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Abigail A Salyers - One of the best experts on this subject based on the ideXlab platform.
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new regulatory gene that contributes to control of bacteroides thetaiotaomicron starch utilization Genes
Journal of Bacteriology, 2001Co-Authors: Kyu Hong Cho, Diedre Cho, Guirong Wang, Abigail A SalyersAbstract:Bacteroides thetaiotaomicron uses starch as a source of carbon and energy. Early steps in the pathway of starch utilization, such as starch binding and starch hydrolysis, are encoded by sus Genes, which have been characterized previously. The sus Structural Genes are expressed only if cells are grown in medium containing maltose or higher oligomers of glucose. Regulation of the sus Structural Genes is mediated by SusR, an activator that is encoded by a gene located next to the sus Structural Genes. A strain with a disruption in susR cannot grow on starch but can still grow on maltose and maltotriose. A search for transposon-generated mutants that could not grow on maltose and maltotriose unexpectedly located a gene, designated malR, which regulates expression of an alpha-glucosidase not controlled by SusR. Although a disruption in susR did not affect expression of the malR controlled gene, a disruption in malR reduced expression of the sus Structural Genes. Thus, MalR appears to participate with SusR in regulation of the sus Genes. Results of transcriptional fusion assays and reverse transcription-PCR experiments showed that malR is expressed constitutively. Moreover, multiple copies of malR provided on a plasmid (5 to 10 copies per cell) more than doubled the amount of alpha-glucosidase activity in cell extracts. Our results demonstrate that the starch utilization system of B. thetaiotaomicron is controlled on at least two levels by the regulatory proteins SusR and MalR.
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Effect of regulatory protein levels on utilization of starch by Bacteroides thetaiotaomicron.
Journal of bacteriology, 1996Co-Authors: J N D'elia, Abigail A SalyersAbstract:Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, appears to utilize starch by first binding the polymer to its surface and then translocating it into the periplasmic space. Several Genes that encode enzymes or outer membrane proteins involved in starch utilization have been identified. These have been called sus Genes, for starch utilization system. Previous studies have shown that sus Structural Genes are regulated at the transcriptional level and their expression is induced by maltose. We report here the identification and characterization of a gene, susR, which appears to be responsible for maltose-dependent regulation of the sus Structural Genes. The deduced amino acid sequence of SusR protein had a helix-turn-helix motif at its carboxy-terminal end, and this region had highest sequence similarity to the corresponding regions of known transcriptional activators. A disruption in susR eliminated the expression of all known sus Structural Genes, as expected if susR encoded an activator of sus gene expression. The expression of susR itself was not affected by the growth substrate and was not autoregulated, suggesting that binding of SusR to maltose might be the step that activates SusR. Three susR-controlled Structural Genes, susA, susB, and susC, are located immediately upstream of susR. These Genes are organized into two transcriptional units, one containing susA and another containing susB and susC. susA was expressed at a lower level than susBC, and susA expression was more sensitive to the gene dosage of susR than was that of the susBC operon. An unexpected finding was that increasing the number of copies of susR in B. thetaiotaomicron increased the rate of growth on starch. This effect could be due to higher levels of susA expression. Whatever the explanation, the level of SusR in the cell appears to be a limiting factor for growth on starch.
Stephen S Whitehead - One of the best experts on this subject based on the ideXlab platform.
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a novel approach for the rapid mutaGenesis and directed evolution of the Structural Genes of west nile virus
Journal of Virology, 2012Co-Authors: Tsaiyu Lin, Stephen S Whitehead, Kimberly A Dowd, Carolyn J Manhart, Steevenson Nelson, Theodore C PiersonAbstract:ABSTRACT Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutaGenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the Structural Genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the Structural Genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (∼10 7 infectious units/ml in 48 h). The suitability of this approach for large-scale mutaGenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the “pr” portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the Structural Genes of this important human pathogen.
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Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the Structural Genes of rDEN4 and rDEN4Δ30 with those of DEN1
Virology Journal, 2007Co-Authors: Joseph E Blaney, Neeraj S Sathe, Christopher T Hanson, Cai-yen Firestone, Brian R Murphy, Stephen S WhiteheadAbstract:Background Antigenic chimeric viruses have previously been generated in which the Structural Genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Δ30(ME) and rDEN3/4Δ30(ME), which contain the membrane (M) precursor and envelope (E) Genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Δ30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Δ30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME Structural Genes of DEN1 Puerto Rico/94 strain. Results Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Δ30(CME), rDEN1/4(ME), and rDEN1/4Δ30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Δ30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Δ30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Δ30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Δ30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Δ30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. Conclusion The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Δ30(ME) should be considered for evaluation in a clinical trial.
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substitution of the Structural Genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes mice and rhesus monkeys
Vaccine, 2003Co-Authors: Stephen S Whitehead, Joseph E Blaney, Kathryn A Hanley, Lara E Gilmore, William R Elkins, Brian R MurphyAbstract:Antigenic chimeric viruses in which the Structural Genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding Genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3' untranslated region. The two DEN2/4 chimeric viruses lacking the delta 30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta 30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta 30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta 30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta 30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta 30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials.
Brian R Murphy - One of the best experts on this subject based on the ideXlab platform.
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Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the Structural Genes of rDEN4 and rDEN4Δ30 with those of DEN1
Virology Journal, 2007Co-Authors: Joseph E Blaney, Neeraj S Sathe, Christopher T Hanson, Cai-yen Firestone, Brian R Murphy, Stephen S WhiteheadAbstract:Background Antigenic chimeric viruses have previously been generated in which the Structural Genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Δ30(ME) and rDEN3/4Δ30(ME), which contain the membrane (M) precursor and envelope (E) Genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Δ30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Δ30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME Structural Genes of DEN1 Puerto Rico/94 strain. Results Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Δ30(CME), rDEN1/4(ME), and rDEN1/4Δ30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Δ30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Δ30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Δ30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Δ30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Δ30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. Conclusion The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Δ30(ME) should be considered for evaluation in a clinical trial.
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substitution of the Structural Genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes mice and rhesus monkeys
Vaccine, 2003Co-Authors: Stephen S Whitehead, Joseph E Blaney, Kathryn A Hanley, Lara E Gilmore, William R Elkins, Brian R MurphyAbstract:Antigenic chimeric viruses in which the Structural Genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding Genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3' untranslated region. The two DEN2/4 chimeric viruses lacking the delta 30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta 30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta 30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta 30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta 30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta 30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials.
Peter M. Muriana - One of the best experts on this subject based on the ideXlab platform.
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A ‘bacteriocin PCR array’ for identification of bacteriocin-related Structural Genes in lactic acid bacteria ☆
Journal of Microbiological Methods, 2011Co-Authors: Sunita Macwana, Peter M. MurianaAbstract:Bacteriocins have been identified in many strains of lactic acid bacteria (LAB) which are a source of natural food preservatives and microbial inhibitors. Our objectives were to use a PCR array of primers to identify bacteriocin Structural Genes in Bac+ LAB. DNA sequence homology at the 5′- and 3′-ends of the various Structural Genes indicated that non-specific priming may allow PCR amplification of heterologous bacteriocin Genes. Successful amplification was obtained by real-time PCR and confirmed by melting curve and agarose gel analysis. Sequence information specific to targeted bacteriocin Structural Genes from the intra-primer regions of amplimers was compared to sequences residing in GenBank. The bacteriocin PCR array allowed the successful amplification of bacteriocin Structural Genes from strains of Lactobacillus, Lactococcus, and Pediococcus including one whose amino acid sequence was unable to be determined by Edman degradation analysis. DNA sequence analysis identified as many as 3 bacteriocin Structural Genes within a given strain, identifying ten unique bacteriocin sequences that were previously uncharacterized (partial homology) and one that was 100% identical to sequences in GenBank. This study provides a rapid approach to sequence and identify bacteriocin Structural Genes among Bac+ LAB using a microplate bacteriocin PCR array.
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A ‘bacteriocin PCR array’ for identification of bacteriocin-related Structural Genes in lactic acid bacteria
Journal of microbiological methods, 2011Co-Authors: Sunita Macwana, Peter M. MurianaAbstract:Bacteriocins have been identified in many strains of lactic acid bacteria (LAB) which are a source of natural food preservatives and microbial inhibitors. Our objectives were to use a PCR array of primers to identify bacteriocin Structural Genes in Bac+ LAB. DNA sequence homology at the 5′- and 3′-ends of the various Structural Genes indicated that non-specific priming may allow PCR amplification of heterologous bacteriocin Genes. Successful amplification was obtained by real-time PCR and confirmed by melting curve and agarose gel analysis. Sequence information specific to targeted bacteriocin Structural Genes from the intra-primer regions of amplimers was compared to sequences residing in GenBank. The bacteriocin PCR array allowed the successful amplification of bacteriocin Structural Genes from strains of Lactobacillus, Lactococcus, and Pediococcus including one whose amino acid sequence was unable to be determined by Edman degradation analysis. DNA sequence analysis identified as many as 3 bacteriocin Structural Genes within a given strain, identifying ten unique bacteriocin sequences that were previously uncharacterized (partial homology) and one that was 100% identical to sequences in GenBank. This study provides a rapid approach to sequence and identify bacteriocin Structural Genes among Bac+ LAB using a microplate bacteriocin PCR array.
Joseph E Blaney - One of the best experts on this subject based on the ideXlab platform.
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Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the Structural Genes of rDEN4 and rDEN4Δ30 with those of DEN1
Virology Journal, 2007Co-Authors: Joseph E Blaney, Neeraj S Sathe, Christopher T Hanson, Cai-yen Firestone, Brian R Murphy, Stephen S WhiteheadAbstract:Background Antigenic chimeric viruses have previously been generated in which the Structural Genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Δ30(ME) and rDEN3/4Δ30(ME), which contain the membrane (M) precursor and envelope (E) Genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Δ30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Δ30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME Structural Genes of DEN1 Puerto Rico/94 strain. Results Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Δ30(CME), rDEN1/4(ME), and rDEN1/4Δ30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Δ30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Δ30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Δ30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Δ30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Δ30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. Conclusion The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Δ30(ME) should be considered for evaluation in a clinical trial.
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substitution of the Structural Genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes mice and rhesus monkeys
Vaccine, 2003Co-Authors: Stephen S Whitehead, Joseph E Blaney, Kathryn A Hanley, Lara E Gilmore, William R Elkins, Brian R MurphyAbstract:Antigenic chimeric viruses in which the Structural Genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding Genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3' untranslated region. The two DEN2/4 chimeric viruses lacking the delta 30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta 30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta 30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta 30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta 30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta 30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials.
