiation nor gamma irradiation modified HpRecA localization, suggesting the existence of a constitutive DNA damage adaptation system. PMID:21949074

  7. 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.

  8. Dynamic Growth and Shrinkage Govern the pH Dependence of RecA Filament Stability

    PubMed Central

    Kim, Sung Hyun; Park, Jeehae; Joo, Chirlmin; Kim, Doseok; Ha, Taekjip

    2015-01-01

    RecA proteins form a long stable filament on a single-stranded DNA and catalyze strand exchange reaction. The stability of RecA filament changes dramatically with pH, yet its detailed mechanism is not known. Here, using a single molecule assay, we determined the binding and dissociation rates of RecA monomers at the filament ends at various pH. The pH-induced rate changes were moderate but occurred in opposite directions for binding and dissociation, resulting in a substantial increase in filament stability in lower pH. The highly charged residues in C-terminal domain do not contribute to the pH dependent stability. The stability enhancement of RecA filament in low pH may help the cell to cope with acidic stress by fine-tuning of the binding and dissociation rates without losing the highly dynamic nature of the filament required for strand exchange. PMID:25608006

  9. Solution structure of DinI provides insight into its mode of RecA inactivation.

    PubMed Central

    Ramirez, B. E.; Voloshin, O. N.; Camerini-Otero, R. D.; Bax, A.

    2000-01-01

    The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA. PMID:11152126

  10. The muc genes of pKM101 are induced by DNA damage.

    PubMed Central

    Elledge, S J; Walker, G C

    1983-01-01

    A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed. Images PMID:6350267

  11. 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.

  12. Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP.

    PubMed

    Xing, Xu; Bell, Charles E

    2004-12-28

    RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks. The binding of RecA to DNA is modulated by adenosine nucleotides. ATP increases the affinity of RecA for DNA, while ADP decreases the affinity. Previously, the crystal structures of E. coli RecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 A, respectively, but the model for the RecA-ADP complex did not include magnesium ion or side chains. Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP-PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 A, respectively. Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of approximately 82 A. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the gamma phosphate of AMP-PNP. There are very few conformational differences between the structures of RecA bound to ADP and AMP-PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66-73 upon nucleotide binding. To interpret the functional significance of the structure of the MnAMP-PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA. Whereas ATPgammaS and ADP-AlF(4) facilitate a robust coprotease activity, ADP and AMP-PNP do not activate RecA at all. We conclude that the crystal structure of the RecA-MnAMP-PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state. PMID:15610008

  13. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.

    PubMed

    Bell, Jason C; Plank, Jody L; Dombrowski, Christopher C; Kowalczykowski, Stephen C

    2012-11-01

    Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5'→3' direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair

  14. Interaction of RecA protein with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene and 4-hydroxyaminoquinoline 1-oxide.

    PubMed Central

    Kojima, M; Suzuki, M; Morita, T; Ogawa, T; Ogawa, H; Tada, M

    1990-01-01

    Interaction of RecA protein of Escherichia coli with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and 4-hydroxyaminoquinoline 1-oxide (4HAQO) was investigated. RecA protein bound more efficiently to modified DNA than to unmodified DNA as judged by filter-binding and gel electrophoresis assay. The binding of RecA protein with modified DNA resulted in the stimulation of ATPase activity and the activation for RecA protein to stimulate the repressor cleavage. These abilities of RecA protein were increased proportionally to the number of adducts in the plasmid DNA (0-5 adducts). Apurinic and alkylated DNA did not activate RecA protein. We suggest that modification of DNA by N-OH-AAF and 4HAQO provides binding sites for RecA protein and may act as an activation signal for SOS response. Images PMID:2140154

  15. Expression of cloned bacteriophage T4 uvsW and uvsY genes in rec+ and rec- Escherichia coli

    SciTech Connect

    DeVries, J.K.; Wallace, S.S.

    1983-09-01

    Chimeric plasmids containing the uvsY uvsW region of the T4 genome were examined for the expression of these genes. Certain of these plasmids were shown to express the uvsY or the uvsW gene products by their ability to complement the UV sensitivity of infecting uvsW or uvsY mutant phage. Also, a chimeric plasmid containing both the uvsW and uvsY genes increases the survival of UV-irradiated, methyl methane sulfonate- or ethyl methane sulfonate-treated recA hosts.

  16. 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

  17. The inactive form of recA protein: the 'compact' structure.

    PubMed Central

    Ruigrok, R W; Bohrmann, B; Hewat, E; Engel, A; Kellenberger, E; DiCapua, E

    1993-01-01

    When recA protein is enzymatically inactive in vitro, it adopts a more compact helical polymer form than that of the active protein polymerized onto DNA in the presence of ATP. Here we describe some aspects of this structure. By cryo-electron microscopy, a pitch of 76 A is found for both the self-polymer and the inactive complex with ssDNA. A smaller pitch of 64 A is observed in conventional electron micrographs. The contour length of complexes with ssDNA was used to estimate the binding stoichiometry in the compact complex, 6 +/- 1 nt/recA. In addition, the compact structure was observed in vivo in Escherichia coli: inclusion bodies produced upon induction of recA expression in an overproducing strain have a fibrous morphology with the structural parameters of the compact polymer. Images PMID:8428597

  18. RecA protein promotes the regression of stalled replication forks in vitro.

    PubMed

    Robu, M E; Inman, R B; Cox, M M

    2001-07-17

    Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria. PMID:11459955

  19. 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.

  20. 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.

  1. X-ray Crystal Structure of the Bacterial Conjugation Factor PsiB, a Negative Regulator of RecA

    SciTech Connect

    Petrova, Vessela; Satyshur, Kenneth A.; George, Nicholas P.; McCaslin, Darrell; Cox, Michael M.; Keck, James L.

    2012-03-16

    During bacterial conjugation, genetic material from one cell is transferred to another as single-stranded DNA. The introduction of single-stranded DNA into the recipient cell would ordinarily trigger a potentially deleterious transcriptional response called SOS, which is initiated by RecA protein filaments formed on the DNA. During F plasmid conjugation, however, the SOS response is suppressed by PsiB, an F-plasmid-encoded protein that binds and sequesters free RecA to prevent filament formation. Among the many characterized RecA modulator proteins, PsiB is unique in using sequestration as an inhibitory mechanism. We describe the crystal structure of PsiB from the Escherichia coli F plasmid. The stucture of PsiB is surprisingly similar to CapZ, a eukaryotic actin filament capping protein. Structure-directed neutralization of electronegative surfaces on PsiB abrogates RecA inhibition whereas neutralization of an electropositive surface element enhances PsiB inhibition of RecA. Together, these studies provide a first molecular view of PsiB and highlight its use as a reagent in studies of RecA activity.

  2. The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.

    PubMed

    Metrick, Michael A; Temple, Joshua E; MacDonald, Gina

    2013-12-31

    The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions.

  3. RuvABC-dependent double-strand breaks in dnaBts mutants require recA.

    PubMed

    Seigneur, M; Ehrlich, S D; Michel, B

    2000-11-01

    Replication fork arrest can cause DNA double-strand breaks (DSBs). These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks. In this work, we study the homologous recombination functions required for RuvABC-mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB. We show that, in the rep mutant, RuvABC-mediated breakage occurs in the absence of the homologous recombination protein RecA. In contrast, in dnaBts mutants, most of the RuvABC-mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB. This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO. Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins. PMID:11069680

  4. Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli.

    PubMed

    Baitin, Dmitry M; Bakhlanova, Irina V; Kil, Yury V; Cox, Michael M; Lanzov, Vladislav A

    2006-08-01

    In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities. PMID:16885449

  5. RecFOR and RecOR as distinct RecA loading pathways.

    PubMed

    Sakai, Akiko; Cox, Michael M

    2009-01-30

    The molecular role of the RecF protein in loading RecA protein onto single-stranded DNA (ssDNA)-binding protein-coated ssDNA has been obscured by the facility with which the RecO and RecR proteins alone perform this function. We now show that RecFOR and RecOR define distinct RecA loading functions that operate optimally in different contexts. RecFOR, but not RecOR, is most effective when RecF(R) is bound near an ssDNA/double-stranded (dsDNA) junction. However, RecF(R) has no enhanced binding affinity for such a junction. RecO and RecR proteins are both required under all conditions in which the RecFOR pathway operates. The RecOR pathway is uniquely distinguished by a required interaction between RecO protein and the ssDNA binding protein C terminus. The RecOR pathway is more efficient for RecA loading onto ssDNA when no proximal dsDNA is available. A merger of new and published results leads to a new model for RecFOR function. PMID:18986990

  6. 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.

  7. Regulatory Oversight of Cell- and Tissue-Based Therapeutic Products and Gene Therapy Products in Singapore.

    PubMed

    Goh, Choon Wee; Kellathur, Srinivasan N; Ong, Lee Lee; Wu, Xiaofeng

    2015-01-01

    The regulatory environment for cell- and tissue-based therapeutic products and gene therapy products is rapidly evolving and drug regulatory agencies are working towards establishing a risk-based system in the regulatory framework. Similarly in Singapore, a risk-based tiered approach has been applied whereby clinical trials and product licence of high-risk cell- and tissue-based therapeutic products (substantially manipulated products, products intended for nonhomologous use or combined products) and gene therapy products are regulated as medicinal products under the Medicines Act. There is no legal definition for cell- and tissue-based therapeutic and gene therapy products. The current working definition for a cell- and tissue-based therapeutic product is an article containing or consisting of an autologous or allogeneic human cell or tissue that are used for or administered to, or intended to be used for or administered to, human beings for the diagnosis, treatment, or prevention of human diseases or conditions. Gene therapy products are included under the current biological medicinal product definition.

  8. [Structure and function of neural plasticity-related gene products].

    PubMed

    Yamagata, K; Sugiura, H; Suzuki, K

    1998-08-01

    We have isolated novel immediate early genes (IEGs) from the hippocampus by differential cloning techniques. These mRNAs are induced by synaptic activity and translated into proteins that may affect neural function. We have analyzed a variety of "effector" immediate early genes. These mRNAs encode: 1) cytoplasmic proteins, such as cyclooxygenase-2, a small G protein, Rheb, and a cytoskeleton-associated protein, Arc; 2) membrane-bound proteins, such as the cell adhesion protein Arcadlin, and a neurite-outgrowth protein, Neuritin; and 3) a secreted protein, Narp. We hypothesize that physiological stimulation induces "effector" proteins that might strengthen synaptic connections of activated synapses. In contrast, pathological conditions such as epilepsy or drug addiction may accelerate overproduction of these gene products, which cause abnormal synapse formation. Gene targeting and in vivo gene transfer techniques are required to prove this hypothesis. PMID:9866829

  9. Pathogens and gene product normalization in the biomedical literature.

    PubMed

    Vishnyakova, Dina; Pasche, Emilie; Teodoro, Douglas; Lovis, Christian; Ruch, Patrick

    2012-01-01

    We present a new approach for pathogens and gene product normalization in the biomedical literature. The idea of this approach was motivated by needs such as literature curation, in particular applied to the field of infectious diseases thus, variants of bacterial species (S. aureus, Staphyloccocus aureus, ...) and their gene products (protein ArsC, Arsenical pump modifier, Arsenate reductase, ...). Our approach is based on the use of an Ontology Look-up Service, a Gene Ontology Categorizer (GOCat) and Gene Normalization methods. In the pathogen detection task the use of OLS disambiguates found pathogen names. GOCat results are incorporated into overall score system to support and to confirm the decisionmaking in normalization process of pathogens and their genomes. The evaluation was done on two test sets of BioCreativeIII benchmark: gold standard of manual curation (50 articles) and silver standard (507 articles) curated by collective results of BCIII participants. For the cross-species GN we achieved the precision of 46% for silver and 27% for gold sets. Pathogen normalization results showed 95% of precision and 93% of recall. The impact of GOCat explicitly improves results of pathogen and gene normalization, basically confirming identified pathogens and boosting correct gene identifiers on the top of the results' list ranked by confidence. A correct identification of the pathogen is able to improve significantly normalization effectiveness and to solve the disambiguation problem of genes.

  10. DNA double strand break end-processing and RecA induce RecN expression levels in Bacillus subtilis.

    PubMed

    Cardenas, Paula P; Gándara, Carolina; Alonso, Juan C

    2014-02-01

    Bacillus subtilis cells respond to double strand breaks (DSBs) with an ordered recruitment of repair proteins to the site lesion, being RecN one of the first responders. In B. subtilis, one of the responses to DSBs is to increase RecN expression rather than modifying its turnover rate. End-processing activities and the RecA protein itself contribute to increase RecN levels after DNA DSBs. RecO is required for RecA filament formation and full SOS induction, but its absence did not significantly affect RecN expression. Neither the absence of LexA nor the phosphorylation state of RecA or SsbA significantly affect RecN expression levels. These findings identify two major mechanisms (SOS and DSB response) used to respond to DSBs, with LexA required for one of them (SOS response). The DSB response, which requires end-processing and RecA or short RecO-independent RecA filaments, highlights the importance of guarding genome stability by modulating the DNA damage responses. PMID:24373815

  11. Efficacy of species-specific recA PCR tests in the identification of Burkholderia cepacia complex environmental isolates.

    PubMed

    Dalmastri, Claudia; Pirone, Luisa; Tabacchioni, Silvia; Bevivino, Annamaria; Chiarini, Luigi

    2005-05-01

    In this study, we evaluated if recA species-specific PCR assays could be successfully applied to identify environmental isolates of the widespread Burkholderia cepacia complex (Bcc) species. A total of 729 Bcc rhizosphere isolates collected in different samplings were assigned to the species B. cepacia genomovar I (61), B. cenocepacia recA lineage IIIB (514), B. ambifaria (124) and B. pyrrocinia (30), by means of recA (RFLP) analysis, and PCR tests were performed to assess sensitivity and specificity of recA species-specific primers pairs. B. cepacia genomovar I specific primers produced the expected amplicon with all isolates of the corresponding species (sensitivity, 100%), and cross-reacted with all B. pyrrocinia isolates. On the contrary, B. cenocepacia IIIB primers did not give the expected amplicon in 164 B. cenocepacia IIIB isolates (sensitivity, 68.1%), and isolates of distinct populations showed different sensitivity. B. ambifaria primers failed to amplify a recA-specific fragment only in a few isolates of this species (sensitivity, 93.5%). The absence of specific amplification in a high number of B. cenocepacia rhizosphere isolates indicates that recA specific PCR assays can lead to an underestimation of environmental microorganisms belonging to this bacterial species. PMID:15869960

  12. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli

    SciTech Connect

    Kenyon, C.J.; Walker, G.C.

    1988-05-01

    Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

  13. Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer.

    PubMed

    Lin, Zhenguo; Kong, Hongzhi; Nei, Masatoshi; Ma, Hong

    2006-07-01

    The bacterial recA gene and its eukaryotic homolog RAD51 are important for DNA repair, homologous recombination, and genome stability. Members of the recA/RAD51 family have functions that have differentiated during evolution. However, the evolutionary history and relationships of these members remains unclear. Homolog searches in prokaryotes and eukaryotes indicated that most eubacteria contain only one recA. However, many archaeal species have two recA/RAD51 homologs (RADA and RADB), and eukaryotes possess multiple members (RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, and recA). Phylogenetic analyses indicated that the recA/RAD51 family can be divided into three subfamilies: (i) RADalpha, with highly conserved functions; (ii) RADbeta, with relatively divergent functions; and (iii) recA, functioning in eubacteria and eukaryotic organelles. The RADalpha and RADbeta subfamilies each contain archaeal and eukaryotic members, suggesting that a gene duplication occurred before the archaea/eukaryote split. In the RADalpha subfamily, eukaryotic RAD51 and DMC1 genes formed two separate monophyletic groups when archaeal RADA genes were used as an outgroup. This result suggests that another duplication event occurred in the early stage of eukaryotic evolution, producing the DMC1 clade with meiosis-specific genes. The RADbeta subfamily has a basal archaeal clade and five eukaryotic clades, suggesting that four eukaryotic duplication events occurred before animals and plants diverged. The eukaryotic recA genes were detected in plants and protists and showed strikingly high levels of sequence similarity to recA genes from proteobacteria or cyanobacteria. These results suggest that endosymbiotic transfer of recA genes occurred from mitochondria and chloroplasts to nuclear genomes of ancestral eukaryotes.

  14. 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

  15. Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments.

    PubMed

    Saladin, Adrien; Amourda, Christopher; Poulain, Pierre; Férey, Nicolas; Baaden, Marc; Zacharias, Martin; Delalande, Olivier; Prévost, Chantal

    2010-10-01

    Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson-Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange.

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

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

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

  17. A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor.

    PubMed

    Murakami, Takeshi; Burian, Jan; Yanai, Koji; Bibb, Mervyn J; Thompson, Charles J

    2011-09-20

    Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4-12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA), it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.

  18. A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor

    PubMed Central

    Murakami, Takeshi; Burian, Jan; Yanai, Koji; Bibb, Mervyn J.; Thompson, Charles J.

    2011-01-01

    Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4–12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA), it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms. PMID:21903924

  19. 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

  20. 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.

  1. 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

  2. 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

  3. RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress.

    PubMed

    Lenhart, Justin S; Brandes, Eileen R; Schroeder, Jeremy W; Sorenson, Roderick J; Showalter, Hollis D; Simmons, Lyle A

    2014-08-01

    RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest. PMID:24891441

  4. RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

    PubMed Central

    Lenhart, Justin S.; Brandes, Eileen R.; Schroeder, Jeremy W.; Sorenson, Roderick J.; Showalter, Hollis D.

    2014-01-01

    RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest. PMID:24891441

  5. Transcriptional regulation of genes related to progesterone production.

    PubMed

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

    2015-01-01

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

  6. Redefining body composition: nutrients hormones, and genes in meat production.

    PubMed

    Wray-Cahen, C D; Kerr, D E; Evock-Clover, C M; Steele, N C

    1998-01-01

    Growth rate and body composition of livestock can be optimized to meet consumer needs for a leaner product and to improve the efficiency of meat-animal production. Optimization strategies have traditionally focused on genetic selection and cost-effective ration formulation to achieve the genetic potential. Advances in understanding the mechanisms of growth and its control have led to additional opportunities for its manipulation. These include nutritional manipulation,the use of growth promotants, and, more recently, the ability to change the genetic potential through genetic engineering. Selection of appropriate candidate genes for manipulation depends on understanding the mechanisms underlying differentiation and growth of embryonic muscle cells. Recent advances in genetic engineering techniques, including gene therapy and germline transgenesis, will likely hasten the genetic progress toward a leaner carcass in domestic livestock. Such strategies may prove to be more beneficial then the controlled enhancement of somatotropin expression.

  7. Chemotaxis in Escherichia coli: Methylation of che gene products

    PubMed Central

    Silverman, Michael; Simon, Melvin

    1977-01-01

    The products of three chemotaxis-specific genes in Escherichia coli, cheM, cheD, and cheZ, are methylated. The cheZ gene codes for the synthesis of a 24,000 molecular weight polypeptide that appears in the cytoplasm. cheM codes for the synthesis of a membrane-bound polypeptide with a molecular weight of 61,000. cheD codes for another membrane-bound polypeptide with an apparent molecular weight of 64,000. CheM- mutants show chemotaxis toward some attractants (Tar- phenotype), while CheD- mutants respond to other attractants (Tsr- phenotype). The double mutant (CheD-, CheM-) does not respond to any attractant or repellent tested. Therefore, these polypeptides play a central role in chemotaxis. They collect information from two subsets of chemoreceptors and act as the last step in the chemoreceptor pathway and the first step in the general processing of signals for transmission to the flagellar rotor. It is suggested that they may be involved in both an initial process that reflects the instantaneous state of the chemoreceptors and in an integrative, adaptive process. Two other genes, cheX and cheW, are required for the methylation of the cheD and cheM gene products. Images PMID:333434

  8. Dickeya species relatedness and clade structure determined by comparison of recA sequences.

    PubMed

    Parkinson, Neil; Stead, David; Bew, Janice; Heeney, John; Tsror Lahkim, Leah; Elphinstone, John

    2009-10-01

    Using sequences from the recA locus, we have produced a phylogeny of 188 Dickeya strains from culture collections and identified species relatedness and subspecies clade structure within the genus. Of the six recognized species, Dickeya paradisiaca, D. chrysanthemi and D. zeae were discriminated with long branch lengths. The clade containing the D. paradisiaca type strain included just one additional strain, isolated from banana in Colombia. Strains isolated from Chrysanthemum and Parthenium species made up most of the clade containing the D. chrysanthemi type strain, and the host range of this species was extended to include potato. The D. zeae clade had the largest number of sequevars and branched into two major sister clades that contained all of the Zea mays isolates, and were identified as phylotypes PI and PII. The host range was increased from six to 13 species, including potato. The recA sequence of an Australian sugar-cane strain was sufficiently distinct to rank as a new species-level branch. In contrast to these species, Dickeya dadantii, D. dianthicola and D. dieffenbachiae were distinguished with shorter branch lengths, indicating relatively closer relatedness. The recA sequence for the type strain of D. dadantii clustered separately from other strains of the species. However, sequence comparison of three additional loci revealed that the D. dadantii type strain grouped together with the six other D. dadantii strains that were sequenced. Analysis of all four loci indicated that the D. dadantii strains were most closely related to D. dieffenbachiae. Three further branches (DUC-1, -2 and -3) were associated with these three species, which all diverged from a common origin and can be considered as a species complex. The large clade containing the D. dianthicola type strain comprised 58 strains and had little sequence diversity. One sequevar accounted for the majority of these strains, which were isolated nearly exclusively from eight hosts from Europe

  9. Plasmid genes required for microcin B17 production.

    PubMed Central

    San Millán, J L; Kolter, R; Moreno, F

    1985-01-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production. PMID:2993228

  10. Plasmid genes required for microcin B17 production.

    PubMed

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  11. Non-covalent interactions between ATP and RecA DNA-repairing proteins: DFT and semiempirical calculations

    NASA Astrophysics Data System (ADS)

    Rodriguez, Jorge

    2015-03-01

    The role of Bacterial RecA in the structural maintenance of genomes and the genetic information they carry has been established. In particular, the RecA DNA-repairing protein from D. Radiodurans, a radiation-resistant bacteria, is crucial for the repair of double strand breaks (DSBs). We have performed semi-empirical free-energy calculations and QM/MM calculations to study their non-covalent interactions with ATP and ADP. Such studies provide insight into the mechanisms of ATP/ADP --> RecA energy transfer and, therefore, about specific functional uses of incoming energy for DNA repairing mechanisms. We present a detailed analysis of the non-covalent interactions which minimize the interaction Gibbs free energies leading to the most stable non-covalent binding sites. Van der Waal, hydrogen bonding and electrostatic interactions has been quantified which provides a detailed insight into the mechanisms of ATP-RecA interaction. Further, possible chemical interactions and functional roles of RecA proteins are explored based on the previously mentioned studies. Acknowledgements: Funded, in part, by DTRA award 106339 (JHR). Dr. Mark C. Palenik and Mrs. Lora Beard are gratefully acknowledged Supported in part by DTRA Award 106339.

  12. Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells.

    PubMed

    Kidane, Dawit; Graumann, Peter L

    2005-08-01

    We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced approximately 30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 microm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions. PMID:16061691

  13. Large-scale structure of RecA protein from Deinococcus radiodurance and its complexes in solution

    NASA Astrophysics Data System (ADS)

    Karelov, D. V.; Lebedev, D. V.; Suslov, A. V.; Shalguev, V. I.; Kuklin, A. I.; Islamov, A. Kh; Lauter, H.; Lanzov, V. A.; Isaev-Ivanov, V. V.

    2008-03-01

    Different conformational states of the filaments formed by RecA protein from a radiation resistant strain Deinococcus radiodurance (RecADr) in solution were investigated using small angle neutron scattering. Scattering by the protein self-polymer was consistent with a long helix model, with the pitch of the helix being lower than that in the crystal structure. Compared to those of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, helical filaments of RecA from D. radiodurance exhibited a lower helical pitch and lower stability at low Mg2+ concentrations or under conditions of elevated ionic strength in the absence of ATP (adenosine triphosphate). Formation of an active filament upon binding of ATPγS and either single- or double-stranded DNA brought about a significant increase in the helix pitch and a moderate decrease in the cross-sectional gyration radius, but resulted in little change in the number of monomers per helix turn. The helix pitch value of the RecADr presynaptic complex was conservative and close to that found for other RecA proteins and their analogs.

  14. 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.

  15. Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers.

    PubMed

    Pobegalov, Georgii; Cherevatenko, Galina; Alekseev, Aleksandr; Sabantsev, Anton; Kovaleva, Oksana; Vedyaykin, Alexey; Morozova, Natalia; Baitin, Dmitrii; Khodorkovskii, Mikhail

    2015-10-23

    Deinococcus radiodurans can survive extreme doses of ionizing radiation due to the very efficient DNA repair mechanisms that are able to cope even with hundreds of double-strand breaks. RecA, the critical protein of homologous recombination in bacteria, is one of the key components of the DNA-repair system. Repair of double-strand breaks requires RecA binding to DNA and assembly of the RecA nucleoprotein helical filaments. The Escherichia coli RecA protein (EcRecA) and its interactions with DNA have been extensively studied using various approaches including single-molecule techniques, while the D. radiodurans RecA (DrRecA) remains much less characterized. However, DrRecA shows some remarkable differences from E. coli homolog. Here we combine microfluidics and single-molecule DNA manipulation with optical tweezers to follow the binding of DrRecA to long double-stranded DNA molecules and probe the mechanical properties of DrRecA nucleoprotein filaments at physiological pH. Our data provide a direct comparison of DrRecA and EcRecA binding to double-stranded DNA under identical conditions. We report a significantly faster filaments assembly as well as lower values of persistence length and contour length for DrRecA nucleoprotein filaments compared to EcRecA. Our results support the existing model of DrRecA forming more frequent and less continuous filaments relative to those of EcRecA.

  16. Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

    PubMed Central

    Fonville, Natalie C.; Bates, David; Hastings, P. J.; Hanawalt, Philip C.; Rosenberg, Susan M.

    2010-01-01

    Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans. PMID:20221259

  17. 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

  18. 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.

  19. Molecular Interaction and Cellular Location of RecA and CheW Proteins in Salmonella enterica during SOS Response and Their Implication in Swarming

    PubMed Central

    Irazoki, Oihane; Aranda, Jesús; Zimmermann, Timo; Campoy, Susana; Barbé, Jordi

    2016-01-01

    In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed. PMID:27766091

  20. Guidelines for the naming of genes, gene products, and mutants in the opportunistic protists.

    PubMed

    Limper, Andrew H; Weiss, Louis M

    2011-01-01

    The opportunistic protists encompass a wide diversity of organisms including Pneumocystis, Toxoplasma, cryptosporidia, microsporidia, and related genera. Recent advances in the molecular biology and cellular biochemistry of these organisms have led to the identification of an ever growing numbers of key genes and their cognate proteins. Until now, these molecules have not been designated using any consistent nomenclature system, leading to considerable confusion. The participants of the 11th International Workshop on Opportunistic Protists met on August 3, 2010 to reach consensus of a nomenclature system for genes, gene products, and mutants in the opportunistic protists. The following summary reports the consensus agreement to move toward a unified nomenclature system for these organisms. The system is adapted from that used for Saccharomyces cerevisiae.

  1. 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.

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

    PubMed Central

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

    2014-01-01

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

  3. 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

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

  4. 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.

  5. Single-molecule studies of the stringency factors and rates governing the polymerization of RecA on double-stranded DNA

    PubMed Central

    Feinstein, Efraim; Danilowicz, Claudia; Conover, Alyson; Gunaratne, Ruwan; Kleckner, Nancy; Prentiss, Mara

    2011-01-01

    RecA is a key protein in homologous recombination. During recombination, one single-stranded DNA (ssDNA) bound to site I in RecA exchanges Watson–Crick pairing with a sequence-matched ssDNA that was part of a double-stranded DNA molecule (dsDNA) bound to site II in RecA. After strand exchange, heteroduplex dsDNA is bound to site I. In vivo, direct polymerization of RecA on dsDNA through site I does not occur, though it does in vitro. The mechanisms underlying the difference have been unclear. We use single-molecule experiments to decouple the two steps involved in polymerization: nucleation and elongation. We find that elongation is governed by a fundamental clock that is insensitive to force and RecA concentration from 0.2 and 6 µM, though rates depend on ionic conditions. Thus, we can probe nucleation site stability by creating nucleation sites at high force and then measuring elongation as a function of applied force. We find that in the presence of ATP hydrolysis a minimum force is required for polymerization. The minimum force decreases with increasing RecA or ATP concentrations. We propose that force reduces the off-rate for nucleation site binding and that nucleation site stability is the stringency factor that prevents in vivo polymerization. PMID:21245047

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

    SciTech Connect

    Wohlbach, Dana J.; Gasch, Audrey P.

    2014-08-05

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

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

    SciTech Connect

    Wohlbach, Dana J.; Gasch, Audrey P.

    2015-09-29

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

  8. The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

    PubMed

    Bork, J M; Cox, M M; Inman, R B

    2001-12-17

    The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein. PMID:11743007

  9. Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

    NASA Astrophysics Data System (ADS)

    Yu, Garmay; A, Shvetsov; D, Karelov; D, Lebedev; A, Radulescu; M, Petukhov; V, Isaev-Ivanov

    2012-02-01

    Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

  10. RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations

    PubMed Central

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-01-01

    Most genetic regulatory mechanisms involve protein–DNA interactions. In these processes, the classical Watson–Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein–DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA–protein interactions. PMID:9770480

  11. RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations.

    PubMed

    Leger, J F; Robert, J; Bourdieu, L; Chatenay, D; Marko, J F

    1998-10-13

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  12. RecA Binding to a Single Double-Stranded DNA Molecule: A Possible Role of DNA Conformational Fluctuations

    NASA Astrophysics Data System (ADS)

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-10-01

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  13. 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.

  14. The SCL gene product: a positive regulator of erythroid differentiation.

    PubMed Central

    Aplan, P D; Nakahara, K; Orkin, S H; Kirsch, I R

    1992-01-01

    The SCL (tal-1, TCL5) gene is a member of the basic domain, helix-loop-helix (bHLH) class of putative transcription factors. We found that (i) the SCL promoter for exon Ia contains a potential recognition site for GATA-binding transcription factors, (ii) SCL mRNA is expressed in all erythroid tissues and cell lines examined, and (iii) SCL mRNA increases upon induced differentiation of murine erythroleukemia (MEL) cells, and inferred that SCL may play a physiologic role in erythroid differentiation. We used gel shift and transfection assays to demonstrate that the GATA motif in the SCL promoter binds GATA-1 (and GATA-2), and also mediates transcriptional transactivation. To identify a role for SCL in erythroid differentiation, we generated stable transfectants of MEL and K562 (a human chronic myelogenous leukemia cell line that can differentiate along the erythroid pathway) cells overexpressing wild-type, antisense or mutant SCL cDNA. Increasing the level of SCL expression in two independent MEL lines (F4-6 and C19, a 745 derivative) and K562 cells increased the rate of spontaneous (i.e. in the absence of inducer) erythroid differentiation. Conversely, induced differentiation was inhibited in MEL transfectants expressing either antisense SCL cDNA or a mutant SCL lacking the basic domain. Our experiments suggest that the SCL gene can be a target for the erythroid transcription factor GATA-1 and that the SCL gene product serves as a positive regulator of erythroid differentiation. Images PMID:1396592

  15. Evaluation of Clostridium ljungdahlii DSM 13528 reference genes in gene expression studies by qRT-PCR.

    PubMed

    Liu, Juanjuan; Tan, Yang; Yang, Xiaohong; Chen, Xiaohua; Li, Fuli

    2013-10-01

    Clostridium ljungdahlii DSM 13528 is a promising platform organism for biofuel production from syngas. Gene expression analysis permits a better understanding of the important molecular biological characteristics of this organism, such as carbon fixation and solvent adaptation. Normalization is a prerequisite for accurate gene expression analysis, but until now, no valid reference genes have been proposed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of C. ljungdahlii DSM 13528. In this study, seven candidate reference genes (gyrA, rho, fotl, rpoA, gukl, recA, 16S rRNA) were selected for qRT-PCR quantification of their expression levels in various culture conditions that corresponded to different carbon sources and stresses. Two analytical programs, geNorm and NormFinder, were used to evaluate reference gene stability. The results showed that gyrA, rho and fotl exhibited the most stable expression levels across all tested samples and can be confidently used as reference genes to normalize the transcriptional data of target genes in qRT-PCR analyses of C. ljungdahlii DSM 13528. This study presents the first attempt to explore the validity of candidate reference genes and provide a set of valid reference genes for normalizing C. ljungdahlii DSM 13528 target gene expression and transcriptome analysis.

  16. 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

  17. RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins.

    PubMed

    Shan, Q; Bork, J M; Webb, B L; Inman, R B; Cox, M M

    1997-02-01

    RecA protein filaments formed on circular (ssDNA) in the presence of ssDNA binding protein (SSB) are generally stable as long as ATP is regenerated. On linear ssDNA, stable RecA filaments are believed to be formed by nucleation at random sites on the DNA followed by filament extension in the 5' to 3' direction. This view must now be enlarged as we demonstrate that RecA filaments formed on linear ssDNA are subject to a previously undetected end-dependent disassembly process. RecA protein slowly dissociates from one filament end and is replaced by SSB. The results are most consistent with disassembly from the filament end nearest the 5' end of the DNA. The bound SSB prevents re-formation of the RecA filaments, rendering the dissociation largely irreversible. The dissociation requires ATP hydrolysis. Disassembly is not observed when the pH is lowered to 6.3 or when dATP replaces ATP. Disassembly is not observed even with ATP when both the RecO and RecR proteins are present in the initial reaction mixture. When the RecO and RecR proteins are added after most of the RecA protein has already dissociated, RecA protein filaments re-form after a short lag. The newly formed filaments contain an amount of RecA protein and exhibit an ATP hydrolysis rate comparable to that observed when the RecO and RecR proteins are included in the initial reaction mixture. The RecO and RecR proteins thereby stabilize RecA filaments even at the 5' ends of ssDNA, a fact which should affect the recombination potential of 5' ends relative to 3' ends. The location and length of RecA filaments involved in recombinational DNA repair is dictated by both the assembly and disassembly processes, as well as by the presence or absence of a variety of other proteins that can modulate either process. PMID:9048946

  18. 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

  19. 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

  20. Transcription of interferon-stimulated genes is induced by adenovirus particles but is suppressed by E1A gene products.

    PubMed Central

    Reich, N; Pine, R; Levy, D; Darnell, J E

    1988-01-01

    Interferon treatment of cell cultures results in the rapid transcriptional induction of a specific set of genes. In this paper we explore the effect of cellular infection by several adenoviruses, both wild type and mutant, on the expression of these genes. Infection with adenovirus induces the transcription of the interferon-stimulated genes in the absence of any protein synthesis. In fact, the inhibition of protein synthesis during a wild-type infection produces enhanced stimulation of transcription of these genes. Experiments with viral mutants indicate the ability to specifically suppress this transcription maps to the E1A gene. In addition, the E1A gene products are capable of suppressing the specific transcriptional induction of interferon-stimulated promoters during cotransfection experiments and therefore presumably during viral infection. The dual effect of adenovirus on the expression of interferon-stimulated genes may represent an example of action and evolutionary reaction between virus and host. Images PMID:2446013

  1. Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

    PubMed

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

    Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

  2. [Collaborative study on regulatory science for facilitating clinical development of gene therapy products for genetic diseases].

    PubMed

    Uchida, Eriko; Igarashi, Yuka; Sato, Yoji

    2014-01-01

    Gene therapy products are expected as innovative medicinal products for intractable diseases such as life-threatening genetic diseases and cancer. Recently, clinical developments by pharmaceutical companies are accelerated in Europe and the United States, and the first gene therapy product in advanced countries was approved for marketing authorization by the European Commission in 2012. On the other hand, more than 40 clinical studies for gene therapy have been completed or ongoing in Japan, most of them are conducted as clinical researches by academic institutes, and few clinical trials have been conducted for approval of gene therapy products. In order to promote the development of gene therapy products, revision of the current guideline and/or preparation of concept paper to address the evaluation of the quality and safety of gene therapy products are necessary and desired to clearly show what data should be submitted before First-in-Human clinical trials of novel gene therapy products. We started collaborative study with academia and regulatory agency to promote regulatory science toward clinical development of gene therapy products for genetic diseases based on lentivirus and adeno-associated virus vectors; National Center for Child Health and Development (NCCHD), Nippon Medical School and PMDA have been joined in the task force. At first, we are preparing pre-draft of the revision of the current gene therapy guidelines in this project.

  3. The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli

    PubMed Central

    Pan, Xuefeng; Leach, David R. F.

    2000-01-01

    A 24 triplet TGG·CCA repeat array shows length- and orientation-dependent propagation when present in the plasmid pUC18. When TGG24 is present as template for leading-strand synthesis, plasmid recovery is normal in all strains tested. However, when it acts as template for lagging-strand synthesis, plasmid propagation is seriously compromised. Plasmids carrying deletions in the 5′ side of this sequence can be isolated and products carrying 15 TGG triplets do not significantly interfere with plasmid propagation. Mutations in sbcCD, mutS and recA significantly improve the recovery of plasmids with TGG24 on the lagging-strand template. These findings suggest that TGG24 can fold into a structure that can interfere with DNA replication in vivo but that TGG15 cannot. Furthermore, since the presence of the MutS and SbcCD proteins are required for propagation interference, it is likely that stabilisation of mismatched base pairs and secondary structure cleavage are implicated. In contrast, there is no correlation of triplet repeat expansion and deletion instability with predicted DNA folding. These results argue for a dissociation of the factors affecting DNA fragility from those affecting trinucleotide repeat expansion–contraction instability. PMID:10931934

  4. Involvement of recF, recO, and recR genes in UV-radiation mutagenesis of Escherichia coli.

    PubMed

    Liu, Y H; Cheng, A J; Wang, T C

    1998-04-01

    The recF, recO, and recR genes were originally identified as those affecting the RecF pathway of recombination in Escherichia coli cells. Several lines of evidence suggest that the recF, recO, and recR genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in recF, recO, or recR greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp- (trpE65) to a Trp+ phenotypes. Introduction of the defective lexA51 mutation [lexA51(Def)] and/or UmuD' into recF, recO, and recR mutants failed to restore normal UVM in the mutants. On the other hand, the presence of recA2020, a suppressor mutation for recF, recO, and recR mutations, restored normal UVM in recF, recO, and recR mutants. These results indicate an involvement of the recF, recO, and recR genes and their products in UVM, possibly by affecting the third role of RecA in UVM. PMID:9537373

  5. Aflatoxin conducive and non-conducive growth conditions reveal new gene associations with aflatoxin production.

    PubMed

    Price, Michael S; Conners, Shannon B; Tachdjian, Sabrina; Kelly, Robert M; Payne, Gary A

    2005-06-01

    Research on aflatoxin (AF) production has traditionally focused on defining the AF biosynthetic pathway with the goal of identifying potential targets for intervention. To understand the effect of nitrogen source, carbon source, temperature, and pH on the regulation of AF biosynthesis, a targeted cDNA microarray consisting of genes associated with AF production over time was employed. Expression profiles for genes involved in AF biosynthesis grouped into five clades. A putative regulon was identified consisting of 20 genes that were induced in the conducive nitrogen and pH treatments and the non-conducive carbon and temperature treatments, as well as four other putative regulons corresponding to each of the four variables studied. Seventeen genes exhibited consistent induction/repression profiles across all the experiments. One of these genes was consistently downregulated with AF production. Overexpression of this gene resulted in repression of AF biosynthesis. The cellular function of this gene is currently unresolved.

  6. Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production.

    PubMed

    Giardina, P C; Foster, L A; Toth, S I; Roe, B A; Dyer, D W

    1995-12-29

    The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced. The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846. Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron. The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

  7. Long-term follow-up of cancer patients treated with gene therapy medicinal products.

    PubMed

    Galli, Maria Cristina

    2012-06-01

    European Union requirements are discussed for the long-term follow-up of advanced therapy medicinal products, as well as how they can be applied to cancer patients treated with gene therapy medicinal products in the context of clinical trials, as described in a specific guideline issued by Gene Therapy Working Party at the European Medicine Agency.

  8. A mutant gene that increases gibberellin production in brassica.

    PubMed

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

    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(3) (GA(3)) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA(1) and GA(3) were estimated by gas chromatography-selected ion monitoring using [(2)H]GA(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(20) and GA(1), and the rate of GA(19) metabolism were simultaneously analyzed at day 7 by feeding [(2)H(2)]GA(19) and measuring metabolites [(2)H(2)]GA(20) and [(2)H(2)]GA(1) and endogenous GA(20) and GA(1), with [(2)H(5)]GA(20) and [(2)H(5)]GA(1) as quantitative internal standards. Levels of GA(1) and GA(20) were 4.6- and 12.9-fold higher, respectively, and conversions to GA(20) and GA(1) were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA(1) biosynthesis in ein, the conversion of [(3)H]GA(20) to [(3)H]GA(1) was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA(1) biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A(1) and A(3). The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation.

  9. 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

  10. 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.

  11. 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.

  12. Form gene clustering method about pan-ethnic-group products based on emotional semantic

    NASA Astrophysics Data System (ADS)

    Chen, Dengkai; Ding, Jingjing; Gao, Minzhuo; Ma, Danping; Liu, Donghui

    2016-09-01

    The use of pan-ethnic-group products form knowledge primarily depends on a designer's subjective experience without user participation. The majority of studies primarily focus on the detection of the perceptual demands of consumers from the target product category. A pan-ethnic-group products form gene clustering method based on emotional semantic is constructed. Consumers' perceptual images of the pan-ethnic-group products are obtained by means of product form gene extraction and coding and computer aided product form clustering technology. A case of form gene clustering about the typical pan-ethnic-group products is investigated which indicates that the method is feasible. This paper opens up a new direction for the future development of product form design which improves the agility of product design process in the era of Industry 4.0.

  13. Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps.

    PubMed

    Webb, B L; Cox, M M; Inman, R B

    1997-10-31

    In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair. PMID:9363943

  14. 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.

  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 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

  17. Distribution and Diversity of Natural Product Genes in Marine and Freshwater Cyanobacterial Cultures and Genomes

    PubMed Central

    Ehrenreich, Ian M.; Waterbury, John B.; Webb, Eric A.

    2005-01-01

    Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms. PMID:16269782

  18. RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli.

    PubMed

    Vlasić, Ignacija; Ivancić-Baće, Ivana; Imesek, Mirna; Mihaljević, Boris; Brcić-Kostić, Krunoslav

    2008-09-01

    The SOS response is an important mechanism which allows Escherichia coli cells to maintain genome integrity. Two key proteins in SOS regulation are LexA (repressor) and RecA (coprotease). The signal for SOS induction is generated at the level of a RecA filament. Depending on the type of DNA damage, a RecA filament is produced by specific activities (helicase, nuclease and RecA loading) of either RecBCD, RecF or a hybrid recombination pathway. It was recently demonstrated that RecA loading activity is essential for the induction of the SOS response after UV-irradiation. In this paper we studied the genetic requirements for SOS induction after introduction of a double-strand break (DSB) by the I-SceI endonuclease in a RecA loading deficient recB mutant (recB1080). We monitored SOS induction by assaying beta-galactosidase activity and compared induction of the response between strains having one or more inactivated mechanisms of RecA loading and their derivatives. We found that simultaneous inactivation of both RecA loading functions (in recB1080 recO double mutant) partially impairs SOS induction after introduction of a DSB. However, we found that the RecJ nuclease is essential for SOS induction after the introduction of a DSB in the recB1080 mutant. This result indicates that RecJ is needed to prepare ssDNA for subsequent loading of RecA protein. It implies that an additional type of RecA loading could exist in the cell. PMID:18445487

  19. MultiFun, a multifunctional classification scheme for Escherichia coli K-12 gene products.

    PubMed

    Serres, M H; Riley, M

    2000-01-01

    An enriched classification system for cellular functions of gene products of Escherichia coli K-12 was developed based on the initial classification by Riley. In the new classification scheme, MultiFun, cellular functions are divided into 10 major categories: Metabolism, Information Transfer, Regulation, Transport, Cell Processes, Cell Structure, Location, Extra-chromosomal Origin, DNA Site, and Cryptic Gene. These major categories are further sub-divided into a hierarchical scheme. Two thousand nine hundred twenty-two gene products of E. coli K-12 were assigned to one or more functions depending on the role they play in the cell. Functional assignments were made to 66% of E. coli gene products, ranging from 1 to 16 assignments per gene product. The expansion of cellular function categories and the assignment to more than one category (multifunction) provides a more complete description of the gene products and their roles and hence better reflects the functional complexity of organisms. We believe this classification system will be useful in the field of genome analysis, both for annotation purposes and for comparative studies. The functional classification scheme and the cellular function assignments made to E. coli gene products can be accessed from the web at the databases GenProtEC (http://genprotec.mbl.edu) and EcoCyc (http://www.ecocyc.org).

  20. The genomisotopic approach: a systematic method to isolate products of orphan biosynthetic gene clusters.

    PubMed

    Gross, Harald; Stockwell, Virginia O; Henkels, Marcella D; Nowak-Thompson, Brian; Loper, Joyce E; Gerwick, William H

    2007-01-01

    With the increasing number of genomes sequenced and available in the public domain, a large number of orphan gene clusters, for which the encoded natural product is unknown, have been identified. These orphan gene clusters represent a tremendous source of novel and possibly bioactive compounds. Here, we describe a "genomisotopic approach," which employs a combination of genomic sequence analysis and isotope-guided fractionation to identify unknown compounds synthesized from orphan gene clusters containing nonribosomal peptide synthetases. Analysis of the Pseudomonas fluorescens Pf-5 genome led to the identification of an orphan gene cluster predicted to code for the biosynthesis of a lipopeptide natural product. Application of the genomisotopic approach to isolate the product of this gene cluster resulted in the discovery of orfamide A, founder of a group of bioactive cyclic lipopeptides.

  1. 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).

  2. 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.

  3. DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17.

    PubMed

    Genilloud, O; Moreno, F; Kolter, R

    1989-02-01

    The 3.8-kilobase segment of plasmid DNA that contains the genes required for production of the DNA replication inhibitor microcin B17 was sequenced. The sequence contains four open reading frames which were shown to be translated in vivo by the construction of fusions to lacZ. The location of these open reading frames fits well with the location of the four microcin B17 production genes, mcbABCD, identified previously through genetic complementation. The products of the four genes have been identified, and the observed molecular weights of the proteins agree with those predicted from the nucleotide sequence. The transcription of these genes was studied by using fusions to lacZ and physical mapping of mRNA start sites. Three promoters were identified in this region. The major promoter for all the genes is a growth phase-regulated OmpR-dependent promoter located upstream of mcbA. A second promoter is located within mcbC and is responsible for a low-level basal expression of mcbD. A third promoter, located within mcbD, promotes transcription in the reverse direction starting within mcbD and extending through mcbC. The resulting mRNA appears to be an untranslated antisense transcript that could play a regulatory role in the expression of these genes.

  4. 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

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

    PubMed

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

    2016-07-07

    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.

  6. 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

  7. High-temperature piezoelectric crystals ReCa4O(BO3)3: a review.

    PubMed

    Yu, Fapeng; Hou, Shuai; Zhao, Xian; Zhang, Shujun

    2014-08-01

    High-temperature sensors are desirable for structural health monitoring and/or nondestructive evaluation of next-generation turbines, more efficient jet engines, and the furnace components of electrical power plants. Of all the investigated high-temperature piezoelectric materials, rare-earth calcium oxyborate crystals ReCa4O(BO3)3 (ReCOB, Re: rare-earth) exhibit attractive advantages for high-temperature piezoelectric sensing. In this paper, the electroelastic properties of different ReCOB piezoelectric crystals are investigated. The crosstalk between various vibration modes are discussed, from which the optimized crystal cuts are achieved. Furthermore, temperature dependences of the electrical resistivity, dielectric, elastic, piezoelectric, and electromechanical properties of ReCOB crystals are studied. Finally, the thermal properties, including thermal expansion, specific heat, and thermal conductivity at elevated temperatures are studied and compared with commercially available high-temperature piezoelectric crystals.

  8. High-temperature piezoelectric crystals ReCa4O(BO3)3: a review.

    PubMed

    Yu, Fapeng; Hou, Shuai; Zhao, Xian; Zhang, Shujun

    2014-08-01

    High-temperature sensors are desirable for structural health monitoring and/or nondestructive evaluation of next-generation turbines, more efficient jet engines, and the furnace components of electrical power plants. Of all the investigated high-temperature piezoelectric materials, rare-earth calcium oxyborate crystals ReCa4O(BO3)3 (ReCOB, Re: rare-earth) exhibit attractive advantages for high-temperature piezoelectric sensing. In this paper, the electroelastic properties of different ReCOB piezoelectric crystals are investigated. The crosstalk between various vibration modes are discussed, from which the optimized crystal cuts are achieved. Furthermore, temperature dependences of the electrical resistivity, dielectric, elastic, piezoelectric, and electromechanical properties of ReCOB crystals are studied. Finally, the thermal properties, including thermal expansion, specific heat, and thermal conductivity at elevated temperatures are studied and compared with commercially available high-temperature piezoelectric crystals. PMID:25073142

  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. Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

    PubMed

    Rangarajan, Savithri; Woodgate, Roger; Goodman, Myron F

    2002-02-01

    In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough. PMID:11929519

  12. 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.

  13. Regulation of Deinococcus radiodurans RecA Protein Function via Modulation of Active and Inactive Nucleoprotein Filament States*

    PubMed Central

    Ngo, Khanh V.; Molzberger, Eileen T.; Chitteni-Pattu, Sindhu; Cox, Michael M.

    2013-01-01

    The RecA protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding protein. The ATPase function of DrRecA nucleoprotein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans. PMID:23729671

  14. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

    PubMed Central

    Ma, Yue-jiao; Sun, Xiao-hong; Xu, Xiao-yan; Zhao, Yong; Pan, Ying-jie; Hwang, Cheng-An; Wu, Vivian C. H.

    2015-01-01

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus. PMID:26659406

  15. 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.

  16. 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.

  17. In silico identification of gene amplification targets for improvement of lycopene production.

    PubMed

    Choi, Hyung Seok; Lee, Sang Yup; Kim, Tae Yong; Woo, Han Min

    2010-05-01

    The identification of genes to be deleted or amplified is an essential step in metabolic engineering for strain improvement toward the enhanced production of desired bioproducts. In the past, several methods based on flux analysis of genome-scale metabolic models have been developed for identifying gene targets for deletion. Genome-wide identification of gene targets for amplification, on the other hand, has been rather difficult. Here, we report a strategy called flux scanning based on enforced objective flux (FSEOF) to identify gene amplification targets. FSEOF scans all the metabolic fluxes in the metabolic model and selects fluxes that increase when the flux toward product formation is enforced as an additional constraint during flux analysis. This strategy was successfully employed for the identification of gene amplification targets for the enhanced production of the red-colored antioxidant lycopene. Additional metabolic engineering based on gene knockout simulation resulted in further synergistic enhancement of lycopene production. Thus, FSEOF can be used as a general strategy for selecting genome-wide gene amplification targets in silico.

  18. Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product

    SciTech Connect

    Santhanam, U.; Ray, A.; Sehgal, P.B. )

    1991-09-01

    The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. The authors transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriate chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or {beta}-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.

  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. 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.

  1. 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

  2. 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.

  3. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  6. Gene product identification and promoter analysis of hig locus of plasmid Rts1.

    PubMed

    Tian, Q B; Hayashi, T; Murata, T; Terawaki, Y

    1996-08-14

    The hig (host inhibition of growth) genes of plasmid Rts1 belong to the plasmid-encoded proteic killer gene family. Compared with other proteic killer genes described so far, hig is unique in that the toxic part (higB) exists upstream of the antidote gene (higA). Here we describe results of the promoter analysis of hig genes together with identification of the proteic gene products of higA and higB. Two promoters were identified in the hig locus; a stronger one, named Phig, is located upstream of higB and a weaker one, PhigA, is upstream of higA within the higB coding region. The Phig activity was negatively regulated by HigA and this regulation was augmented by HigB, whereas PhigA was not subjected to such a regulation.

  7. Purification of the gam gene-product of bacteriophage Mu and determination of the nucleotide sequence of the gam gene.

    PubMed Central

    Akroyd, J E; Clayson, E; Higgins, N P

    1986-01-01

    The gam gene of bacteriophage Mu encodes a protein which protects linear double stranded DNA from exonuclease degradation in vitro and in vivo. We purified the Mu gam gene product to apparent homogeneity from cells in which it is over-produced from a plasmid clone. The purified protein is a dimer of identical subunits of 18.9 kd. It can aggregate DNA into large, rapidly sedimenting complexes and is a potent exonuclease inhibitor when bound to DNA. The N-terminal amino acid sequence of the purified protein was determined by automated degradation and the nucleotide sequence of the Mu gam gene is presented to accurately map its position in the Mu genome. Images PMID:2945162

  8. Identification of the intracellular polyhydroxyalkanoate depolymerase gene of Paracoccus denitrificans and some properties of the gene product.

    PubMed

    Gao, D; Maehara, A; Yamane, T; Ueda, S

    2001-03-15

    Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene (phaZ) was identified near the PHA synthase gene (phaC) of P. denitrificans. Cell extract of Escherichia coli carrying lacZ--phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D(--)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg(2+) ion inhibited PHB degradation. However, the inhibitory effect by Mg(2+) ion was not observed using the cell extract of P. denitrificans.

  9. 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

  10. 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

  11. Gene regulatory networks controlling hematopoietic progenitor niche cell production and differentiation in the Drosophila lymph gland.

    PubMed

    Tokusumi, Yumiko; Tokusumi, Tsuyoshi; Shoue, Douglas A; Schulz, Robert A

    2012-01-01

    Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. In this report, we utilize a PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. We assessed the effect of abrogating the function of 820 genes as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, we characterized loss- and gain-of-function phenotypes. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for

  12. 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

  13. Regulation of a Novel Gene Cluster Involved in Secondary Metabolite Production in Streptomyces coelicolor▿ †

    PubMed Central

    Hindra; Pak, Patricia; Elliot, Marie A.

    2010-01-01

    Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in Streptomyces coelicolor. The overexpression of a gene cluster comprising four protein-encoding genes (abeABCD) and an antisense RNA-encoding gene (α-abeA) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (abeR) encoding a predicted Streptomyces antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the abe genes to be differentially regulated and controlled at multiple levels. Upstream of abeA was a promoter that directed the transcription of abeABCD at a low but constitutive level. The expression of abeBCD was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within abeA. Expressed divergently to the abeBCD promoter was α-abeA, whose expression mirrored that of abeBCD but did not require activation by AbeR. Instead, α-abeA transcript levels were subject to negative control by the double-strand-specific RNase, RNase III. PMID:20675485

  14. Regulation of the Escherichia coli glyA gene by the purR gene product.

    PubMed

    Steiert, J G; Rolfes, R J; Zalkin, H; Stauffer, G V

    1990-07-01

    The purine regulon repressor protein, PurR, was shown to be a purine component involved in glyA regulation in Escherichia coli. Expression of glyA, encoding serine hydroxymethyltransferase activity, was elevated in a purR mutant compared with a wild-type strain. When the purR mutant was transformed with a plasmid carrying the purR gene, the serine hydroxymethyltransferase levels returned to the wild-type level. The PurR protein bound specifically to a DNA fragment carrying the glyA control region, as determined by gel retardation. In a DNase I protection assay, a 24-base-pair region was protected from DNase I digestion by PurR. The glyA operator sequence for PurR binding is similar to that reported for several pur regulon genes.

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

    PubMed

    Gaballa, A; Koedam, N; Cornelis, P

    1996-08-01

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

  16. Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product.

    PubMed

    Chen, G T; Axley, M J; Hacia, J; Inouye, M

    1992-03-01

    The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H. PMID:1533438

  17. Gene protein products of SA11 simian rotavirus genome.

    PubMed Central

    Arias, C F; López, S; Espejo, R T

    1982-01-01

    When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments. Images PMID:6283128

  18. 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.

  19. Expression of MAGE-1 and -3 genes and gene products in human hepatocellular carcinoma

    PubMed Central

    Kariyama, K; Higashi, T; Kobayashi, Y; Nouso, K; Nakatsukasa, H; Yamano, T; Ishizaki, M; Kaneyoshi, T; Toshikuni, N; Ohnishi, T; Fujiwara, K; Nakayama, E; Terracciano, L; Spagnoli, G C; Tsuji, T

    1999-01-01

    MAGE gene family encodes peptides recognized by autologous cytotoxic T lymphocytes in a major histocompatibility complex (MHC) class-I restricted fashion. In the present study, we have performed reverse-transcription polymerase chain reaction (RT-PCR) for the genes, as well as immunohistochemical analysis and Western blotting of MAGE-1 and -3 proteins in 33 surgically resected hepatocellular carcinomas (HCCs). MAGE-1 and -3 mRNAs were constitutively expressed exclusively in 78 and 42% of HCCs respectively. On immunohistochemistry with monoclonal antibodies, 77B for MAGE-1 and 57B for MAGE-3, MAGE-1 and -3 proteins were recognized in cytoplasm of only six among 33 (18%) and two of 29 HCCs (7%) respectively. The distribution pattern was mostly focal in HCC nodules. By contrast, the Western blot analysis revealed that the MAGE-1 (46 kDa) and -3 proteins (48 kDa) were expressed in 80 and 60% of 15 HCCs examined respectively. The proteins of MAGE-1 and -3 were also expressed exclusively in HCCs regardless of the histological grading and clinical staging. Our results indicate that the detection of the genes by RT-PCR or the proteins by Western blotting is useful for differentiating early HCCs from non-cancerous lesions, and that the peptides derived from MAGE-1 and -3 proteins might be suitable targets for immunotherapy of human HCC. © 1999 Cancer Research Campaign PMID:10576668

  20. QUANTIFICATION OF RECA GENE EXPRESSION AS AN INDICATOR OF REPAIR POTENTIAL IN MARINE BACTERIOPLANKTON COMMUNITIES OF ANTARCTICA.

    EPA Science Inventory

    Marine bacteria in surface waters must cope daily with the damaging effects of exposure to solar radiation (containing both UV-A and UV-B wavelengths), which produces lesions in their DNA. As the stratospheric ozone layer is depleted, these coping mechanisms are likely to play an...

  1. Genes of the RNASE5 pathway contain SNP associated with milk production traits in dairy cattle

    PubMed Central

    2013-01-01

    Background Identification of the processes and mutations responsible for the large genetic variation in milk production among dairy cattle has proved challenging. One approach is to identify a biological process potentially involved in milk production and to determine the genetic influence of all the genes included in the process or pathway. Angiogenin encoded by angiogenin, ribonuclease, RNase A family 5 (RNASE5) is relatively abundant in milk, and has been shown to regulate protein synthesis and act as a growth factor in epithelial cells in vitro. However, little is known about the role of angiogenin in the mammary gland or if the polymorphisms present in the bovine RNASE5 gene are associated with lactation and milk production traits in dairy cattle. Given the high economic value of increased protein in milk, we have tested the hypothesis that RNASE5 or genes in the RNASE5 pathway are associated with milk production traits. First, we constructed a “RNASE5 pathway” based on upstream and downstream interacting genes reported in the literature. We then tested SNP in close proximity to the genes of this pathway for association with milk production traits in a large dairy cattle dataset. Results The constructed RNASE5 pathway consisted of 11 genes. Association analysis between SNP in 1 Mb regions surrounding these genes and milk production traits revealed that more SNP than expected by chance were associated with milk protein percent (P < 0.05 significance). There was no significant association with other traits such as milk fat content or fertility. Conclusions These results support a role for the RNASE5 pathway in milk production, specifically milk protein percent, and indicate that polymorphisms in or near these genes explain a proportion of the variation for this trait. This method provides a novel way of understanding the underlying biology of lactation with implications for milk production and can be applied to any pathway or gene set to test whether

  2. 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}.

  3. 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.

  4. Split-gene system for hybrid wheat seed production.

    PubMed

    Kempe, Katja; Rubtsova, Myroslava; Gils, Mario

    2014-06-24

    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.

  5. 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

  6. 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

  7. 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

  8. Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

    PubMed Central

    Paradkar, A. S.; Mosher, R. H.; Anders, C.; Griffin, A.; Griffin, J.; Hughes, C.; Greaves, P.; Barton, B.; Jensen, S. E.

    2001-01-01

    Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine ɛ-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine. PMID:11319114

  9. 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

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

    DOE PAGES

    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

  11. 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.

  12. Contrasts in codon usage of latent versus productive genes of Epstein-Barr virus: data and hypotheses.

    PubMed Central

    Karlin, S; Blaisdell, B E; Schachtel, G A

    1990-01-01

    Epstein-Barr virus (EBV) has two different modes of existence: latent and productive. There are eight known genes expressed during latency (and hardly at all during the productive phase) and about 70 other ("productive") genes. It is shown that the EBV genes known to be expressed during latency display codon usage strikingly different from that of genes that are expressed during lytic growth. In particular, the percentage of S3 (G or C in codon site 3) is persistently lower (about 20%) in all latent genes than in nonlatent genes. Moreover, S3 is lower in each multicodon amino acid form. Also, the percentage of S in silent codon sites 1 of leucine and arginine is lower in latent than in nonlatent genes. The largest absolute differences in amino acid usage between latent and nonlatent genes emphasize codon types SSN and WWN (W means nucleotide A or T and N is any nucleotide). Two principal explanations to account for the EBV latent versus productive gene codon disparity are proposed. Latent genes have codon usage substantially different from that of host cell genes to minimize the deleterious consequences to the host of viral gene expression during latency. (Productive genes are not so constrained.) It is also proposed that the latency genes of EBV were acquired recently by the viral genome. Evidence and arguments for these proposals are presented. PMID:2166815

  13. 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.

  14. New LIC Vectors For Production of Proteins from Genes Containing Rare Codons

    PubMed Central

    Eschenfeldt, William H.; Makowska-Grzyska, Magdalena; Stols, Lucy; Donnelly, Mark; Jedrzejczak, Robert; Joachimiak, Andrzej

    2014-01-01

    In the effort to produce proteins coded by diverse genomes, structural genomics projects often must express genes containing codons that are rare in the production strain. To address this problem, genes expressing tRNAs corresponding to those codons are typically coexpressed from a second plasmid in the host strain, or from genes incorporated into production plasmids. Here we describe the modification of a series of LIC pMCSG vectors currently used in the high-throughput production of proteins to include crucial tRNA genes covering rare codons for Arg (AGG/AGA) and Ile (AUA). We also present variants of these new vectors that allow analysis of ligand binding or co-expression of multiple proteins introduced through two independent LIC steps. Additionally, to accommodate the cloning of multiple large proteins, the size of the plasmids was reduced by approximately one kilobase through the removal of non-essential DNA from the base vector. Production of proteins from core vectors of this series validated the desired enhanced capabilities: higher yields of proteins expressed from genes with rare codons occurred in most cases, biotinylated derivatives enabled detailed automated ligand binding analysis, and multiple proteins introduced by dual LIC cloning were expressed successfully and in near balanced stoichiometry, allowing tandem purification of interacting proteins. PMID:24057978

  15. 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.

  16. 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

  17. 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.

  18. The mechanism of recA polA lethality: Suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli

    SciTech Connect

    Cao, Yang; Kogoma, Tokio

    1995-04-01

    The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5{prime} {yields} 3{prime} exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF{sup +} is essential for this suppression pathway, recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by {Delta}recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of {Delta}recA polA25::spc cells to UV damage by {approximately}10{sup 4}-fold. lexA(Def) also restores P1 transduction proficiency to the {Delta}recA polA25::spc mutant to a level that is 7.3% of the recA{sup +} wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants. 52 refs., 7 figs., 5 tabs.

  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. RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli.

    PubMed

    Ivancić-Baće, Ivana; Peharec, Petra; Moslavac, Suncana; Skrobot, Nikolina; Salaj-Smic, Erika; Brcić-Kostić, Krunoslav

    2003-02-01

    The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' --> 3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF(-) background the recB1080 mutant is recombination deficient, whereas in a recF(+) genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D(-)) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' --> 3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR. PMID:12618388

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

    PubMed

    Przystałowska, Hanna; Zeyland, Joanna; Kośmider, Alicja; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Relationship of polypeptide products of the transforming gene of Rous sarcoma virus and the homologous gene of vertebrates.

    PubMed

    Sefton, B M; Hunter, T; Beemon, K

    1980-04-01

    All vertebrate cells have been shown to contain a gene, sarc, that has some homology with the transforming gene of Rous sarcoma virus, src. We have compared the polypeptide products of the sarc gene, p60(sarc), of human, mouse, and chicken cells with the polymorphic polypeptide product of the src gene, p60(src), of several strains of Rous sarcoma virus by two-dimensional peptide mapping. p60(sarc) from chicken cells was clearly related to every viral p60(src). Eleven of its 13 methionine-containing tryptic peptides were present in some viral p60(src). Conversely, the other two peptides were not present in any p60(src) we have examined so far. The 11 peptides from p60(sarc) of chickens that were shared with viral p60(src), however, were not all present in any single viral p60(src). These 11 peptides most closely resemble those in the p60(src)s of B77 virus and the Prague strain of Rous sarcoma virus. These data are consistent with the hypothesis that cellular sarc is the progenitor of viral src. The p60(sarc)s of human, mouse, and chicken cells were so similar in tryptic peptide composition that they were more closely related to each other than were some viral p60(src)s. The two mammalian p60(sarc)s differed from avian p60(sarc) most notably in that they lacked a peptide that chicken p60(sarc) shares with all the viral p60(src)s. The similarity of these maps suggests that the sequence of the p60(sarc) polypeptide has diverged very little during evolution. This may imply that p60(sarc) is an essential cellular component.

  8. 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

  9. 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.

  10. High-temperature piezoelectric single crystal ReCa(4)O(BO(3))(3) for sensor applications.

    PubMed

    Zhang, Shujun; Fei, Yiting; Frantz, Eric; Snyder, David W; Chai, Bruce H T; Shrout, Thomas R

    2008-12-01

    Large-size and high-quality ReCa(4)O(BO(3))(3) (ReCOB, Re = rare earth) single crystals were grown by the Czochralski pulling method. In this work, the electrical properties were investigated at room temperature and elevated temperature for YCa(4)O(BO(3))(3) (YCOB). The dielectric permittivity, piezoelectric strain coefficient, and electromechanical coupling were found to be on the order of 11, 6.5 pC/N, and 12.5%, respectively, with a high piezoelectric voltage coefficient around 0.067 Vm/N. The electrical resistivity of YCOB was found to be 2 x 10(8) Ohm.m at 800 degrees C, with Q values of 4,500 at 950 degrees C. The frequency/temperature coefficient of YCOB was found to be -75 to -85ppm/K in the temperature range of 30 to 950 degrees C, depending on the crystal orientations. Together with their temperature-independent properties, ReCOB crystals are promising candidates for sensing applications at elevated temperatures.

  11. The discovery of error-prone DNA polymerase V and its unique regulation by RecA and ATP.

    PubMed

    Goodman, Myron F

    2014-09-26

    My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.

  12. DNA compaction in the early part of the SOS response is dependent on RecN and RecA.

    PubMed

    Odsbu, Ingvild; Skarstad, Kirsten

    2014-05-01

    The nucleoids of undamaged Escherichia coli cells have a characteristic shape and number, which is dependent on the growth medium. Upon induction of the SOS response by a low dose of UV irradiation an extensive reorganization of the nucleoids occurred. Two distinct phases were observed by fluorescence microscopy. First, the nucleoids were found to change shape and fuse into compact structures at midcell. The compaction of the nucleoids lasted for 10-20 min and was followed by a phase where the DNA was dispersed throughout the cells. This second phase lasted for ~1 h. The compaction was found to be dependent on the recombination proteins RecA, RecO and RecR as well as the SOS-inducible, SMC (structural maintenance of chromosomes)-like protein RecN. RecN protein is produced in high amounts during the first part of the SOS response. It is possible that the RecN-mediated 'compact DNA' stage at the beginning of the SOS response serves to stabilize damaged DNA prior to recombination and repair. PMID:24615185

  13. The Discovery of Error-prone DNA Polymerase V and Its Unique Regulation by RecA and ATP

    PubMed Central

    Goodman, Myron F.

    2014-01-01

    My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts. PMID:25160630

  14. DNA compaction in the early part of the SOS response is dependent on RecN and RecA.

    PubMed

    Odsbu, Ingvild; Skarstad, Kirsten

    2014-05-01

    The nucleoids of undamaged Escherichia coli cells have a characteristic shape and number, which is dependent on the growth medium. Upon induction of the SOS response by a low dose of UV irradiation an extensive reorganization of the nucleoids occurred. Two distinct phases were observed by fluorescence microscopy. First, the nucleoids were found to change shape and fuse into compact structures at midcell. The compaction of the nucleoids lasted for 10-20 min and was followed by a phase where the DNA was dispersed throughout the cells. This second phase lasted for ~1 h. The compaction was found to be dependent on the recombination proteins RecA, RecO and RecR as well as the SOS-inducible, SMC (structural maintenance of chromosomes)-like protein RecN. RecN protein is produced in high amounts during the first part of the SOS response. It is possible that the RecN-mediated 'compact DNA' stage at the beginning of the SOS response serves to stabilize damaged DNA prior to recombination and repair.

  15. Enhanced succinic acid productivity by expression of mgtCB gene in Escherichia coli mutant.

    PubMed

    Wang, Jing; Yang, Le; Wang, Dan; Dong, Lichun; Chen, Rachel

    2016-04-01

    In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg(2+) concentration and mgtB to enhance the transport of Mg(2+) into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)2 and NH3·H2O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97 g L(-1) h(-1)) compared with that by using the engineering strain with the overexpression of mgtA gene. PMID:26711444

  16. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

  17. 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

  18. 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.

  19. 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

  20. 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. ...

  1. 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

  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.

  3. Prevalence of antibiotic resistance genes in staphylococci isolated from ready-to-eat meat products.

    PubMed

    Podkowik, M; Bystroń, J; Bania, J

    2012-01-01

    Prevalence of mecA, blaZ, tetO/K/M, ermA/B/C, aph, and vanA/B/C/D genes conferring resistance to oxacillin, penicillin, tetracycline, erythromycin, gentamicin, and vancomycin was investigated in 65 staphylococcal isolates belonging to twelve species obtained from ready-to-eat porcine, bovine, and chicken products. All coagulase negative staphylococci (CNS) and S. aureus isolates harbored at least one antibiotic resistance gene. None of the S. aureus possessed more than three genes, while 25% of the CNS isolates harbored at least four genes encoding resistance to clinically used antibiotics. In 15 CNS isolates the mecA gene was detected, while all S. aureus isolates were mecA-negative. We demonstrate that in ready-to-eat food the frequency of CNS harboring multiple antibiotic resistance genes is higher than that of multiple resistant S. aureus, meaning that food can be considered a reservoir of bacteria containing genes potentially contributing to the evolution of antibiotic resistance in staphylococci.

  4. Aerobic conditions increase isoprenoid biosynthesis pathway gene expression levels for carotenoid production in Enterococcus gilvus.

    PubMed

    Hagi, Tatsuro; Kobayashi, Miho; Nomura, Masaru

    2015-06-01

    Some lactic acid bacteria that harbour carotenoid biosynthesis genes (crtNM) can produce carotenoids. Although aerobic conditions can increase carotenoid production and crtNM expression levels, their effects on the pathways that synthesize carotenoid precursors such as mevalonate and isoprene are not completely understood. In this study, we investigated whether aerobic conditions affected gene expression levels involved in the isoprenoid biosynthesis pathway that includes the mevalonate and isoprene biosynthesis pathways in Enterococcus gilvus using real-time quantitative reverse transcription PCR. NADH oxidase (nox) and superoxide dismutase (sod) gene expression levels were investigated as controls for aerobic conditions. The expression levels of nox and sod under aerobic conditions were 7.2- and 8.0-fold higher, respectively, than those under anaerobic conditions. Aerobic conditions concomitantly increased the expression levels of crtNM carotenoid biosynthesis genes. HMG-CoA synthase gene expression levels in the mevalonate pathway were only slightly increased under aerobic conditions, whereas the expression levels of HMG-CoA reductase and five other genes in the isoprene biosynthesis pathways were 1.2-2.3-fold higher than those under anaerobic conditions. These results demonstrated that aerobic conditions could increase the expression levels of genes involved in the isoprenoid biosynthesis pathway via mevalonate in E. gilvus.

  5. 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

  6. Identification of an Extradiol Dioxygenase Involved in Tetralin Biodegradation: Gene Sequence Analysis and Purification and Characterization of the Gene Product

    PubMed Central

    Andújar, Eloísa; Hernáez, María José; Kaschabek, Stefan R.; Reineke, Walter; Santero, Eduardo

    2000-01-01

    A genomic region involved in tetralin biodegradation was recently identified in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1,2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7,8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (Vmax, 40.5 U mg−1; Km, 18.6 μM). The enzyme shows even higher activity with 1,2-dihydroxynaphthalene and also significant activity toward 1,2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1,2-dihydroxynaphthalene dioxygenases. PMID:10633115

  7. Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

    PubMed

    Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

    2015-03-27

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

  8. Direct Capture and Heterologous Expression of Salinispora Natural Product Genes for the Biosynthesis of Enterocin

    PubMed Central

    2015-01-01

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora’s promising secondary metabolome. PMID:25382643

  9. 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.

  10. 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.

  11. Modern plant metabolomics: Advanced natural product gene discoveries, improved technologies, and future prospects

    DOE PAGES

    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

  12. Correlation of Methane Production and Functional Gene Transcriptional Activity in a Peat Soil ▿

    PubMed Central

    Freitag, Thomas E.; Prosser, James I.

    2009-01-01

    The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37°C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25°C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient = 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems. PMID:19749064

  13. Correlation of methane production and functional gene transcriptional activity in a peat soil.

    PubMed

    Freitag, Thomas E; Prosser, James I

    2009-11-01

    The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37 degrees C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25 degrees C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient = 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems.

  14. The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production.

    PubMed

    Stegmann, Efthimia; Rausch, Christian; Stockert, Sigrid; Burkert, Daniel; Wohlleben, Wolfgang

    2006-09-01

    The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.

  15. 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

  16. 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

  17. Uncovering the gene knockout landscape for improved lycopene production in E. coli.

    PubMed

    Alper, Hal; Stephanopoulos, Gregory

    2008-04-01

    Systematic and combinatorial genetic approaches for the identification of gene knockout and overexpression targets have been effectively employed in the improvement of cellular phenotypes. Previously, we demonstrated how two of these tools, metabolic modeling and transposon mutagenesis, can be combined to identify strains of interest spanning the metabolic landscape of recombinant lycopene production in Escherichia coli. However, it is unknown how to best select multiple-gene knockout targets. Hence, this study seeks to understand how the overall order of gene selection, or search trajectory, biases the exploration and topology of the metabolic landscape. In particular, transposon mutagenesis and selection were employed in the background of eight different knockout genotypes. Collectively, 800,000 mutants were analyzed in hopes of exhaustively identifying all advantageous gene knockout targets. Several interesting observations, including clusters of gene functions, recurrence, and divergent genotypes, demonstrate the complexity of mapping only one genotype to one phenotype. One particularly interesting mutant, the DeltahnrDeltayliE genotype, exhibited a drastically improved lycopene production capacity in basic minimal medium in comparison to the best strains identified in previous studies.

  18. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    PubMed

    Przystałowska, Hanna; Zeyland, Joanna; Szymanowska-Powałowska, Daria; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol₁,₃-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming

  19. A shortest-path graph kernel for estimating gene product semantic similarity

    PubMed Central

    2011-01-01

    Background Existing methods for calculating semantic similarity between gene products using the Gene Ontology (GO) often rely on external resources, which are not part of the ontology. Consequently, changes in these external resources like biased term distribution caused by shifting of hot research topics, will affect the calculation of semantic similarity. One way to avoid this problem is to use semantic methods that are "intrinsic" to the ontology, i.e. independent of external knowledge. Results We present a shortest-path graph kernel (spgk) method that relies exclusively on the GO and its structure. In spgk, a gene product is represented by an induced subgraph of the GO, which consists of all the GO terms annotating it. Then a shortest-path graph kernel is used to compute the similarity between two graphs. In a comprehensive evaluation using a benchmark dataset, spgk compares favorably with other methods that depend on external resources. Compared with simUI, a method that is also intrinsic to GO, spgk achieves slightly better results on the benchmark dataset. Statistical tests show that the improvement is significant when the resolution and EC similarity correlation coefficient are used to measure the performance, but is insignificant when the Pfam similarity correlation coefficient is used. Conclusions Spgk uses a graph kernel method in polynomial time to exploit the structure of the GO to calculate semantic similarity between gene products. It provides an alternative to both methods that use external resources and "intrinsic" methods with comparable performance. PMID:21801410

  20. 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

  1. IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    2016-01-01

    The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA (+) cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel

  2. 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

  3. The SKP1-Like Gene Family of Arabidopsis Exhibits a High Degree of Differential Gene Expression and Gene Product Interaction during Development

    PubMed Central

    Dezfulian, Mohammad H.; Soulliere, Danielle M.; Dhaliwal, Rajdeep K.; Sareen, Madhulika; Crosby, William L.

    2012-01-01

    The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. Phylogenetic analysis based on deduced polypeptide sequence organizes the family of ASK proteins into 7 clades. The complexity of the ASK gene family, together with the close structural similarity among its members raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo as well as the in planta assessment of individual ASK-F-Box protein interactions using bimolecular fluorescent complementation techniques in combination with confocal imagery in live cells. The results indicate significant functional divergence of steady state transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies will prove important for defining the functional intersection of expression, localization and gene product interaction that better predicts the formation of discrete SCF complexes, as a prelude to investigating their molecular mode of action. PMID:23226441

  4. Coupling with Packaging Explains Apparent Nonreciprocality of Chi-Stimulated Recombination of Bacteriophage Lambda by Reca and Recbc Functions

    PubMed Central

    Kobayashi, Ichizo; Stahl, Mary M.; Fairfield, Frederic R.; Stahl, Franklin W.

    1984-01-01

    Chi (χ, 5'-GCTGGTGG) is a recombinator in RecA- and RecBC-mediated recombination in Escherichia coli. In vegetative recombination between two bacteriophage lambda strains, one with and the other without Chi (a+χ+b- x a-χob+), the χ-containing recombinant (a-χ+b -) is less abundant than the non-χ-containing recombinant (a+χob+). Previously this was taken was evidence for nonreciprocality of χ-stimulated exchange. This inequality, however, is now seen to result from an event at cos (λ's packaging origin) that both activates Chi and initiates DNA packaging. An event at rightward cos leads to activation of leftward χ on the same chromosome for an exchange to its left. From the resulting circulating dimer (—cos-a+-χo-b +-cos-a --χ+-b-—), the cos that activated χ is more likely to be used for rightward packaging initiation than is the cos from the other parent. Consistent with this coupling model is "biased packaging" in λ carrying two cos sites per monomer genome. When their maturation is dependent on dimerization by χ-stimulated exchange, the phage particles result more often from packaging from the cos that activates χ than from packaging from the other cos. Since Chi activation and packaging can be uncoupled, we infer that some early and reversible step in packaging activates χ. A strong candidate for this step is a double-strand break at cos that provides an oriented entry site for a recombinase. PMID:6239809

  5. Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

    PubMed Central

    2012-01-01

    Background Plasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC) using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in E. coli (strain VH33) allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the recA, deoR, nupG and endA genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions. Results The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X). The triple mutant VH33 Δ (recA deoR nupG) accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g), whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation

  6. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  7. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  8. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters

    PubMed Central

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F.

    2015-01-01

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this “dead” cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  9. High-Throughput Fluorescent Tagging of Full-Length Arabidopsis Gene Products in Planta1

    PubMed Central

    Tian, Guo-Wei; Mohanty, Amitabh; Chary, S. Narasimha; Li, Shijun; Paap, Brigitte; Drakakaki, Georgia; Kopec, Charles D.; Li, Jianxiong; Ehrhardt, David; Jackson, David; Rhee, Seung Y.; Raikhel, Natasha V.; Citovsky, Vitaly

    2004-01-01

    We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization. PMID:15141064

  10. 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

  11. Gene expression and flavonolignan production in fruits and cell cultures of Silybum marianum.

    PubMed

    Torres, María; Corchete, Purificación

    2016-03-15

    The hepatoprotectant flavonolignan silymarin (Sm) is synthesized through 4-coumaroyl-CoA, which enters both the flavonoid and the monolignol pathway giving the two immediate precursors taxifolin (Tx) and coniferyl alcohol (CA), respectively. Sm formation occurs via oxidative radicalization of Tx and CA and is accumulated at high levels at final stages of maturation of Silybum marianum fruits. By contrast, Sm production is severely reduced in cell cultures of this species, although suspensions are able to excrete Sm compounds into the medium upon elicitation with methyl jasmonate (MeJA) or cyclodextrins (CD). Knowledge of gene expression is important to understand Sm dynamics and to develop strategies aimed at increasing production by means of cell cultures but, to date, only one gene of the pathway (chalcone synthase, SmCHS) has been cloned. Therefore, to elucidate the relationship between expression of Sm pathway genes and production of these metabolites, four cDNA fragments of genes putatively involved in flavonolignan biosynthesis, chalcone isomerase, flavanone 3-hydroxylase, flavonol 3'-hydroxylase and cinnamyl alcohol dehydrogenase, were isolated from Sm producing S. marianum fruits and their expression, together with that of the SmCHS, were studied both in fruits at different maturation stages and in elicited cell suspensions. Combined results at both transcript expression and metabolite levels at three different stages of fruit maturation revealed that the formation of the flavonoid moiety precedes flavonolignan biosynthesis, being Sm accumulation associated to expression of the monolignol pathway. There was not detectable accumulation of transcripts in cell suspensions, however, elicitation with MeJA or CD notably induced expression of the studied fragments. These results indicate that the five genes expressed during maturation of S. marianum fruits may contribute to observed increases in flavonolignan accumulation upon treatment of cell cultures with

  12. Stimulated stromal cells induce gamma-globin gene expression in erythroid cells via nitric oxide production

    PubMed Central

    Čokić, Vladan P.; Beleslin-Čokić, Bojana B.; Smith, Reginald D.; Economou, Antaeus P.; Wahl, Larry M.; Noguchi, Constance T.; Schechter, Alan N.

    2009-01-01

    Objective We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that the stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. Materials and Methods Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In co-culture studies of erythroid and stromal cells we measured globin gene expression during stimulation by NO inducers. Results Hydroxyurea (30–100 μM) induced NOS-dependent production of NO in human macrophages (up to 1.2 μM). Co-culture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 μM) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to 2 fold) in human macrophage/erythroid cell co-cultures. Co-culture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4 fold) in the presence of hydroxyurea (40 μM). Conclusion These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells. PMID:19576950

  13. Identification of pKM101-encoded loci specifying potentially lethal gene products.

    PubMed Central

    Winans, S C; Walker, G C

    1985-01-01

    Two pKM101-encoded loci (designated kilA and kilB) have been identified which elaborate products that are potentially lethal to the bacterial cell. The lethal effects of each of these products is inhibited by two other plasmid-encoded loci, designated korA and korB (for kil override). Both korA and korB are required to control the lethality of either kil gene. In the presence of korA and korB both kil genes have other phenotypes: kilB is necessary for conjugal transfer, whereas kilA is responsible for the small-colony morphology on defined media that is characteristic of pKM101-containing strains (the Slo phenotype). PMID:3881396

  14. 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

  15. Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene.

    PubMed

    Kahraman, Huseyin; Aytan, Emel; Kurt, Ash Giray

    2011-09-01

    The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.

  16. 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

  17. 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

  18. Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control

    PubMed Central

    Perkin, Lindsey C.; Adrianos, Sherry L.; Oppert, Brenda

    2016-01-01

    Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs. PMID:27657138

  19. Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control.

    PubMed

    Perkin, Lindsey C; Adrianos, Sherry L; Oppert, Brenda

    2016-09-19

    Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.

  20. Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control.

    PubMed

    Perkin, Lindsey C; Adrianos, Sherry L; Oppert, Brenda

    2016-01-01

    Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs. PMID:27657138

  1. Expression of the HMGI(Y) gene products in human neuroblastic tumours correlates with differentiation status

    PubMed Central

    Giannini, G; Kim, C J; Marcotullio, L Di; Manfioletti, G; Cardinali, B; Cerignoli, F; Ristori, E; Zani, M; Frati, L; Screpanti, I; Gulino, A

    2000-01-01

    HMGI and HMGY are splicing variants of the HMGI(Y) gene and together with HMGI-C, belong to a family of DNA binding proteins involved in maintaining active chromatin conformation and in the regulation of gene transcription. The expression of the HMGI(Y) gene is maximal during embryonic development, declines in adult differentiated tissues and is reactivated in most transformed cells in vitro and in many human cancers in vivo. The HMGI(Y) genomic locus is frequently rearranged in mesenchymal tumours, suggesting a biological role for HMGI(Y) gene products in tumour biology. HMGIs are both target and modulators of retinoic acid activity. In fact, HMGI(Y) gene expression is differentially regulated by retinoic acid in retinoid-sensitive and -resistant neuroblastoma cells, while HMGI-C participates in conferring retinoic acid resistance in some neuroblastoma cells. In this paper we show that HMGI and HMGY isoforms are equally regulated by retinoic acid in neuroblastoma cell lines at both RNA and protein levels. More importantly our immunohistochemical analysis shows that, although HMGI(Y) is expressed in all neuroblastic tumours, consistently higher levels are observed in less differentiated neuroblastomas compared to more differentiated ganglioneuromas, indicating that HMGI(Y) expression should be evaluated as a potential diagnostic and prognostic marker in neuroblastic tumours. © 2000 Cancer Research Campaign http://www.bjcancer.com PMID:11076660

  2. Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus Isolates in Fishery Products in Iran

    PubMed Central

    Arfatahery, Noushin; Davoodabadi, Abolfazl; Abedimohtasab, Taranehpeimaneh

    2016-01-01

    Staphylococcus aureus is one of the most common causes of seafood-borne diseases worldwide, which are attributable to the contamination of food by preformed enterotoxins. In this study, a total of 206 (34.3%) Staphylococcus aureus strains were obtained from 600 fish and shrimp samples and were tested for their antimicrobial susceptibility. We assessed the prevalence of the genes responsible for the staphylococcal enterotoxins (SEA, SEB) and toxic shock syndrome toxin 1 (TSST-1) genes. The results indicated that 34% of aqua food samples were contaminated with S. aureus, and 23.8% of these isolates were mec-A-positive. Sixty-four percent of the strains isolated from contaminated seafood was enterotoxigenic S. aureus, and 28.2% of SEs were MRSA-positive. The most prevalent genotype was characterized by the presence of the sea gene (45.2%), followed by the seb gene (18.5%), and the tst gene encoding TSST-1 was found in eight strains (3.9%). Of the 206 S. aureus isolates, 189 strains (84.9%) were resistant to at least one antibiotic. Given the frequent outbreaks of enterotoxigenic MRSA, it is necessary to make revisions to mandatory programmes to facilitate improved hygiene practices during fishing, aquaculture, processing, and sales to prevent the contamination of fishery products in Iran. PMID:27694813

  3. 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.

  4. The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans.

    PubMed

    Tougou, Makoto; Yamagishi, Noriko; Furutani, Noriyuki; Kaku, Koichiro; Shimizu, Tsutomu; Takahata, Yoshihito; Sakai, Jun-ichi; Kanematsu, Seiji; Hidaka, Soh

    2009-05-01

    We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

  5. A mutation in the Xanthomonas oryzae pv. oryzae wxoD gene affects xanthan production and chemotaxis.

    PubMed

    Nam, Jae-Young; Kim, Hong-Il; Lee, Chang-Soo; Park, Young-Jin

    2013-11-01

    Xanthomonas oryzae pv. oryzae causes bacterial blight in rice (Oryza sativa L.). The effect of a mutation in the wxoD gene, that encodes a putative O-antigen acetylase, on xanthan production as well as bacterial chemotaxis was investigated. The mutation increased xanthan production by 52 %. The mutant strain was non-motile on semi-solid agar swarm plates. In addition, several genes involved in chemotaxis, including the cheW, cheV, cheR, and cheD genes, were down-regulated by a mutation in the wxoD gene. Thus, the mutation in the wxoD gene affects xanthan production as well as bacterial chemotaxis. However, the wxoD gene is not essential for the virulence of X. oryzae.

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

    PubMed

    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.

  7. 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

  8. A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

    PubMed

    Díaz-Magaña, Amada; Alva-Murillo, Nayeli; Chávez-Moctezuma, Martha P; López-Meza, Joel E; Ramírez-Díaz, Martha I; Cervantes, Carlos

    2015-07-01

    The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA-  mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA-  mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA-  mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

  9. Products of vasopressin gene expression in small-cell carcinoma of the lung.

    PubMed Central

    Friedmann, A. S.; Malott, K. A.; Memoli, V. A.; Pai, S. I.; Yu, X. M.; North, W. G.

    1994-01-01

    Small-cell neuroendocrine carcinoma of the lung is known to express products related to the vasopressin gene, although these products have been reported to sometimes differ from those generated by neurones of the hypothalamo-neurohypophyseal system. To further investigate vasopressin gene expression in neuroendocrine carcinomas, we performed immunohistochemistry on 24 histologically classified small-cell carcinomas using antibodies directed against different regions of the vasopressin precursor. All of the tumours examined contained at least two parts of the vasopressin precursor, suggesting that vasopressin might have a biological role in these tumours and indicating a role for these products in tumour diagnosis and treatment. Sixty-seven per cent of the tumours contained immunoreactivity for all major regions of the precursor: vasopressin, vasopressin-associated human neurophysin, the bridging region between the hormone and the neurophysin, and vasopressin-associated human glycopeptide. However, 33% of the tumours examined appeared to express only part of the vasopressin precursor, as evidenced by the absence of immunoreactivity for the neurophysin and/or the glycopeptide. These results support the proposition that both normal and abnormal vasopressin gene expression occurs in small-cell carcinoma of the lung. Images Figure 1 Figure 2 Figure 3 PMID:8297723

  10. 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

  11. Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication.

    PubMed Central

    Colgrove, R; Simon, G; Ganem, D

    1989-01-01

    The potential of the hepadnavirus X gene product to activate gene expression in trans was tested through a series of cotransfections of X expression vectors with a variety of potential targets for transactivation. The X gene products from human hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus are all equally active in augmenting the expression of a wide array of target promoters in both permissive and nonpermissive cells. Using the HBV genome itself as the source of X protein, we demonstrate that transactivation of HBV and heterologous genes occurs when X protein is expressed in its native state during productive infection of permissive cells. Run-on transcription analysis indicates that this transactivation occurs at the level of primary transcription. Images PMID:2788226

  12. Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities.

    PubMed

    Mackie, Roderick I; Koike, Satoshi; Krapac, Ivan; Chee-Sanford, Joanne; Maxwell, Scott; Aminov, Rustam 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 microg/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

  13. 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

  14. Dairy products, polymorphisms in the vitamin D receptor gene and colorectal adenoma recurrence.

    PubMed

    Hubner, Richard A; Muir, Kenneth R; Liu, Jo-Fen; Logan, Richard F A; Grainge, Matthew J; Houlston, Richard S

    2008-08-01

    Vitamin D receptor (VDR) activation inhibits proliferation and angiogenesis in the colorectal epithelium, and inhibits metastasis of colorectal tumors. Polymorphisms in the VDR gene alter receptor cellular levels and functioning, and may confer altered susceptibility to colorectal neoplasia. We aimed to investigate the influence of VDR polymorphisms and dietary factors impacting on vitamin D metabolism on colorectal adenoma (CRA) recurrence. Data on dietary intakes of calcium, vitamin D and dairy products were collected from 853 participants in the United Kingdom Colorectal Adenoma Prevention trial, a randomized trial of aspirin and folate for CRA recurrence prevention. The VDR Cdx2, FokI, BsmI, ApaI and TaqI polymorphisms were genotyped in 546 participants with available DNA, and gene-diet interaction analyses performed in 480. Dairy product intake was inversely related to CRA recurrence risk independent of calcium and vitamin D [relative risk (RR) = 0.64; 95% confidence intervals (CIs): 0.47-0.88, for subjects in the highest compared to lowest intake tertile, p(trend) = 0.005]. Milk accounted for 60% of dairy product intake, and on analysis of milk and nonmilk dairy products separately recurrence risk in individuals in the highest tertile of milk intake was half that of lowest tertile individuals (RR = 0.52; 95% CI: 0.38-0.72, p(trend) = 3.2 x 10(-5)), whereas nonmilk dairy products did not influence recurrence. VDR polymorphism genotypes and haplotypes did not directly alter recurrence risk, but the reduction in risk associated with high dairy product intake was confined to individuals with ApaI aA/AA genotype (p(interaction) = 0.02). These findings indicate dairy products, and in particular milk, have chemopreventive activity against CRA recurrence. PMID:18470879

  15. Manipulation of IL-10 gene expression by Toxoplasma gondii and its products

    PubMed Central

    Pestechian, Nader; Khanahmad Shahreza, Hosein; Faridnia, Roghiyeh; Kalani, Hamed

    2016-01-01

    Background: This study was designed to evaluate whether or not T. gondii and its derivatives can change the gene expression level of IL-10 in murine leukocytes in vivo. Methods: Fifty BALB/c mice were divided into 5 groups, four of which received the excretory/secretory product (ESP) from cell culture medium, the ESP from cell free medium, the Toxoplasma lysate product (TLP) and the active tachyzoites, respectively. The fifth group was considered as control and received PBS. The peritoneal leukocytes from the mice were collected. Their total RNA were extracted and converted to cDNA and the gene expression levels of IL-10 in the samples were evaluated by quantitative real time-PCR using the REST-2009 software. Results: The findings showed a decrease in the expression level of IL-10 in the TLP group (p=0.004). Moreover, the IL-10 gene expression level was upregulated in the group of the ESP from cell culture medium (p=0.04) and the active tachyzoite group (p=0.04). The expression of IL-10 gene in the group of ESP from cell-free medium was not significant compared to the control one (p=0.45). Conclusion: T. gondii and its derivatives are able to increase (the active T. gondii tachyzoite and the ESP from cell culture medium) and decrease (the TLP) the gene expression level of IL-10 in a murine model. The question remains to be examined in further study about which molecules are involved in this process. PMID:27683651

  16. Manipulation of IL-10 gene expression by Toxoplasma gondii and its products

    PubMed Central

    Pestechian, Nader; Khanahmad Shahreza, Hosein; Faridnia, Roghiyeh; Kalani, Hamed

    2016-01-01

    Background: This study was designed to evaluate whether or not T. gondii and its derivatives can change the gene expression level of IL-10 in murine leukocytes in vivo. Methods: Fifty BALB/c mice were divided into 5 groups, four of which received the excretory/secretory product (ESP) from cell culture medium, the ESP from cell free medium, the Toxoplasma lysate product (TLP) and the active tachyzoites, respectively. The fifth group was considered as control and received PBS. The peritoneal leukocytes from the mice were collected. Their total RNA were extracted and converted to cDNA and the gene expression levels of IL-10 in the samples were evaluated by quantitative real time-PCR using the REST-2009 software. Results: The findings showed a decrease in the expression level of IL-10 in the TLP group (p=0.004). Moreover, the IL-10 gene expression level was upregulated in the group of the ESP from cell culture medium (p=0.04) and the active tachyzoite group (p=0.04). The expression of IL-10 gene in the group of ESP from cell-free medium was not significant compared to the control one (p=0.45). Conclusion: T. gondii and its derivatives are able to increase (the active T. gondii tachyzoite and the ESP from cell culture medium) and decrease (the TLP) the gene expression level of IL-10 in a murine model. The question remains to be examined in further study about which molecules are involved in this process.

  17. 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.

  18. 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

  19. Identification and characterization of a novel yeast gene: the YGP1 gene product is a highly glycosylated secreted protein that is synthesized in response to nutrient limitation.

    PubMed Central

    Destruelle, M; Holzer, H; Klionsky, D J

    1994-01-01

    Nutrient starvation in the yeast Saccharomyces cerevisiae leads to a number of physiological changes that accompany entry into stationary phase. The expression of genes whose products play a role in stress adaptation is regulated in a manner that allows the cell to sense and respond to changing environmental conditions. We have identified a novel yeast gene, YGP1, that displays homology to the sporulation-specific SPS100 gene. The expression of YGP1 is regulated by nutrient availability. The gene is expressed at a basal level during "respiro-fermentative" (logarithmic) growth. When the glucose concentration in the medium falls below 1%, the YGP1 gene is derepressed and the gene product, gp37, is synthesized at levels up to 50-fold above the basal level. The glucose-sensing mechanism is independent of the SNF1 pathway and does not operate when cells are directly shifted to a low glucose concentration. The expression of YGP1 also responds to the depletion of nitrogen and phosphate, indicating a general response to nutrient deprivation. These results suggest that the YGP1 gene product may be involved in cellular adaptations prior to stationary phase and may be a useful marker protein for monitoring early events associated with the stress response. Images PMID:8139573

  20. The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisin production.

    PubMed

    Brown, Daren W; Butchko, Robert A E; Busman, Mark; Proctor, Robert H

    2007-07-01

    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 the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Deltafum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

  1. Relationships of growth hormone gene and milk protein polymorphisms to milk production traits in Simmental cattle.

    PubMed

    Falaki, M; Prandi, A; Corradini, C; Sneyers, M; Gengler, N; Massart, S; Fazzini, U; Burny, A; Portetelle, D; Renaville, R

    1997-02-01

    The importance of milk proteins and the positive effect of administration of growth hormone (GH) on milk production, and the presence in some dairy cattle lines of greater GH concentrations prompted us to examine the presence of restriction fragment length polymorphism at the GH gene using the restriction enzyme TaqI and to investigate associations between this polymorphism in Simmental cows and bulls, as well as milk protein variants in Simmental cows, and milk production traits. Blood and milk were sampled from 279 Italian Simmental cows and semen was collected from 148 bulls of the same breed. Two fragment bands, denoted A and B, of 6200 and 5200 bp respectively, were examined and three patterns, AA, AB and BB, were found in both animal samples. All variants previously reported in other studies, for kappa, beta, and alpha s1-caseins, and beta-lactoglobulin, were found in the cows' samples. For the cows' samples, a BLUP (Best Linear Unbiased Predictor) analysis of results was performed using a REML (Restricted Maximum Likelihood) program and known heritabilities, whereas for bulls we have performed a General Linear Model analysis. The effect of GH gene polymorphism, using TaqI restriction enzyme, on milk production traits was not significant, but bulls of BB pattern had a higher breeding value for milk yield than AA bulls (P < 0.05). For the kappa-casein genotypic effects, cows of AB genotype gave milk with 1.53 +/- 0.70 g/kg less fat than cows of AA genotype. In addition, breeding values for milk protein content were significantly higher in BB bulls, with 0.87 +/- 0.32 and 0.71 +/- 0.34 g/kg more milk protein than AA and AB bulls respectively. Thus, our results revealed a GH gene polymorphism and indicated significant effects of milk protein polymorphisms on milk production traits in the Italian Simmental breed.

  2. Hepatic Gene Transfer as a Means of Tolerance Induction to Transgene Products

    PubMed Central

    LoDuca, Paul A.; Hoffman, Brad E.; Herzog, Roland W.

    2010-01-01

    The liver is a preferred target organ for gene therapy not only for liver-specific diseases but also for disorders that require systemic delivery of a protein. Diseases that could benefit from hepatic gene transfer include hemophilia, metabolic disorders, lysosomal storage disorders, and others. For a successful delivery of the transgene and sustained expression, the protocol must avoid immune responses in order to be efficacious. A growing number of studies have demonstrated that liver-directed transfer can induce transgene product-specific immune tolerance. Tolerance obtained via this route requires optimal engineering of the vector to eliminate transgene expression in antigen presenting cells while restricting high levels of therapeutic expression to hepatocytes. Innate immune responses may prevent tolerance induction, cause toxicity, and have to be minimized. Discussed in our review is the crucial role of CD4+CD25+ regulatory T cells in tolerance to the hepatocyte-derived gene product, the immunobiology of the liver and our current understanding of its tolerogenic properties, current and proposed research as to the mechanisms behind the liver’s unique cellular environment, as well as development of the tools for tolerance induction such as advanced vector systems. PMID:19355868

  3. Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

    SciTech Connect

    Napp, S.J.; Da Silva, N.A. )

    1993-04-01

    Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product [beta]-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and [beta]-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both [beta]-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and urid-k mutations were very stable; after more than 50 generation of growth in complex medium, stability values of 99-100% were measured.

  4. Nucleic acid binding property of the gene products of rice stripe virus.

    PubMed

    Liang, Delin; Ma, Xiangqiang; Qu, Zhicai; Hull, Roger

    2005-10-01

    GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function. PMID:16025246

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Bacillus subtilis RecA and its accessory factors, RecF, RecO, RecR and RecX, are required for spore resistance to DNA double-strand break

    PubMed Central

    Vlašić, Ignacija; Mertens, Ramona; Seco, Elena M.; Carrasco, Begoña; Ayora, Silvia; Reitz, Günther; Commichau, Fabian M.; Alonso, Juan C.; Moeller, Ralf

    2014-01-01

    Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing. PMID:24285298

  11. Bacillus subtilis RecA and its accessory factors, RecF, RecO, RecR and RecX, are required for spore resistance to DNA double-strand break.

    PubMed

    Vlašić, Ignacija; Mertens, Ramona; Seco, Elena M; Carrasco, Begoña; Ayora, Silvia; Reitz, Günther; Commichau, Fabian M; Alonso, Juan C; Moeller, Ralf

    2014-02-01

    Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing. PMID:24285298

  12. 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.

  13. 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.

  14. Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

    PubMed

    Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

    2010-07-01

    An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

  15. Treatment of pea (Pisum sativum L.) protoplasts with DNA-damaging agents induces a 39-kilodalton chloroplast protein immunologically related to Escherichia coli RecA.

    PubMed Central

    Cerutti, H; Ibrahim, H Z; Jagendorf, A T

    1993-01-01

    Organisms must have efficient mechanisms of DNA repair and recombination to prevent alterations in their genetic information due to DNA damage. There is evidence for DNA repair and recombination in plastids of higher plants, although very little is known at the biochemical level. Many chloroplast proteins are of eubacterial ancestry, suggesting that the same could be true for the components of a DNA repair and recombination system. A 39-kD protein, immunologically related to Escherichia coli RecA, is present in chloroplasts of pea (Pisum sativum L.). Bandshift gel assays suggest that it binds single-stranded DNA. Its steady-state level is increased by several DNA-damaging agents. These results are consistent with it being a plastid homolog of E. coli RecA protein, presumably involved in DNA repair and recombination, and with the existence of an SOS-like response in pea leaf cells. Experiments with protein synthesis inhibitors suggest that the 39-kD chloroplast protein is encoded in the nucleus. PMID:8108495

  16. Increased biomass production and glycogen accumulation in apcE gene deleted Synechocystis sp. PCC 6803

    PubMed Central

    2014-01-01

    The effect of phycobilisome antenna-truncation in the cyanobacterium Synechocystis sp. PCC 6803 on biomass production and glycogen accumulation have not yet been fully clarified. To investigate these effects here, the apcE gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of Synechocystis sp. PCC 6803. Biomass production of the apcE-deleted strain under photoautotrophic and atmospheric air conditions was 1.6 times higher than that of strain PCC 6803 (1.32 ± 0.01 versus 0.84 ± 0.07 g cell-dry weight L−1, respectively) after 15 days of cultivation. In addition, the glycogen content of the apcE-deleted strain (24.2 ± 0.7%) was also higher than that of strain PCC 6803 (11.1 ± 0.3%). Together, these results demonstrate that antenna truncation by deleting the apcE gene was effective for increasing biomass production and glycogen accumulation under photoautotrophic and atmospheric air conditions in Synechocystis sp. PCC 6803. PMID:24949254

  17. 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

    2015-03-24

    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.

  18. 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.

  19. Genetic association between SNPs in the DGAT1 gene and milk production traits in Murrah buffaloes.

    PubMed

    de Freitas, Ana Cláudia; de Camargo, Gregório Miguel Ferreira; Stafuzza, Nedenia Bonvino; Aspilcueta-Borquis, Rusbel Raul; Venturini, Guilherme Costa; Dias, Marina Mortati; Cardoso, Diercles Francisco; Tonhati, Humberto

    2016-10-01

    This study identified polymorphisms in the DGAT1 gene in Murrah buffaloes and investigated the associations to milk production and quality traits (milk, fat and protein yields and percentages, somatic cell count). Genomic DNA was extracted from hair follicles collected from the tail of 196 females. Three SNPs were identified in DGAT1 gene by sequencing. Statistical analyses were performed to verify the linkage and the association between polymorphisms and traits. The estimated value of r (2) between two SNPs in exon 17 (g.11,783G > A and g.11,785 T > C) was 0.029. SNP g.11,785 T > C was significantly associated (P < 0.05) to fat and protein percentage. Dominance effect was significant for milk and fat yields and protein percentage (P < 0.05). The additive effect of the SNP g.11,785 T > C was significant for protein production and somatic cell count (P < 0.05). It indicates that assisted marker selection might be done with considerations to balance production and udder health.

  20. Genetic association between SNPs in the DGAT1 gene and milk production traits in Murrah buffaloes.

    PubMed

    de Freitas, Ana Cláudia; de Camargo, Gregório Miguel Ferreira; Stafuzza, Nedenia Bonvino; Aspilcueta-Borquis, Rusbel Raul; Venturini, Guilherme Costa; Dias, Marina Mortati; Cardoso, Diercles Francisco; Tonhati, Humberto

    2016-10-01

    This study identified polymorphisms in the DGAT1 gene in Murrah buffaloes and investigated the associations to milk production and quality traits (milk, fat and protein yields and percentages, somatic cell count). Genomic DNA was extracted from hair follicles collected from the tail of 196 females. Three SNPs were identified in DGAT1 gene by sequencing. Statistical analyses were performed to verify the linkage and the association between polymorphisms and traits. The estimated value of r (2) between two SNPs in exon 17 (g.11,783G > A and g.11,785 T > C) was 0.029. SNP g.11,785 T > C was significantly associated (P < 0.05) to fat and protein percentage. Dominance effect was significant for milk and fat yields and protein percentage (P < 0.05). The additive effect of the SNP g.11,785 T > C was significant for protein production and somatic cell count (P < 0.05). It indicates that assisted marker selection might be done with considerations to balance production and udder health. PMID:27469895

  1. Novel Fc gamma receptor I family gene products in human mononuclear cells.

    PubMed Central

    Porges, A J; Redecha, P B; Doebele, R; Pan, L C; Salmon, J E; Kimberly, R P

    1992-01-01

    Unlike the human Fc gamma RII and Fc gamma RIII families, which exhibit considerable diversity at both the nucleic acid and protein levels, the human Fc gamma RI family has only a single recognized product expressed as a 70-kD cell surface receptor with high affinity for monomeric IgG (hFc gamma RIa1). Using both polymerase chain reaction-based amplification and Northern hybridization, we document multiple interferon-gamma-inducible hFc gamma RI RNA transcripts in human mononuclear cells and neutrophils. The sequences of two of these Fc gamma RI related transcripts indicate that they are alternatively spliced products of a second Fc gamma RI family gene, termed Fc gamma RIB. The cDNA derived from the larger of these transcripts, termed hFc gamma RIb1, encodes a surface molecule that is not recognized by Fc gamma RI specific monoclonal antibodies when transfected into COS-7 cells. Unlike the interferon-gamma-inducible hFc gamma RIA gene product, hFc gamma RIb1 does not bind monomeric IgG with high affinity. However, both hFc gamma RIa1 and hFc gamma RIb1 do bind aggregated human IgG. Previously unrecognized diversity within the hFc gamma RI family includes an interferon-gamma-inducible, putative low affinity Fc gamma receptor that may play an important role in the mechanism by which Fc gamma receptors participate in the humoral immune response. Images PMID:1430234

  2. 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

  3. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins

    SciTech Connect

    Strebel, K.; Beck, E.; Strohmaier, K.; Schaller, H.

    1986-03-01

    Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambdaPL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in /sup 35/S-labeled extracts from foot-and-mouth disease virus-infected BHK cells. This allowed us to locate unequivocally all mature foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and mouth disease virus gene products not previously identified in vivo or in vitro.

  4. 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.

  5. Expression of herpes simplex virus beta and gamma genes integrated in mammalian cells and their induction by an alpha gene product.

    PubMed Central

    Sandri-Goldin, R M; Goldin, A L; Holland, L E; Glorioso, J C; Levine, M

    1983-01-01

    The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed alpha, beta, and gamma, whose synthesis is regulated in a cascade fashion. alpha products are synthesized first during infection, and they are required for synthesis of beta and gamma proteins. To examine the expression of several HSV-1 beta and gamma genes in the absence of alpha functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only beta and gamma genes (0.315 to 0.421 map units). We found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the beta and gamma classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 alpha gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 beta and gamma genes can be transcribed in the absence of alpha functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the alpha gene product ICP4. Images PMID:6318078

  6. 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

  7. 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...

  8. Analysis of Tox5 gene expression in Gibberella pulicaris strains with different trichothecene production phenotypes.

    PubMed

    Hohn, T M; Desjardins, A E; McCormick, S P

    1993-08-01

    The Tox5 gene encodes trichodiene synthase, the first unique enzyme in the trichothecene biosynthetic pathway. In Gibberella pulicaris R-6380, the level of Tox5 mRNA was found to increase 47-fold in early stationary phase. Sequence analysis of the Tox5 promoter regions from geographically distinct strains of G. pulicaris revealed the existence of two Tox5 alleles (Tox5-1 and Tox5-2). All G. pulicaris strains that produce high levels of trichothecenes in liquid culture carry a 42-nucleotide (nt) tandem repeat sequence (Tox5-1) in the Tox5 promoter region, whereas strains that produce low levels of trichothecenes carry a single copy of this sequence (Tox5-2). A genetic cross between high- and low-level trichothecene producers resulted in the cosegregation of higher-level trichothecene production with the Tox5-1 allele. To determine the importance of the 42-nt repeat sequence in the regulation of Tox5 expression, reporter gene constructs carrying either the Tox5-1 or the Tox5-2 promoter region fused to the beta-galactosidase gene of Escherichia coli were introduced into the high-level-trichothecene-producing strain, R-6380. Expression of reporter gene activity in transformants was found to be regulated in a manner similar to Tox5 expression but appeared to be independent of the 42-nt sequence copy number. These results indicate that transcriptional controls play an important role in the regulation of Tox5 expression and that genes involved in trichothecene biosynthesis in G. pulicaris may be linked to Tox5. PMID:8368827

  9. 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.

  10. 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

  11. Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives.

    PubMed

    Sala, Francesco; Manuela Rigano, M; Barbante, Alessandra; Basso, Barbara; Walmsley, Amanda M; Castiglione, Stefano

    2003-01-30

    Stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g. tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral or parental administration. This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plants. The use of plant-derived vaccines may overcome some of the major problems encountered with traditional vaccination against infectious diseases, autoimmune diseases and tumours. They also offer a convenient tool against the threat of bio-terrorism. State of the art, experimental strategies, safety and perspectives are discussed in this article.

  12. 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

  13. 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.

  14. A systems biology approach for the identification of target genes for the improvement of itaconic acid production in Aspergillus species

    PubMed Central

    2013-01-01

    Background In this paper, a clone based transcriptome analysis towards the identification of genes related to itaconic acid production in Aspergillus terreus was carried out as an extension of a previously published a clone-based transcriptome analysis from a set of batch fermentation experiments. Also a publically available A. niger transcriptome dataset from cultures similar to those of the A. terreus data set was analyzed to evaluate the specificity of the approach followed for A. terreus. Results Besides the itaconic acid gene cluster (cis-aconitate decarboxylase, mitochondrial tri-carboxylic acid transporter and major facilitator superfamily transporter) discovered previously, additional genes of interest were identified in the A. terreus transcriptome data correlating to itaconic acid production, including 6 genes encoding enzymes in glycolysis and the pentose phosphate pathway, 4 genes functioning in vitamins synthesis, and a gene encoding a copper transporter. Only three of the 83 low pH specific genes identified from the A. niger dataset corresponded to high itaconic acid / low pH expressed genes identified from the A. terreus data set. However, in all three cases, the regulation of pH dependent gene expression was completely different between the two species. Conclusions An extended clone based transcriptome analysis using a clone based transcription array to identify genes correlating with itaconic acid production revealed novel genes both in the central metabolism and in other more secondary pathways such as vitamin biosynthesis and Cu2+ transport, providing targets for further metabolic and process engineering to optimize itaconic acid production. PMID:24304666

  15. 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.

  16. SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution.

    PubMed

    Currin, Andrew; Swainston, Neil; Day, Philip J; Kell, Douglas B

    2017-01-01

    Gene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries. We summarize the integrated use of GeneGenie to design DNA and oligonucleotide sequences, followed by the procedure for assembling these accurately and efficiently using SpeedyGenes.

  17. SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution.

    PubMed

    Currin, Andrew; Swainston, Neil; Day, Philip J; Kell, Douglas B

    2017-01-01

    Gene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries. We summarize the integrated use of GeneGenie to design DNA and oligonucleotide sequences, followed by the procedure for assembling these accurately and efficiently using SpeedyGenes. PMID:27671932

  18. 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

  19. Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI.

    PubMed Central

    Maneewannakul, S; Maneewannakul, K; Ippen-Ihler, K

    1992-01-01

    The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed. Images PMID:1355084

  20. 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.

  1. 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

  2. Expression of NF2 gene product merlin in arachnoid villi and meningiomas.

    PubMed

    Sakuda, K; Kohda, Y; Matsumoto, T; Park, C; Seto, A; Tohma, Y; Hasegawa, M; Kida, S; Nitta, H; Yamashima, T; Yamashita, J

    1996-11-01

    Neurofibromatosis type 2 (NF2) gene encodes a novel 595 amino acid protein named merlin. Recently, Ruttledge et al demonstrated inactivation of NF2 gene in approximately 60% of sporadically occurring meningiomas. Merlin is thought to physiologically exist beneath the cell membrane, and to form a part of modulation in signal transduction, for example, information concerning contact inhibition. In NF2-related tumors, it is supposed that the mutation of merlin results in loss of this signal transduction leading to tumorigenesis. In this paper, we investigated the expression of NF2 gene product merlin in arachnoid villi and meningiomas. The immunohistochemical staining of merlin showed a striking contrast between arachnoid villi and meningiomas. In arachnoid cells, merlin was labeled in the whole cytoplasm, but not within the nuclei. In contrast, in meningiomas, immunoreactivity of merlin was mainly seen in the nuclei. These results suggest that arachnoid cells with normal merlin are capable of normal signal transduction, whereas meningioma cells with mutated merlin show impairment of signal transduction which may lead to tumorigenesis.

  3. 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

  4. Gene mutations that promote adrenal aldosterone production, sodium retention, and hypertension

    PubMed Central

    Moraitis, Andreas G; Rainey, William E; Auchus, Richard J

    2014-01-01

    Primary aldosteronism (PA) is the most common form of secondary hypertension, found in about 5% of all hypertension cases, and up to 20% of resistant hypertension cases. The most common forms of PA are an aldosterone-producing adenoma and idiopathic (bilateral) hyperaldosteronism. Rare genetic forms of PA exist and, until recently, the only condition with a known genetic mechanism was familial hyperaldosteronism type 1, also known as glucocorticoid-remediable aldosteronism (FHA1/GRA). FHA type 3 has now been shown to derive from germline mutations in the KCNJ5 gene, which encodes a potassium channel found on the adrenal cells. Remarkably, somatic mutations in KCNJ5 are found in about one-third of aldosterone-producing adenomas, and these mutations are likely to be involved in their pathogenesis. Finally, mutations in the genes encoding an L-type calcium channel (CACNA1D) and in genes encoding a sodium–potassium adenosine triphosphatase (ATP1A1) or a calcium adenosine triphosphatase (ATP2B3) are found in other aldosterone-producing adenomas. These findings provide a working model, in which adenoma formation and/or aldosterone production in many cases derives from increased calcium entry, which drives the pathogenesis of primary aldosteronism. PMID:24399884

  5. A Genetic Strategy to Measure Circulating Drosophila Insulin Reveals Genes Regulating Insulin Production and Secretion

    PubMed Central

    Park, Sangbin; Alfa, Ronald W.; Topper, Sydni M.; Kim, Grace E. S.; Kockel, Lutz; Kim, Seung K.

    2014-01-01

    Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, we developed a genetic tool to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. Our system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics. PMID:25101872

  6. c-Myb influences HIV type 1 gene expression and virus production.

    PubMed

    Churchill, M J; Ramsay, R G; Rhodes, D I; Deacon, N J

    2001-11-01

    c-Myb is expressed in proliferating T cells. Fifteen c-Myb-binding sites can be identified in the HIV-1 long terminal repeat (LTR), suggesting that c-Myb may regulate HIV-1 gene expression and virus replication. Increasing the cellular levels of c-Myb by transient transfection of CEM cells resulted in a 10- to 20-fold activation of HIV-1 LTR-driven gene expression and mutation of one high-affinity Myb-binding site within the LTR reduced this activation by 60 to 70%. Conversely, inhibition of c-Myb expression in MT-2 cells by treatment with c-myb antisense oligonucleotides decreased HIV-1 replication by 85%, as measured by reverse transcriptase activity and cytopathic effects. The effect of c-myb antisense oligonucleotides on HIV-1 gene expression and virus particle production appeared to be independent of cell proliferation, but dependent on the presence of c-Myb activity mediated through the HIV-1 LTR. These data show that c-myb expression affects HIV-1 replication in CD4(+) T cells.

  7. 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

  8. 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

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

    PubMed

    Watanabe, Natsumi; Yano, Kazuo; Tsuyuki, Kenichiro; Okano, Teruo; Yamato, Masayuki

    2015-01-01

    The first commercially approved human gene therapy in the Western world is Glybera (alipogene tiparvovec), which is an adenoassociated viral vector encoding the lipoprotein lipase gene. Glybera was recommended for marketing authorization by the European Medicines Agency in 2012. The European Medicines Agency had only ever reviewed three marketing authorization applications for gene therapy medicinal products. Unlike in the case of Glybera, the applications of the first two products, Cerepro and Contusugene Ladenovec Gendux/Advexin, both of which were for cancer diseases, were withdrawn. In this report, we studied the European public assessment reports of the three gene therapy products. During the assessment process, Glybera was re-examined and reviewed for a fourth time. We therefore researched the re-examination procedure of the European Union regulatory process. Approximately 25% of the new medicinal products initially given negative opinions from the Committee for Medicinal Products for Human Use were ultimately approved after re-examination from 2009 to 2013. The indications of most medicines were changed during the re-examination procedure, and the products were later approved with a mode of approval. These results suggested that the re-examination system in the European Union contributed to the approval of both several new drugs and the first gene therapy product. PMID:26052534

  10. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

    PubMed Central

    2011-01-01

    Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial

  11. 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

  12. 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

  13. 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

  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

    2016-08-19

    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.

  15. 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

  16. A mutation in the aroE gene affects pigment production, virulence, and chemotaxis in Xanthomonas oryzae pv. oryzae.

    PubMed

    Kim, Hong-Il; Noh, Tae-Hwan; Lee, Chang-Soo; Park, Young-Jin

    2015-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice. To study its function, a random insertion mutation library of Xoo was constructed using the Tn5 transposon. A mutant strain with decreased virulence against the susceptible rice cultivar IR24 was isolated from the library (aroE mutant), which also had extremely low pigment production. Thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and sequence analysis of the mutant revealed that the transposon was inserted into the aroE gene (encoding shikimate dehydrogenase). To investigate gene expression changes in the pigment- and virulence-deficient mutant, DNA microarray analysis was performed, which showed downregulation of 20 genes involved in the chemotaxis of Xoo. Our findings reveal that mutation of the aroE gene affects virulence and pigment production, as well as expression of genes involved in Xoo chemotaxis.

  17. Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep

    PubMed Central

    Vitezica, Zulma G; Moreno, Carole R; Lantier, Frederic; Lantier, Isabelle; Schibler, Laurent; Roig, Anne; François, Dominique; Bouix, Jacques; Allain, Daniel; Brunel, Jean-Claude; Barillet, Francis; Elsen, Jean-Michel

    2007-01-01

    In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapinière INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance) and production (wool and carcass) traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL) detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes. PMID:17612481

  18. 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.

  19. A Brain Membrane Protein Similar to the Rat src Gene Product

    NASA Astrophysics Data System (ADS)

    Scheinberg, David A.; Strand, Mette

    1981-01-01

    We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 μ M). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.

  20. Expression of the trichodiene synthase gene of Fusarium sporotrichioides in Escherichia coli results in sesquiterpene production.

    PubMed

    Hohn, T M; Plattner, R D

    1989-11-15

    Trichodiene synthase is a sesquiterpene cyclase involved in the biosynthesis of trichothecene mycotoxins. We report that insertion of the unaltered trichodiene synthase gene of Fusarium sporotrichioides into the Escherichia coli expression vector pDR540 produced an inactive polypeptide with a molecular weight approximately 2000 greater than that of trichodiene synthase. This result is consistent with the presence of an intron in the trichodiene synthase gene, and prompted us to specifically delete a putative 60-nucleotide intron sequence. Insertion of the intron-deleted open reading frame into pDR540 resulted in the production of active enzyme. Trichodiene synthase activity in crude extracts from induced cultures was 0.07 nmol/min/mg of protein and represented 0.05-0.10% of the total cell protein. A cross-reactive protein was present with the same apparent molecular weight as the subunit of native trichodiene synthase. The recombinant enzyme was partially purified and shown to have properties closely resembling those of the native enzyme. Trichodiene was detected in ethyl acetate extracts from induced cultures at a concentration of 60 micrograms/liter after 4.5 h. These findings support the primary structure recently reported for trichodiene synthase and demonstrate that the expression of a sesquiterpene cyclase in E. coli results in sesquiterpene production. PMID:2817906

  1. 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.

  2. Homer1 gene products orchestrate Ca2+-permeable AMPA receptor distribution and LTP expression

    PubMed Central

    Rozov, Andrei; Zivkovic, Aleksandar R.; Schwarz, Martin K.

    2012-01-01

    We studied the role of Homer1 gene products on the presence of synaptic Ca2+-permeable AMPA receptors (AMPARs) and long-term potentiation (LTP) generation in hippocampal CA1 pyramidal neurons, using mice either lacking all Homer1 isoforms (Homer1 KO) or overexpressing the immediate early gene (IEG) product Homer1a (H1aTG). We found that Homer1 KO caused a significant redistribution of the AMPAR subunit GluA2 from the dendritic compartment to the soma. Furthermore, deletion of Homer1 enhanced the AMPAR-mediated component of glutamatergic currents at Schaffer collateral synapses as demonstrated by increased AMPA/NMDA current ratios. Meanwhile, LTP generation appeared to be unaffected. Conversely, sustained overexpression of Homer1a strongly reduced AMPA/NMDA current ratios and polyamine sensitivity of synaptic AMPAR, indicating that the proportion of synaptic GluA2-containing AMPAR increased relative to WT. LTP maintenance was abolished in H1aTG. Notably, overexpression of Homer1a in Homer1 KO or GluA2 KO mice did not affect LTP expression, suggesting activity-dependent interaction between Homer1a and long Homer1 isoforms with GluA2-containing AMPAR. Thus, Homer1a is essential for the activity-dependent regulation of excitatory synaptic transmission. PMID:23133416

  3. 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

  4. 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.

  5. Lifespan profiles of Alzheimer's disease–associated genes and their products in monkeys and mice

    PubMed Central

    Dosunmu, Remi; Wu, Jinfang; Adwan, Lina; Maloney, Bryan; Basha, Md. Riyaz; McPherson, Christopher A.; Harry, G. Jean; Rice, Deborah C.; Zawia, Nasser H.; Lahiri, Debomoy K.

    2009-01-01

    Alzheimer's disease (AD) is characterized by plaques of amyloid–beta (Aβ) peptide, cleaved from amyloid–β precursor protein (AβPP). Our hypothesis is that lifespan profiles of AD-associated mRNA and protein levels in monkeys would differ from mice, and that differential lifespan expression profiles would be useful to understand human AD pathogenesis. We compared profiles of AβPP mRNA, AβPP protein, and Aβ levels in rodents and primates. We also tracked a transcriptional regulator of the AβPP gene, specificity protein 1 (SP1), and the β amyloid precursor cleaving enzyme (BACE1). In mice, AβPP and Sp1 mRNA and their protein products were elevated late in life; Aβ levels declined in old age. In monkeys, Sp1, AβPP, and BACE1 mRNA declined in old age, while protein products and Aβ levels rose. Proteolytic processing in both species did not match production of Aβ. In primates, AβPP and Sp1 mRNA levels coordinate, but an inverse relationship exists with corresponding protein products, as well as Aβ levels. Comparison of human DNA and mRNA sequences to monkey and mouse counterparts revealed structural features that may explain differences in transcriptional and translational processing. These findings are important for selecting appropriate models for AD and other age–related diseases. PMID:19584442

  6. 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

  7. 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

  8. Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products.

    PubMed

    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.

  9. Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products.

    PubMed

    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. Nucleotide sequence of the pnd gene in plasmid R483 and role of the pnd gene product in plasmolysis.

    PubMed

    Ono, K; Akimoto, S; Ohnishi, Y

    1987-01-01

    The pnd gene of R plasmid R483, like the srnB gene of the F plasmid, increases the degradation of stable RNA in Escherichia coli. The nucleotide sequence of the pnd locus was determined and compared with that of the srnB locus. The genes have open reading frames that are 54% homologous, and both have an upstream inverted repeat sequence. The pnd gene expression seems to decrease the osmotic barrier of the cytoplasmic membrane, since no plasmolytic vacuoles were formed in the cells carrying the gene when the cells were exposed to hypertonic sucrose solution. This result suggests that RNase I in the periplasm passes through the altered membrane to degrade stable RNA in the cytoplasm.

  11. Influence of the spxB gene on competence in Streptococcus pneumoniae.

    PubMed

    Bättig, Patrick; Mühlemann, Kathrin

    2008-02-01

    In Streptococcus pneumoniae expression of pyruvate oxidase (SpxB) peaks during the early growth phase, coincident with the time of natural competence. This study investigated whether SpxB influences parameters of competence, such as spontaneous transformation frequency, expression of competence genes, and DNA release. Knockout of the spxB gene in strain D39 abolished spontaneous transformation (compared to a frequency of 6.3 x 10(-6) in the parent strain [P < 0.01]). It also reduced expression levels of comC and recA as well as DNA release from bacterial cells significantly during the early growth phase, coincident with the time of spontaneous competence in the parent strain. In the spxB mutant, supplementation with competence-stimulating peptide 1 (CSP-1) restored transformation (rate, 1.8 x 10(-2)). This speaks against the role of SpxB as a necessary source of energy for competence. Neither supplementation with CSP-1 nor supplementation with the SpxB products H2O2 and acetate altered DNA release. Supplementation of the parent strain with catalase did not reduce DNA release significantly. In conclusion, the pneumococcal spxB gene influences competence; however, the mechanism remains elusive. PMID:18065543

  12. 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

  13. 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.

  14. 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

  15. New PHA products using unrelated carbon sources

    PubMed Central

    Matias, Fernanda; de Andrade Rodrigues, Maria Filomena

    2011-01-01

    Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used. PMID:24031764

  16. A model for the abrogation of the SOS response by an SOS protein: a negatively charged helix in DinI mimics DNA in its interaction with RecA

    PubMed Central

    Voloshin, Oleg N.; Ramirez, Benjamin E.; Bax, Ad; Camerini-Otero, R. Daniel

    2001-01-01

    DinI is a recently described negative regulator of the SOS response in Escherichia coli. Here we show that it physically interacts with RecA and prevents the binding of single-stranded DNA to RecA, which is required for the activation of the latter. DinI also displaces ssDNA from a stable RecA–DNA cofilament, thus eliminating the SOS signal. In addition, DinI inhibits RecA-mediated homologous DNA pairing, but has no effect on actively proceeding strand exchange. Biochemical data, together with the molecular structure, define the C-terminal α-helix in DinI as the active site of the protein. In an unusual example of molecular mimicry, a negatively charged surface on this α-helix, by imitating single-stranded DNA, interacts with the loop L2 homologous pairing region of RecA and interferes with the activation of RecA. PMID:11230150

  17. Autoregulatory Functioning of a Drosophila Gene Product That Establishes and Maintains the Sexually Determined State

    PubMed Central

    Cline, Thomas W.

    1984-01-01

    Sxl appears to head a regulatory gene hierarchy that controls Drosophila sexual dimorphism in response to the X chromosome/autosome balance. Only XXAA cells normally have Sxl+ activity. It maintains both the female morphogenetic sequence and a level of X-linked dosage-compensated gene expression compatible with diplo-X cell survival. In the absence of this activity, male sexual development and dosage-compensated gene hyperactivation ensure. Loss-of-function Sxl mutations generally have female-specific lethal effects caused by upsets in dosage compensation. New female-viable Sxl mutant alleles and combinations which lack Sxl's female sex determination function, yet still provide sufficient dosage compensation function for diplo-X survival, are described here. Consequently, such mutants cause genotypic females to develop as phenotypic males. Some of these sex-transforming Sxl mutants do not require the maternally produced da+ activity that is normally essential for the functioning of zygotic Sxl alleles. In this paper, products of these unusual alleles are shown to act in trans to induce the expression of zygotic Sxl+ alleles that would otherwise be unable to function due to a lack of maternal da+ activity. This result indicates a third function for Sxl+ product: a positive autoregulatory role. Controls for the autoregulation experiments demonstrated the sex-trans-forming epigenetic effect of the da mutation for the first time in diploids. In these experiments the female-specific zygotic lethal effects that normally would have accompanied loss of maternal da+ activity were suppressed by mutations known to block dosage-compensation gene hyperactivation—the autosomal, male-specific lethals. Three types of abnormal sexual phenotypes were produced in the experiments described here, each with important implications for the mechanism of sex determination: (1) a true intersex phenotype produced by one particular Sxl allele shows that Sxl+ must be involved in the cellular

  18. Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis.

    PubMed

    Rodríguez, D; Esteban, M; Rodríguez, J R

    1995-08-01

    Vaccinia virus (VV) A17L gene encodes a 23-kDa protein that is proteolytically cleaved to generate a 21-kDa product that is incorporated into the viral particles. We have previously shown that the 21-kDa protein forms a stable complex with the VV 14-kDa envelope protein and suggested that the 21-kDa protein may serve to anchor the 14-kDa protein to the envelope of the virion (D. Rodríguez, J. R. Rodríguez, and M. Esteban, J. Virol. 67:3435-3440, 1993). To study the role of the 21-kDa protein in virion assembly, in this investigation we generated a VV recombinant, VVindA17L, that contains an inducible A17L gene regulated by the E. coli repressor/operator system. In the absence of the inducer, shutoff of the A17L gene was complete, and this shutoff correlated with a reduction in virus yields of about 3 log units. Although early and late viral polypeptides are normally synthesized in the absence of the A17L gene product, proteolytic processing of the major p4a and p4b core proteins was clearly impaired under these conditions. Electron microscopy examination of cells infected in the absence of isopropylthiogalactopyranoside (IPTG) revealed that virion morphogenesis was completely arrested at a very early stage, even prior to the formation of crescent-shaped membranes, which are the first distinguishable viral structures. Only electron-dense structures similar to rifampin bodies, but devoid of membranes, could be observed in the cytoplasm of cells infected with VVindA17L under nonpermissive conditions. Considering the most recent assembly model presented by Sodeik et al. (B. Sodeik, R. W. Doms, M. Ericsson, G. Hiller, C. E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths, J. Cell Biol. 121:521-541, 1993), we propose that this protein is targeted to the intermediate compartment and is involved in the recruitment of these membranes to the viral factories, where it forms the characteristic crescent structures that subsequently result in the formation of

  19. Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene.

    PubMed

    Schubert, Justyna; Podkowik, Magdalena; Bystroń, Jarosław; Bania, Jacek

    2016-10-17

    Twenty Staphylococcus aureus strains harboring seh gene, including one carrying also sec gene and 11 sea gene, were grown in BHI+YE broth and milk and were tested for SEA, SEC and SEH production. All strains decreased pH of BHI+YE broth at 24h and increased them at 48h. Seventeen S. aureus strains grown in milk changed pH for no >0.3 unit until 48h. Three other S. aureus strains significantly decreased pH during growth in milk. All S. aureus produced SEH in BHI+YE broth in amounts ranging from 95 to 1292ng/ml, and from 170 to 4158ng/ml at 24 and 48h, respectively. SEH production in milk by 17 strains did not exceed 23ng/ml at 24h and 36ng/ml at 48h. Three S. aureus strains able to decrease milk pH produced 107-3029ng/ml and 320-4246ng/ml of SEH in milk at 24 and 48h, respectively. These strains were grown in milk and BHI+YE broth with pH stabilized at values near neutral leading to a significant decrease of SEH production. Representative weak SEH producers were grown in milk at reduced pH resulting in moderate increase in SEH production. SEA was produced in milk by 10S. aureus strains at 24-151ng/ml at 24h, and 31-303ng/ml at 48h. SEA production in milk was higher or comparable as in BHI+YE broth in 3 strains and lower for remaining strains. Production of SEC by sec-positive S. aureus strains was lower in milk than in BHI+YE broth, ranging from 131 to 2319ng/ml at 24 and 48h in milk and 296-30,087ng/ml in BHI+YE at 24 and 48h. Both lacE and lacG transcripts involved in lactose metabolism were significantly up-regulated in milk in strong SEH producers. In these strains hld, rot and sarA transcripts were up-regulated in milk as compared to weak SEH producers. Stabilization of milk pH at a value of raw milk significantly down-regulated hld, rot and sarA RNA in strong SEH producers. Milk was generally found unfavorable for enterotoxin production. However, certain S. aureus strains were not restricted in SEH and SEA expression in milk, unlike SEC which remained down

  20. Production of host shutoff-defective mutants of herpes simplex virus type 1 by inactivation of the UL13 gene.

    PubMed

    Overton, H; McMillan, D; Hope, L; Wong-Kai-In, P

    1994-07-01

    Two mutants of HSV-1(SC16) carrying disrupted UL13 genes have been generated independently by recombination of wild-type genomic DNA with a plasmid-cloned copy of the UL13 gene containing multiple stop codons. The two mutants were shown to be deficient in UL13 gene expression by Western blotting of infected cells. A revertant virus, in which UL13 expression was restored to a near-normal level, was generated by recombination of one of the UL13-negative mutants with a plasmid carrying the wild-type UL13 gene. The replication of the two UL13-negative viruses in cell culture was somewhat reduced compared to their wild-type parent, and the viruses were unable to produce shutoff of host protein synthesis. The replication of the revertant virus was intermediate between that of the UL13-negative and wild-type viruses, as was its ability to produce host shutoff. Cells infected with the UL13-negative mutants were shown to contain much lower levels than normal of the UL41 gene product, which is known to be required for virion host shutoff. However, there was no significant difference between levels of the UL41 gene product in wild-type and mutant virions. The UL13-negative viruses exhibited different patterns of protein phosphorylation from wild-type virus when infected cells were metabolically labeled with [32P]-orthophosphate and when lysates of infected cells and of virions were subjected to in vitro phosphorylation. However, the UL41 gene product could still be phosphorylated in lysates of UL13-negative virions. We conclude that the UL13 gene is necessary to produce the virion host shutoff effect, but it seems unlikely that the role of UL13 is simply to activate the UL41 gene product by phosphorylation.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. Improvement of clavulanic acid production in Streptomyces clavuligerus by genetic manipulation of structural biosynthesis genes.

    PubMed

    Jnawali, Hum Nath; Yoo, Jin Cheol; Sohng, Jae Kyung

    2011-06-01

    To enhance clavulanic acid production, four structural clavulanic acid biosynthesis genes, carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2) and proclavaminate amidinohydrolase (pah2), were amplified from Streptomyces clavuligerus genomic DNA. They were cloned in the pSET152 integration and pIBR25 expression vectors containing the strong ermE* promoter to generate pHN18 and pHN19, respectively, and both plasmids were introduced into S. clavuligerus by protoplast transformation. Clavulanic acid production was increased by 8.7-fold (to ~310 mg/l) in integrative pHN18 transformants and by 5.1-fold in pHN19 transformants compared to controls. Transcriptional analyses showed that the expression levels of ceas2, bls2, cas2 and pah2 were markedly increased in both transformants as compared with wild-type. The elevation of the ceas2, bls2, cas2 and pah2 transcripts was consistent with the enhanced production of clavulanic acid.

  6. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis

    PubMed Central

    Piriya, P. Sobana; Vasan, P. Thirumalai; Padma, V. S.; Vidhyadevi, U.; Archana, K.; Vennison, S. John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production. PMID:22919503

  7. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis.

    PubMed

    Piriya, P Sobana; Vasan, P Thirumalai; Padma, V S; Vidhyadevi, U; Archana, K; Vennison, S John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production.

  8. Molecular detection of adulteration in chicken products based on mitochondrial 12S rRNA gene.

    PubMed

    Abuzinadah, Osama H A; Yacoub, Haitham Ahmed; El Ashmaoui, Hassan M; Ramadan, Hassan A I

    2015-06-01

    The aim of this study is to detect the fraudulent in chicken products constitutes in order to protect consumers in Saudi Arabia from illegal substitutions. Two different approaches were used in this study, direct sequencing of specific fragments of amplified mitochondrial 12S rRNA gene in addition to species-specific PCR primers for confirmation of the obtained Blast search results. The results showed that all processed chicken products were identified as chicken (Gallus gallus) by 90-98% homology depending on obtained sequence quality. Samples labeled with chicken luncheon (samples tested in this study) were identified as turkey meat (Meleagris gallopavo) by 98% homology, suggesting adulteration with inedible parts of turkey in chicken luncheon ingredients. The results showed also that not only chicken luncheon was mixed with inedible parts of turkey but also all chicken products tested in this study (chicken balls, chicken burger, chicken sausage and chicken mined meat) contained this turkey meat. Applying methods used in this study could be useful for accurate and rapid identification of commercial processed meat.

  9. Differential expression of sets of highly homologous variable region gene products in selected and preimmune repertoires of inbred mouse strains

    PubMed Central

    1986-01-01

    Using mAb that selectively recognize the various allelic forms of the VHT15 and Vk21D-E genes' products, we analyzed the influence of VH and Vk polymorphism on the probability of expression of these gene segments. Our data show that the frequency to which the VHT15 gene product becomes available in the preimmune repertoire is strongly influenced by the polymorphism of the relevant structural gene, suggesting therefore that VH genes cannot be randomly used in the various strains. Contrary to this, the frequency of Vk21D-E+ clones is similar in all mouse strains tested, and in all cases is higher than the frequency of VHT15 clones. This observation strongly suggests that Vk genes can be randomly expressed, and/or that their number is lower than that of their VH counterpart. Finally, analysis of the specificity associated to the expression of the VHT15 segment revealed that VH polymorphism strongly influences not only the probability of expression of each V gene, but also the specificity of the antibodies on which these VH genes are used. PMID:3084699

  10. 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.

  11. 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

  12. 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

  13. The Interaction of cos with Chi Is Separable from DNA Packaging in recA- recBC-Mediated Recombination of Bacteriophage Lambda

    PubMed Central

    Kobayashi, Ichizo; Stahl, Mary M.; Leach, David; Stahl, Franklin W.

    1983-01-01

    Chi (5'-GCTGGTGG) is a recombinator in RecA- RecBC-mediated recombination in Escherichia coli. In bacteriophage λ vegetative recombination, Chi is fully active only when it is correctly oriented with respect to cos, the site that defines the ends of the packaged chromosome. Here we demonstrate that packaging from cos is not necessary for this cos-Chi interaction. Our evidence suggests that correctly oriented cos is an activator of Chi. cos, as an activator, is (1) dominant over cos-, (2) active opposite an extensive heterology, (3) able to interact with Chi only when on the same (cis) chromosome, and (4) able to interact with Chi at distances as far as ≥ 20 kb. Thus, cos and Chi form a two-component recombinator system for general recombination. cos may serve as an asymmetric entry site for a recombination enzyme that recognizes Chi in an asymmetric way. PMID:6225696

  14. Bacillus subtilis DprA Recruits RecA onto Single-stranded DNA and Mediates Annealing of Complementary Strands Coated by SsbB and SsbA*

    PubMed Central

    Yadav, Tribhuwan; Carrasco, Begoña; Hejna, James; Suzuki, Yuki; Takeyasu, Kunio; Alonso, Juan C.

    2013-01-01

    Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation. PMID:23779106

  15. Bacillus subtilis DprA recruits RecA onto single-stranded DNA and mediates annealing of complementary strands coated by SsbB and SsbA.

    PubMed

    Yadav, Tribhuwan; Carrasco, Begoña; Hejna, James; Suzuki, Yuki; Takeyasu, Kunio; Alonso, Juan C

    2013-08-01

    Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation. PMID:23779106

  16. RecFOR proteins target RecA protein to a DNA gap with either DNA or RNA at the 5' terminus: implication for repair of stalled replication forks.

    PubMed

    Morimatsu, Katsumi; Wu, Yun; Kowalczykowski, Stephen C

    2012-10-12

    The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1-2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000-2000 nucleotides downstream (5' → 3') of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5'-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks. PMID:22902627

  17. Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant.

    PubMed

    Thoms, B; Wackernagel, W

    1987-04-01

    We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease. PMID:3031020

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    PubMed

    McMillan, Mary; Pereg, Lily

    2014-01-01

    Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

  3. 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

  4. 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

  5. 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.

  6. Analysis of β-galactosidase production and their genes of two strains of Lactobacillus bulgaricus.

    PubMed

    Zhang, Wen; Wang, Chuan; Huang, Cheng-Yu; Yu, Qian; Liu, Heng-Chuan; Zhang, Chao-Wu; Pei, Xiao-Fang; Xu, Xin; Wang, Guo-Qing

    2012-06-01

    A bacterial β-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these β-galactosidase genes and their resulting production levels are poorly characterized. The β-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa β-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher β-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).

  7. Active movements of the chromatoid body. A possible transport mechanism for haploid gene products.

    PubMed

    Parvinen, M; Parvinen, L M

    1979-03-01

    Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.

  8. Relationship between brassinosteroids and genes controlling stomatal production in the Arabidopsis hypocotyl.

    PubMed

    Fuentes, Sonia; Cañamero, Roberto C; Serna, Laura

    2012-01-01

    Stomata are excellent model systems for examining the mechanisms that regulate cell fate determination and pattern formation. It has recently been demonstrated that brassinosteroids control stomatal development by regulating both the MAPK kinase kinase YODA and the basic helix-loop-helix transcriptional factor SPEECHLESS. Here, we show that these plant regulators positively regulate stomatal formation in the hypocotyl and also accelerate their development. Hormone tests, reporter gene studies and mutant analyses revealed that brassinosteroids act upstream of the transcriptional factors CAPRICE and GLABRA2. These plant regulators control an earlier stage of stomatal production than those regulated by the membrane receptor TOO MANY MOUTHS. This work highlights differences in the genetic control of stomatal development between cotyledons or leaves and hypocotyls.

  9. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

    PubMed

    Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J; Ivison, David; Généreux, Catherine; Dubus, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J; Lloyd, Adrian J; Roper, David I; Schofield, Christopher J; Andersson, Inger; McDonough, Michael A

    2013-08-01

    Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

  10. Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product.

    PubMed

    Hannibal, Luciana; Kim, Jihoe; Brasch, Nicola E; Wang, Sihe; Rosenblatt, David S; Banerjee, Ruma; Jacobsen, Donald W

    2009-08-01

    The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B(12)). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [(57)Co]-AdoCbl and MeCbl analogs consisting of [(57)Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [(57)Co]-AdoCbl was converted to [(57)Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5'-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [(57)Co]-propylcobalamin to [(57)Co]-AdoCbl and [(57)Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.

  11. PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella.

    PubMed Central

    Smith, E F; Lefebvre, P A

    1997-01-01

    The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components, we generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed flagella, and the entire central apparatus is missing in isolated axonemes. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued transformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid sequence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate chains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates that the PF20 protein is localized along the length of the C2 microtubule on the intermicrotubule bridges connecting the two central microtubules. We suggest that the PF20 gene product is a new member of the family of WD repeat proteins and is required for central microtubule assembly and/or stability and flagellar motility. Images PMID:9188098

  12. Genetic Characterization of the Homeodomain-Independent Activity of the Drosophila Fushi Tarazu Gene Product

    PubMed Central

    Hyduk, D.; Percival-Smith, A.

    1996-01-01

    The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results. PMID:8852847

  13. Processing of alkylcobalamins in mammalian cells: a role for the MMACHC (cblC) gene product

    PubMed Central

    Hannibal, Luciana; Kim, Jihoe; Brasch, Nicola E.; Wang, Sihe; Rosenblatt, David S.; Banerjee, Ruma; Jacobsen, Donald W.

    2009-01-01

    The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B12). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [57Co]-AdoCbl and MeCbl analogs consisting of [57Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [57Co]-AdoCbl was converted to [57Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5′-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from 3 patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [57Co]-propylcobalamin to [57Co]-AdoCbl and [57Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells. PMID:19447654

  14. 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.

  15. 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

  16. Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

    PubMed Central

    Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

    2013-01-01

    Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

  17. Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast.

    PubMed

    Misra, Ashish; Conway, Matthew F; Johnnie, Joseph; Qureshi, Tabish M; Lige, Bao; Derrick, Anne M; Agbo, Eddy C; Sriram, Ganesh

    2013-01-01

    Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo (13)C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast.

  18. 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

  19. 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

  20. Spatial and Temporal Variations in Chitinolytic Gene Expression and Bacterial Biomass Production during Chitin Degradation

    PubMed Central

    Baty, Ace M.; Eastburn, Callie C.; Techkarnjanaruk, Somkiet; Goodman, Amanda E.; Geesey, Gill G.

    2000-01-01

    Growth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day−1 were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day−1 on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading genes chiA and chiB. Cells displaying high levels of chiA-chiB expression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population. PMID:10919823

  1. The Production and Characteristics of a Mouse's Embryonic Stem Cell Lineage, Transfected by the Glia Neurotrophic Factor and Gene Fused with the Green Fluorescent Protein Gene

    PubMed Central

    Arsenieva, E. L.; Kuzmin, I. V.; Manuilova, E. S.; Novosadova, E. V.; Murkin, E. V.; Pavlova, G. V.; Tarantul, V. Z.

    2009-01-01

    The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones. PMID:22649595

  2. 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

  3. 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

  4. Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei.

    PubMed

    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.

  5. 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

  6. Sequence analysis of the Alcaligenes eutrophus chromosomally encoded ribulose bisphosphate carboxylase large and small subunit genes and their gene products.

    PubMed Central

    Andersen, K; Caton, J

    1987-01-01

    The nucleotide sequence of the chromosomally encoded ribulose bisphosphate carboxylase/oxygenase (RuBPCase) large (rbcL) and small (rbcS) subunit genes of the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was determined. We found that the two coding regions are separated by a 47-base-pair intergenic region, and both genes are preceded by plausible ribosome-binding sites. Cotranscription of the rbcL and rbcS genes has been demonstrated previously. The rbcL and rbcS genes encode polypeptides of 487 and 135 amino acids, respectively. Both genes exhibited similar codon usage which was highly biased and different from that of other organisms. The N-terminal amino acid sequence of both subunit proteins was determined by Edman degradation. No processing of the rbcS protein was detected, while the rbcL protein underwent a posttranslational loss of formylmethionyl. The A. eutrophus rbcL and rbcS proteins exhibited 56.8 to 58.3% and 35.6 to 38.5% amino acid sequence homology, respectively, with the corresponding proteins from cyanobacteria, eucaryotic algae, and plants. The A. eutrophus and Rhodospirillum rubrum rbcL proteins were only about 32% homologous. The N- and C-terminal sequences of both the rbcL and the rbcS proteins were among the most divergent regions. Known or proposed active site residues in other rbcL proteins, including Lys, His, Arg, and Asp residues, were conserved in the A. eutrophus enzyme. The A. eutrophus rbcS protein, like those of cyanobacteria, lacks a 12-residue internal sequence that is found in plant RuBPCase. Comparison of hydropathy profiles and secondary structure predictions by the method described by Chou and Fasman (P. Y. Chou and G. D. Fasman, Adv. Enzymol. 47:45-148, 1978) revealed striking similarities between A. eutrophus RuBPCase and other hexadecameric enzymes. This suggests that folding of the polypeptide chains is similar. The observed sequence homologies were consistent with the notion that both the rbcL and rbcS genes of the

  7. 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

  8. 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.

  9. Expression of genes responsible for ethylene production and wilting are differently regulated in carnation (Dianthus caryophyllus L.) petals.

    PubMed

    Kosugi; Shibuya; Tsuruno; Iwazaki; Mochizuki; Yoshioka; Hashiba; Satoh

    2000-09-01

    Carnation petals exhibit autocatalytic ethylene production and wilting during senescence. The autocatalytic ethylene production is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes, whereas the wilting of petals is related to the expression of the cysteine proteinase (CPase) gene. So far, it has been believed that the ethylene production and wilting are regulated in concert in senescing carnation petals, since the two events occurred closely in parallel with time. In the present study, we investigated the expression of these genes in petals of a transgenic carnation harboring a sense ACC oxidase transgene and in petals of carnation flowers treated with 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS). In petals of the transgenic carnation flowers, treatment with exogenous ethylene caused accumulation of the transcript for CPase and in-rolling (wilting), whereas it caused no or little accumulation of the transcripts for ACC oxidase and ACC synthase and negligible ethylene production. In petals of the flowers treated with DPSS, the transcripts for ACC synthase and ACC oxidase were accumulated, but no significant change in the level of the transcript for CPase was observed. These results suggest that the expression of ACC synthase and ACC oxidase genes, which leads to ethylene production, is differentially regulated from the expression of CPase, which leads to wilting, in carnation petals.

  10. 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

  11. 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

  12. 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

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

    PubMed Central

    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

  14. Analysis of the sequence and gene products of the transfer region of the F sex factor.

    PubMed Central

    Frost, L S; Ippen-Ihler, K; Skurray, R A

    1994-01-01

    Bacterial conjugation results in the transfer of DNA of either plasmid or chromosomal origin between microorganisms. Transfer begins at a defined point in the DNA sequence, usually called the origin of transfer (oriT). The capacity of conjugative DNA transfer is a property of self-transmissible plasmids and conjugative transposons, which will mobilize other plasmids and DNA sequences that include a compatible oriT locus. This review will concentrate on the genes required for bacterial conjugation that are encoded within the transfer region (or regions) of conjugative plasmids. One of the best-defined conjugation systems is that of the F plasmid, which has been the paradigm for conjugation systems since it was discovered nearly 50 years ago. The F transfer region (over 33 kb) contains about 40 genes, arranged contiguously. These are involved in the synthesis of pili, extracellular filaments which establish contact between donor and recipient cells; mating-pair stabilization; prevention of mating between similar donor cells in a process termed surface exclusions; DNA nicking and transfer during conjugation; and the regulation of expression of these functions. This review is a compendium of the products and other features found in the F transfer region as well as a discussion of their role in conjugation. While the genetics of F transfer have been described extensively, the mechanism of conjugation has proved elusive, in large part because of the low levels of expression of the pilus and the numerous envelope components essential for F plasmid transfer. The advent of molecular genetic techniques has, however, resulted in considerable recent progress. This summary of the known properties of the F transfer region is provided in the hope that it will form a useful basis for future comparison with other conjugation systems. PMID:7915817

  15. Characterization of Genes Responsible for the CO-Linked Hydrogen Production Pathway in Rubrivivax gelatinosus▿

    PubMed Central

    Vanzin, Gary; Yu, Jianping; Smolinski, Sharon; Tek, Vekalet; Pennington, Grant; Maness, Pin-Ching

    2010-01-01

    Upon exposure to carbon monoxide, the purple nonsulfur photosynthetic bacterium Rubrivivax gelatinosus produces hydrogen concomitantly with the oxidation of CO according to the equation CO + H2O ↔ CO2 + H2. Yet little is known about the genetic elements encoding this reaction in this organism. In the present study, we use transposon mutagenesis and functional complementation to uncover three clustered genes, cooL, cooX, and cooH, in Rubrivivax gelatinosus putatively encoding part of a membrane-bound, multisubunit NiFe-hydrogenase. We present the complete amino acid sequences for the large catalytic subunit and its electron-relaying small subunit, encoded by cooH and cooL, respectively. Sequence alignment reveals a conserved region in the large subunit coordinating a binuclear [NiFe] center and a conserved region in the small subunit coordinating a [4Fe-4S] cluster. Protein purification experiments show that a protein fraction of 58 kDa molecular mass could function in H2 evolution mediated by reduced methyl viologen. Western blotting experiments show that the two hydrogenase subunits are detectable and accumulate only when cells are exposed to CO. The cooX gene encodes a putative Fe-S protein mediating electron transfer to the hydrogenase small subunit. We conclude that these three Rubrivivax proteins encompass part of a membrane-bound, multisubunit NiFe-hydrogenase belonging to the energy-converting hydrogenase (Ech) type, which has been found among diverse microbes with a common feature in coupling H2 production with proton pumping for energy generation. PMID:20400563

  16. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

    SciTech Connect

    Yamano, Shigeru; Tatsuno, Jun; Gonzalez, F.J. )

    1990-02-06

    Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a {lambda}gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu{sup 160}{yields}His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a {lambda}{sub max} at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIa mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. These data establish that the CYP2A3 gene product is primarily responsible for coumarin 7-hydroxylase activity in human liver. The level of expression of this activity varied up to 40-fold between livers. Levels of IIA mRNA also varied significantly between liver specimens, and three specimens had no detectable mRNA.

  17. 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.

  18. Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

    PubMed

    Kingston, Anthony W; Roussel-Rossin, Chloé; Dupont, Claire; Raleigh, Elisabeth A

    2015-01-01

    In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F'-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10(-12) CFU/recipient per hour.

  19. Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12

    PubMed Central

    Kingston, Anthony W.; Raleigh, Elisabeth A.

    2015-01-01

    In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10-12 CFU/recipient per hour. PMID:26162088

  20. Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

    PubMed

    Kingston, Anthony W; Roussel-Rossin, Chloé; Dupont, Claire; Raleigh, Elisabeth A

    2015-01-01

    In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F'-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10(-12) CFU/recipient per hour. PMID:26162088

  1. 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

  2. Experimental Model to Study the Role of Retinoblastoma Gene Product (pRb) for Determination of Adipocyte Differentiation.

    PubMed

    Popov, B V; Shilo, P S; Zhidkova, O V; Zaichik, A M; Petrov, N S

    2015-06-01

    Using stable constitutive expression of retinoblastoma gene product (pRb) in polypotent mesenchymal 10T1/2 cells we obtained stable cell lines hyperexpressing functionally active or inactive mutant pRb. The cells producing active exogenous pRb demonstrated high sensitivity to adipocyte differentiation inductors, whereas production of inactive form of the exogenous protein suppressed adipocyte differentiation. The obtained lines can serve as the experimental model for studying the role of pRb in determination of adipocyte differentiation.

  3. The natural product domain seeker NaPDoS: a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity.

    PubMed

    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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

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

    PubMed

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

    2015-03-01

    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.

  10. Functional analysis of the fsoC gene product of the F7(1) (fso) fimbrial gene cluster.

    PubMed

    Riegman, N; Acton, D; Sakkers, R; van Die, I; Hoekstra, W; Bergmans, H

    1990-01-01

    Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F7(1) (fso) P-fimbrial gene cluster do not completely block fimbrial biogenesis. fsoC mutants still express small amounts of fimbriae of normal length, which carry the non-adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.

  11. lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity.

    PubMed Central

    Toder, D S; Ferrell, S J; Nezezon, J L; Rust, L; Iglewski, B H

    1994-01-01

    The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced. Its function and significance have been studied by genetic approaches (D. S. Toder, M. J. Gambello, and B. H. Iglewski, Mol. Microbiol. 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J. E. Peters and D. R. Galloway, J. Bacteriol. 172:2236-2240, 1990). To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1. Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity. Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present. Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity. We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64. Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB. Images PMID:8132339

  12. 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 0.35 g l(-1) and 1.40 g l(-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.

  13. 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 0.35 g l(-1) and 1.40 g l(-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

  14. 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

  15. Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker.

    PubMed

    Khan, Raham Sher; Ntui, Valentine Otang; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2011-04-01

    The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

  16. 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

  17. Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400.

    PubMed

    Baysse, C; Matthijs, S; Pattery, T; Cornelis, P

    2001-11-27

    A Pseudomonas fluorescens Tn5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis. Since this mutant was leaky, a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor delta-aminolevulinate (ALA). Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth. A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin. The DeltahemH mutant was characterized and the corresponding hemH gene sequenced. Antibodies against P. fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant. The DeltahemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans. These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production. PMID:11728716

  18. 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.

  19. The electron transfer flavoprotein fixABCX gene products from Azospirillum brasilense show a NifA-dependent promoter regulation.

    PubMed

    Sperotto, Raul Antonio; Gross, Jeferson; Vedoy, Cleber; Passaglia, Luciane Maria Pereira; Schrank, Irene Silveira

    2004-10-01

    The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric p fixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Improvement of L-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes.

    PubMed

    Hou, Xiaohu; Ge, Xiangyang; Wu, Di; Qian, He; Zhang, Weiguo

    2012-01-01

    Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.

  6. 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

  7. Regulation of transcription of the adenovirus EII promoter by gene products: Absence of sequence specificity

    SciTech Connect

    Kingston, R.E.; Kaufman, R.J.; Sharp, P.A.

    1984-10-01

    During adenovirus infection, the EII promoter is positively regulated by products of the EIa region. The authors have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when cell lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that EIa activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the EII promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the EIa region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EII sequences required for stimulation. EIa activity stimulates all mutaant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. They suggest that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into cells. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the EII promoter. The stimulatory effects of EIa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.

  8. 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

  9. CDX2 hox gene product in a rat model of esophageal cancer

    PubMed Central

    2009-01-01

    Background Barrett's mucosa is the precursor of esophageal adenocarcinoma. The molecular mechanisms behind Barrett's carcinogenesis are largely unknown. Experimental models of longstanding esophageal reflux of duodenal-gastric contents may provide important information on the biological sequence of the Barrett's oncogenesis. Methods The expression of CDX2 hox-gene product was assessed in a rat model of Barrett's carcinogenesis. Seventy-four rats underwent esophago-jejunostomy with gastric preservation. Excluding perisurgical deaths, the animals were sacrificed at various times after the surgical treatment (Group A: <10 weeks; Group B: 10–30 weeks; Group C: >30 weeks). Results No Cdx2 expression was detected in either squamous epithelia of the proximal esophagus or squamous cell carcinomas. De novo Cdx2 expression was consistently documented in the proliferative zone of the squamous epithelium close to reflux ulcers (Group A: 68%; Group B: 64%; Group C: 80%), multilayered epithelium and intestinal metaplasia (Group A: 9%; Group B: 41%; Group C: 60%), and esophageal adenocarcinomas (Group B: 36%; Group C: 35%). A trend for increasing overall Cdx2 expression was documented during the course of the experiment (p = 0.001). Conclusion De novo expression of Cdx2 is an early event in the spectrum of the lesions induced by experimental gastro-esophageal reflux and should be considered as a key step in the morphogenesis of esophageal adenocarcinoma. PMID:19664209

  10. 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

  11. Localization of ocular albinism-1 gene product GPR143 in the rat central nervous system.

    PubMed

    Masukawa, Daiki; Nakamura, Fumio; Koga, Motokazu; Kamiya, Marina; Chen, Sandy; Yamashita, Naoya; Arai, Nobutaka; Goshima, Yoshio

    2014-11-01

    L-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be a precursor of dopamine, and itself being an inert amino acid. Previously, we have proposed DOPA as a neurotransmitter candidate in the central nervous system (CNS). Recent findings have suggested DOPA as an endogenous agonist of a G-protein coupled receptor, ocular albinism 1 gene product (OA1), which is highly expressed in the retinal pigmental epithelium. However, whether OA1 functions as a receptor for DOPA in vivo, and whether this receptor-ligand interaction is responsible for a wide variety of DOPA actions have not been determined yet. To gain insight into the functional implication of OA1, we perform immunohistochemical examination with anti-OA1 antibody to localize OA1 in the adult rat brain. We observed OA1 immunoreactive cells in the hippocampus, cerebral cortex, cerebellum cortex, striatum, substantia nigra, hypothalamic median eminence and supraoptic nucleus, nucleus tractus solitarii and caudal ventrolateral medulla and rostral ventrolateral medulla, medial habenular nucleus and olfactory bulb. This study reveals, for the first time, the unique distribution pattern of OA1-immunoreactive neurons and/or cells in the rat CNS. PMID:25108060

  12. Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways.

    PubMed

    Gomez-Escribano, Juan Pablo; Bibb, Mervyn J

    2014-02-01

    Heterologous gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the heterologous production of structurally diverse actinomycete natural products.

  13. Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

    PubMed

    Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

    2015-02-01

    Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus