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Cook, L. C., and G. M. Dunny. "Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis." Antimicrobial Agents and Chemotherapy 57, no. 4 (February 4, 2013): 1850–56. http://dx.doi.org/10.1128/aac.02010-12.
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ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.
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Smith, M. Alex, and Michael J. Bidochka. "Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size." Canadian Journal of Microbiology 44, no. 4 (April 1, 1998): 351–55. http://dx.doi.org/10.1139/w98-020.
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Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective environments are important to the understanding of the evolution of the bacteria-plasmid associations and the appreciation of the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.Key words: pBluescript, plasmid, stress, fitness, starvation.
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Slavcev, Roderick A., and Barbara E. Funnell. "Identification and Characterization of a Novel Allele of Escherichia coli dnaB Helicase That Compromises the Stability of Plasmid P1." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1227–37. http://dx.doi.org/10.1128/jb.187.4.1227-1237.2005.
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ABSTRACT Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34°C. Shifting dnaB277 cells to 42°C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.
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Percival-Smith, A., and J. Segall. "Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 7 (July 1987): 2484–90. http://dx.doi.org/10.1128/mcb.7.7.2484.
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We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.
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Percival-Smith, A., and J. Segall. "Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 7 (July 1987): 2484–90. http://dx.doi.org/10.1128/mcb.7.7.2484-2490.1987.
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We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.
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Vila, Pau, Jose L. Corchero, Antoni Benito, and Antonio Villaverde. "Ammonium-Mediated Reduction of Plasmid Copy Number and Recombinant Gene Expression in Escherichia coli." Biotechnology Progress 10, no. 6 (November 1994): 648–51. http://dx.doi.org/10.1021/bp00030a010.
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Robertson, Gregory T., Ann Reisenauer, Rachel Wright, Rasmus B. Jensen, Allen Jensen, Lucille Shapiro, and R. Martin Roop. "The Brucella abortus CcrM DNA Methyltransferase Is Essential for Viability, and Its Overexpression Attenuates Intracellular Replication in Murine Macrophages." Journal of Bacteriology 182, no. 12 (June 15, 2000): 3482–89. http://dx.doi.org/10.1128/jb.182.12.3482-3489.2000.
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ABSTRACT The CcrM DNA methyltransferase of the α-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes inB. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carryccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrMcopy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.
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Kazic, T., and D. E. Berg. "Context effects in the formation of deletions in Escherichia coli." Genetics 126, no. 1 (September 1, 1990): 17–24. http://dx.doi.org/10.1093/genetics/126.1.17.
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Abstract We have examined the frequency with which identical deletions are formed in different chromosomal contexts. A panel of six mutant bla genes containing palindrome/direct repeat structures were moved from pBR322 to three locations: at lambda att, at chromosomal lac, and at F'lac. Deletion of the palindromes and one of the direct repeats results in reversion to Ampr. The frequency of deletion for all alleles declines beyond the reduction in copy number when they are moved from the multicopy plasmid environment to a single-copy chromosome. The magnitude of the declines varies in an allele-specific and location-specific manner. Our data support the hypothesis that context can influence the frequency of mutation independent of the immediate DNA sequence.
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McGrath, Stephen, Jos F. M. L. Seegers, Gerald F. Fitzgerald, and Douwe van Sinderen. "Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis." Applied and Environmental Microbiology 65, no. 5 (May 1, 1999): 1891–99. http://dx.doi.org/10.1128/aem.65.5.1891-1899.1999.
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ABSTRACT A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.
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Kwong, Stephen M., Ronald A. Skurray, and Neville Firth. "Replication Control of Staphylococcal Multiresistance Plasmid pSK41: an Antisense RNA Mediates Dual-Level Regulation of Rep Expression." Journal of Bacteriology 188, no. 12 (June 15, 2006): 4404–12. http://dx.doi.org/10.1128/jb.00030-06.
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ABSTRACT Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (P rnaI ) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when P rnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.
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Ederth, J., L. Isaksson, and F. Abdulkarim. "Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase." Molecular Genetics and Genomics 267, no. 5 (July 2002): 587–92. http://dx.doi.org/10.1007/s00438-002-0689-y.
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Smulczyk-Krawczyszyn, Aleksandra, Dagmara Jakimowicz, Beata Ruban-Ośmiałowska, Anna Zawilak-Pawlik, Jerzy Majka, Keith Chater, and Jolanta Zakrzewska-Czerwińska. "Cluster of DnaA Boxes Involved in Regulation of Streptomyces Chromosome Replication: from In Silico to In Vivo Studies." Journal of Bacteriology 188, no. 17 (September 1, 2006): 6184–94. http://dx.doi.org/10.1128/jb.00528-06.
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ABSTRACT In Streptomyces coelicolor, replication is initiated by the DnaA protein in the centrally located oriC region and proceeds bidirectionally until the replication forks reach the ends of the linear chromosome. We identified three clusters of DnaA boxes (H69, H24, and D78) which are in a relatively short segment of the chromosome centered on the oriC region. Of the clusters analyzed, D78 exhibited the highest affinity for the DnaA protein; the affinity of DnaA for the D78 cluster was about eightfold higher than the affinity for oriC. The high-affinity DnaA boxes appear to be involved in the control of chromosome replication. Deletion of D78 resulted in more frequent chromosome replication (an elevated ratio of origins to chromosome ends was observed) and activated aerial mycelium formation, leading to earlier colony maturation. In contrast, extra copies of D78 (delivered on a plasmid) caused slow colony growth, presumably because of a reduction in the frequency of initiation of chromosome replication. This suggests that the number of high-affinity DnaA boxes is relatively constant in hyphal compartments and that deletion of D78 therefore permits an increased copy number of either the chromosomal origin region or a plasmid harboring the D78 cluster. This system conceivably influences the timing of decisions to initiate aerial mycelial formation and sporulation.
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Grassman, Elke, Tomoyasu Higashimoto, Justin Rohrbach, Danielle Hall, David A. Williams, Lilith Reeves, and Punam Malik. "Pulmozyme® Treatment of Vectors Produced by Transient Transfection Reduces Residual Plasmid DNA on Human CD34+ Hematopoietic Progenitor Cells without Loss of Transduction or Engraftment Efficiency." Blood 112, no. 11 (November 16, 2008): 4626. http://dx.doi.org/10.1182/blood.v112.11.4626.4626.
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Abstract A significant percentage of clinical grade safety-enhanced gamma-retrovirus and lentivirus vector supernatants are currently produced by transient transfection. These products contain high concentrations of plasmid DNA that pose the risk of transfer into subjects, and preclude accurate estimation of transduction efficiency. Most envelope pseudotyped vectors are too labile for DNAse treatment or other purification steps and adding non-clinical grade reagents into any production step further complicates product qualification. Here we present development of a method to remove residual plasmid as a part of the transduction procedure of human CD34+ cells or mouse bone marrow progenitors, using the DNAse Pulmozyme®. Using a standard clinical transduction protocol, CD34+ were transduced with SRS11.EFS.IL2RG.pre* or SRS11.EFS.EGFP. pre* gamma-retroviral vectors pseudotyped with GALV envelope or a mock control. Retronectin coated flasks preloaded with vector were either treated with Pulmozyme® or no Pulmozyme®. Cells were transduced on two consecutive days and collected 4 –24 hrs after the 2nd transduction (day1). CFU were plated and enumerated and proportion of transduced progenitors quantified by real time PCR (qPCR) using wPRE primers. For the EGFP containing vector, gene transfer was determined by flow cytometry in cultures that were continued for up to 14 days. DNA was extracted and tested by qPCR on days 1, 7, and 14. Residual plasmid DNA was quantified using GALV primers. Transduction was also estimated on the bulk cultures using qPCR. In all qPCR reactions human ApoB was quantified concurrently to asses the cell number. Pulmozyme® treatment reduced GALV copy numbers in cells one day following transduction by up to 20-fold. After 7 days in culture, there was no residual plasmid detectable in all groups. More importantly, Pulmozyme® treatment did not alter the transduction efficiency of the progenitors in any of the experiments, as evaluated by either wPRE qPCR on Day-7 DNA (4.3 vs. 4.2, 6.8 vs. 7.6 and 2.0 vs. 1.9 vector copies per cell for Pulmozyme® treated and non-treated respectively), colony PCR (69.4 vs. 66.7% vector positive CFUs) or flow cytometry (81.8 vs. 85.7% EGFP positive cells). We also analyzed the engraftment potential of cells transduced using Pulmozyme®-treated versus non-treated vector after transplanting cells in NOD/SCID and NOD/SCID-IL2Rg null (NOG) mice. Analysis of mice at 6 weeks post transplantation showed no significant reduction in human cell engraftment in the Pulmozyme® and the average copy number per human cell in NOG bone marrow was 0.65 vs. 0.52 and in NOD/SCID bone marrow 1.03 vs. 0.67 (Pulmozyme® treated versus non-treated, respectively). We also tested Pulmozyme® treatment in mouse experiments using gamma-retroviral vector pseudotyped with ecotropoc envelope with a similar result. We conclude that treatment of gamma-retroviral vector supernatant with Pulmozyme® after vector preload on Retronectin® coated plates reduces vector and packaging plasmids and does not inhibit transduction, clonogenic potential, or engraftment. The removal of excess plasmid from transiently produced virus supernatant without affecting the virus transducibility allows accurate estimation of transduction efficiencies prior to transplant and will conceivably reduce the toxicity to primary cells from excess plasmid. These studies can be directly translated to preclinical animal safety studies and clinical trials.
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DeVito, Joseph A., and Sheldon Morris. "Exploring the Structure and Function of the Mycobacterial KatG Protein Using trans-Dominant Mutants." Antimicrobial Agents and Chemotherapy 47, no. 1 (January 2003): 188–95. http://dx.doi.org/10.1128/aac.47.1.188-195.2003.
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ABSTRACT In order to probe the structure and function of the mycobacterial catalase-peroxidase enzyme (KatG), we employed a genetic approach using dominant-negative analysis of katG merodiploids. Transformation of Mycobacterium bovis BCG with various katG point mutants (expressed from low-copy-number plasmids) resulted in reductions in peroxidase and catalase activities as measured in cell extracts. These reductions in enzymatic activity usually correlated with increased resistance to the antituberculosis drug isoniazid (INH). However, for the N138S trans-dominant mutant, the catalase-peroxidase activity was significantly decreased while the sensitivity to INH was retained. trans-dominance required katG expression from multicopy plasmids and could not be demonstrated with katG mutants integrated elsewhere on the wild-type M. bovis BCG chromosome. Reversal of the mutant phenotype through plasmid exchange suggested the catalase-peroxidase deficiency occurred at the protein level and that INH resistance was not due to a second site mutation(s). Electrophoretic analysis of KatG proteins from the trans-dominant mutants showed a reduction in KatG dimers compared to WT and formation of heterodimers with reduced activity. The mutants responsible for these defects cluster around proposed active site residues: N138S, T275P, S315T, and D381G. In an attempt to identify mutants that might delimit the region(s) of KatG involved in subunit interactions, C-terminal truncations were constructed (with and without the D381G dominant-negative mutation). None of the C-terminal deletions were able to complement a ΔkatG strain, nor could they cause a dominant-negative effect on the WT. Taken together, these results suggest an intricate association between the amino- and carboxy-terminal regions of KatG and may be consistent with a domain-swapping mechanism for KatG dimer formation.
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Bouchard, Julie D., Eric Dion, Frédéric Bissonnette, and Sylvain Moineau. "Characterization of the Two-Component Abortive Phage Infection Mechanism AbiT from Lactococcus lactis." Journal of Bacteriology 184, no. 22 (November 15, 2002): 6325–32. http://dx.doi.org/10.1128/jb.184.22.6325-6332.2002.
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ABSTRACT During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10−5 and 10−7) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.
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Hoekema, A., R. A. Kastelein, M. Vasser, and H. A. de Boer. "Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression." Molecular and Cellular Biology 7, no. 8 (August 1987): 2914–24. http://dx.doi.org/10.1128/mcb.7.8.2914.
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The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.
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Hoekema, A., R. A. Kastelein, M. Vasser, and H. A. de Boer. "Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression." Molecular and Cellular Biology 7, no. 8 (August 1987): 2914–24. http://dx.doi.org/10.1128/mcb.7.8.2914-2924.1987.
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The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.
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Shlapobersky, Mark, Joshua O. Marshak, Lichun Dong, Meei-li Huang, Qun Wei, Alice Chu, Alain Rolland, Sean Sullivan, and David M. Koelle. "Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection." Journal of General Virology 93, no. 6 (June 1, 2012): 1305–15. http://dx.doi.org/10.1099/vir.0.040055-0.
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The herpes simplex type 2 (HSV-2) envelope glycoprotein (gD2) was evaluated as a potential antigen candidate for a plasmid DNA (pDNA)-based HSV-2 vaccine. The pDNA was formulated with Vaxfectin, a cationic lipid-based adjuvant, and tested in a murine HSV-2 lethal challenge model. gD2 was expressed as full-length (FL) and secreted (S) gD2 forms. A 0.1 µg pDNA dose was tested to distinguish treatment conditions for survival and a 100 µg pDNA dose was tested to distinguish treatment conditions for reduction in vaginal and latent HSV-2 copies. Vaxfectin-formulated gD2 pDNA significantly increased serum IgG titres and survival for both FL gD2 and S gD2 compared with gD2 pDNA alone. Mice immunized with FL gD2 formulated with Vaxfectin showed reduction in vaginal and dorsal root ganglia (DRG) HSV-2 copies. The stringency of this protection was further evaluated by testing Vaxfectin-formulated FL gD2 pDNA at a high 500 LD50 inoculum. At this high viral challenge, the 0.1 µg dose of FL gD2 Vaxfectin-formulated pDNA yielded 80 % survival compared with no survival for FL gD2 pDNA alone. Vaxfectin-formulated FL gD2 pDNA, administered at a 100 µg pDNA dose, significantly reduced HSV-2 DNA copy number, compared with FL gD2 DNA alone. In addition, 40 % of mice vaccinated with adjuvanted FL pDNA had no detectable HSV-2 viral genomes in the DRG, whereas all mice vaccinated with gD2 pDNA alone were positive for HSV-2 viral genomes. These results show the potential contribution of Vaxfectin-gD2 pDNA to a future multivalent HSV-2 vaccine.
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Manabe, Kenji, Yasushi Kageyama, Takuya Morimoto, Tadahiro Ozawa, Kazuhisa Sawada, Keiji Endo, Masatoshi Tohata, Katsutoshi Ara, Katsuya Ozaki, and Naotake Ogasawara. "Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874." Applied and Environmental Microbiology 77, no. 23 (September 30, 2011): 8370–81. http://dx.doi.org/10.1128/aem.06136-11.
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ABSTRACTGenome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novelBacillus subtilisstrain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of therocDEF-rocRregion, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of therocDEF-rocRregion caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without therocDEF-rocRregion, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of therocDEF-rocRregion and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement ofB. subtilisreduced-genome strains as cell factories for the production of heterologous enzymes.
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Ruiz, Françoise, Laurence Vayssié, Catherine Klotz, Linda Sperling, and Luisa Madeddu. "Homology-dependent Gene Silencing inParamecium." Molecular Biology of the Cell 9, no. 4 (April 1998): 931–43. http://dx.doi.org/10.1091/mbc.9.4.931.
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Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for allParamecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant.
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Holmes, Victor F., Jianzhong He, Patrick K. H. Lee, and Lisa Alvarez-Cohen. "Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes." Applied and Environmental Microbiology 72, no. 9 (September 2006): 5877–83. http://dx.doi.org/10.1128/aem.00516-06.
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ABSTRACT While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.
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Fire, A., D. Albertson, S. W. Harrison, and D. G. Moerman. "Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle." Development 113, no. 2 (October 1, 1991): 503–14. http://dx.doi.org/10.1242/dev.113.2.503.
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We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than ‘sense’ RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.
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Ehrhardt, Anja, Hui Xu, and Mark A. Kay. "Episomal Persistence of Recombinant Adenoviral Vector Genomes during the Cell Cycle In Vivo." Journal of Virology 77, no. 13 (July 1, 2003): 7689–95. http://dx.doi.org/10.1128/jvi.77.13.7689-7695.2003.
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ABSTRACT Previously we showed that recombinant adenoviral helper-dependent (HD) vectors result in long-term transgene expression levels in vivo which slowly declined by 95% over a period of 1 year. In this study, we further establish that this was not predominantly immune mediated. To determine if cell turnover was responsible for the loss of transgene expression, we induced rapid hepatocyte cell cycling in mouse liver, by performing a surgical two-thirds partial hepatectomy. We observed a 55 and 65% reduction in transgene expression levels and a 50 and 71% loss of vector genomes for the HD vector and the first-generation adenoviral vector. In sharp contrast, in nonviral, episomal plasmid DNA-injected mice, transgene expression levels and DNA copy numbers decreased by 95 and 99%, respectively. These findings suggest that cell division alone was not the primary reason for the slow decrease in transgene expression levels and that recombinant adenoviral vectors have a more robust mechanism for maintaining persistence during cell cycling. Several potential mechanisms are proposed.
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Holmgren, Sigrid C., Nicole A. Patterson, Michelle A. Ozbun, and Paul F. Lambert. "The Minor Capsid Protein L2 Contributes to Two Steps in the Human Papillomavirus Type 31 Life Cycle." Journal of Virology 79, no. 7 (April 1, 2005): 3938–48. http://dx.doi.org/10.1128/jvi.79.7.3938-3948.2005.
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ABSTRACT Prior studies, which have relied upon the use of pseudovirions generated in heterologous cell types, have led to sometimes conflicting conclusions regarding the role of the minor capsid protein of papillomaviruses, L2, in the viral life cycle. In this study we carry out analyses with true virus particles assembled in the natural host cell to assess L2's role in the viral infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, which was either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes and to amplify its genome to high copy number, support late viral gene expression, and cause formation of virus particles in human keratinocytes that had been induced to undergo terminal differentiation. These results indicate that aspects of both the nonproductive and productive phases of the viral life cycle occur normally in the absence of functional L2. However, upon the analysis of the virus particles generated, we found an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions. Furthermore, there was an over-100-fold reduction in the infectivity of L2-deficient virus. Because the latter deficiency cannot be accounted for solely by the 10-fold decrease in encapsidation, we conclude that L2 contributes to at least two steps in the production of infectious virus.
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Galarion, Luiza H., Merianne Mohamad, Zeyad Alzeyadi, Christopher P. Randall, and Alex J. O’Neill. "A platform for detecting cross-resistance in antibacterial drug discovery." Journal of Antimicrobial Chemotherapy 76, no. 6 (March 23, 2021): 1467–71. http://dx.doi.org/10.1093/jac/dkab063.
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Abstract Background To address the growing antibiotic resistance problem, new antibacterial drugs must exert activity against pathogens resistant to agents already in use. With a view to providing a rapid means for deselecting antibacterial drug candidates that fail to meet this requirement, we report here the generation and application of a platform for detecting cross-resistance between established and novel antibacterial agents. Methods This first iteration of the cross-resistance platform (CRP) consists of 28 strains of defined resistance genotype, established in a uniform genetic background (the SH1000 strain of the clinically significant pathogen Staphylococcus aureus). Most CRP members were engineered through introduction of constitutively expressed resistance determinants on a low copy-number plasmid, with a smaller number selected as spontaneous resistant mutants. Results Members of the CRP collectively exhibit resistance to many of the major classes of antibacterial agent in use. We employed the CRP to test two antibiotics that have been proposed in the literature as potential drug candidates: γ-actinorhodin and batumin. No cross-resistance was detected for γ-actinorhodin, whilst a CRP member resistant to triclosan exhibited a 32-fold reduction in susceptibility to batumin. Thus, a resistance phenotype that already exists in clinical strains mediates profound resistance to batumin, implying that this compound is not a promising antibacterial drug candidate. Conclusions By detecting cross-resistance between established and novel antibacterial agents, the CRP offers the ability to deselect compounds whose activity is substantially impaired by existing resistance mechanisms. The CRP therefore represents a useful addition to the antibacterial drug discovery toolbox.
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Talluri, Srikanth, Mehmet Kemal Samur, Leutz Buon, Stekla A. Megan, Purushothama Nanjappa, Rao Prabhala, Masood A. Shammas, and Nikhil C. Munshi. "Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma." Blood 128, no. 22 (December 2, 2016): 803. http://dx.doi.org/10.1182/blood.v128.22.803.803.
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Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.
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Yamamoto, Shogo, Wataru Gunji, Hiroaki Suzuki, Hiroshi Toda, Masako Suda, Toru Jojima, Masayuki Inui, and Hideaki Yukawa. "Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions." Applied and Environmental Microbiology 78, no. 12 (April 13, 2012): 4447–57. http://dx.doi.org/10.1128/aem.07998-11.
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ABSTRACTWe previously reported thatCorynebacterium glutamicumstrain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encodinggapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements inC. glutamicumglucose metabolism under oxygen deprivation. In addition togapA, overexpressing pyruvate kinase-encodingpykand phosphofructokinase-encodingpfkenabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encodinggpiin strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed thatgapAoverexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.
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Buetti, E. "Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity." Molecular and Cellular Biology 14, no. 2 (February 1994): 1191–203. http://dx.doi.org/10.1128/mcb.14.2.1191.
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In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting.
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Buetti, E. "Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity." Molecular and Cellular Biology 14, no. 2 (February 1994): 1191–203. http://dx.doi.org/10.1128/mcb.14.2.1191-1203.1994.
Full textAbstract:
In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting.
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Hay, Iain D., Uwe Remminghorst, and Bernd H. A. Rehm. "MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa." Applied and Environmental Microbiology 75, no. 4 (December 16, 2008): 1110–20. http://dx.doi.org/10.1128/aem.02416-08.
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ABSTRACT Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic ΔmucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.
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Ash, Caroline. "Plasmid copy number promotes plague." Science 353, no. 6298 (July 28, 2016): 458.7–459. http://dx.doi.org/10.1126/science.353.6298.458-g.
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Toplin, Julie, Kristy Drafahl, Jonathan Eibl, Clark Fjeld, Thomas Yager, Staci McAdams, Kevin Hawkins, and Chad Galderisi. "Development and Validation Of Highly Sensitive Mrdx BCR-ABL Test For Monitoring Deep Molecular Response In Patients With Chronic Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 2617. http://dx.doi.org/10.1182/blood.v122.21.2617.2617.
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Abstract Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKI) has led to progressively lower levels of disease burden and higher rates of complete molecular responses. Very few assays with an analytical sensitivity of 4.5-logs (0.00316% BCR-ABL/ABL) have been validated, and assay standardization at these levels has been problematic (EUTOS, 2007). In order to have an International Scale (IS) standardized assay that can precisely and accurately detect 4.5 log reductions in BCR-ABL/ABL ratios, the test system design must be tightly controlled to ensure this critical performance metric can be reproducibly met. The MolecularMD MRDxTM BCR-ABL Test was developed and validated to be an accurate, reproducible, and highly sensitive IS-standardized solution to measure minimal residual disease (MRD) in CML patients using either PAXgene Blood RNA or EDTA blood collection tubes. The MRDx Test is a one-step quantitative real-time polymerase chain reaction (RT-qPCR) test that allows for quantitation of BCR-ABL e13/14a2 (b2/3a2) transcripts (covering ≥ 95% of CML patients) and ABL transcripts in RNA extracted from peripheral blood. To achieve a sensitivity of 4.5 logs, several one step enzyme systems were evaluated against RNA samples that spanned the potential dynamic range of the assay (MR1.0 to MR5.0: MR1.0=1 log molecular response (MR), or 10% IS; MR5.0=5 log MR, or 0.001% IS). The one step enzyme system with the best sensitivity and precision was chosen based on the performance of three distinct lots. In addition, in order to better control for the variation of the reverse transcription, in vitro transcribed RNA calibrators were developed to create a standard curve for copy number determination rather than the more commonly used plasmid calibrators that may not accurately reflect the same processing as the patient sample RNA. The limit of detection (LOD) for blood drawn into PAXgene Blood RNA tubes was validated by testing a dilution series created from a baseline CML patient blood sample diluted into non-diseased subject blood. Creating this type of dilution series allows for the determination of the LOD in the actual clinical sample matrix as compared using cell line dilution series that have much higher BCR-ABL and ABL copy numbers than routine patient specimens. Based on the analysis of each level of sample tested over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.7 (Mean=MR4.9, 95% CI=MR5.2 to MR4.7) using the more conservative upper bound of the 95% confidence interval. The precision was evaluated based on the standard deviation of the log10 BCR-ABL/ABL ratio and was found to be ≤ 0.20 SD at MR5.0 and above. The LOD for blood drawn into EDTA blood collection tubes was validated by creation of a dilution series from a baseline CML patient RNA sample diluted into RNA isolated from blood of non-diseased subjects. Based on the number of samples with detectable BCR-ABL in at least 95% of replicates over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.9 (Mean=MR5.0, 95% CI=MR5.1 to MR4.9) using the more conservative upper bound of the 95% confidence interval with precision being ≤ 0.22 SD at MR5.0 and above. The accuracy of the BCR-ABL copy numbers, ABL copy numbers, and BCR-ABL/ABL ratio using the MRDx BCR-ABL Test was evaluated for ten patient samples using one step droplet digital PCR (ddPCR) as a reference method. For the BCR-ABL copy numbers, ABL copy numbers, and the BCR-ABL/ABL ratio, the bias of the MRDx Test relative to ddPCR by Bland-Altman method was 0.053, 0.030, and 0.023 respectively. The MRDx BCR-ABL Test reports on the IS with a conversion factor of 1.0 by use of the WHO International Standard 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR; secondary standards were created for bi-annual monitoring of the assay conversion factor to ensure accurate reporting of patient results on the IS. Confirmation of the conversion factor was done by an independent laboratory using different real-time PCR instruments and multiple technicians. Based on the validation data, the MRDx BCR-ABL Test is an accurate, reproducible, and highly sensitive IS-standardized solution to the growing need for a reliable and robust quantitative BCR-ABL assay that can be used for the monitoring of minimal residual disease in CML patients. Disclosures: Toplin: MolecularMD: Employment. Drafahl:MolecularMD: Employment. Eibl:MolecularMD: Employment. Fjeld:MolecularMD: Employment. Yager:MolecularMD: Employment. McAdams:MolecularMD: Employment. Hawkins:MolecularMD: Employment. Galderisi:MolecularMD: Employment.
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Genther, Sybil M., Stephanie Sterling, Stefan Duensing, Karl Münger, Carol Sattler, and Paul F. Lambert. "Quantitative Role of the Human Papillomavirus Type 16 E5 Gene during the Productive Stage of the Viral Life Cycle." Journal of Virology 77, no. 5 (March 1, 2003): 2832–42. http://dx.doi.org/10.1128/jvi.77.5.2832-2842.2003.
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ABSTRACT Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.
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Schmidt, Torsten, Karl Friehs, and Erwin Flaschel. "Rapid determination of plasmid copy number." Journal of Biotechnology 49, no. 1-3 (August 1996): 219–29. http://dx.doi.org/10.1016/0168-1656(96)01519-2.
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Shammas, Masood A., Leutz Buon, Jianhong Lin, Mehmet K. Samur, Jaymin M. Patel, Ankit Vahia, Purushothama Nanjappa, et al. "Nuclease Activity Is Associated with Genomic Instability As Well As Survival in Myeloma; Underlying Mechanisms and Significance." Blood 126, no. 23 (December 3, 2015): 2420. http://dx.doi.org/10.1182/blood.v126.23.2420.2420.
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Abstract Genomic instability leads to acquisition of mutational changes which underlie development and progression of cancer, including development of drug resistance and poor clinical outcome. Understanding mechanisms of genomic instability is therefore necessary to develop promising strategies for prevention and treatment of disease. Homologous recombination (HR), the most precise DNA repair mechanism, has been previously described to be dysregulated in multiple myeloma (MM) mediating genomic instability. Since nuclease activity, by producing free ends of DNA, can induce DNA recombination leading to genomic rearrangements, we investigated prognostic significance of nuclease activity and nuclease gene expression in MM. We first developed a nuclease gene signature correlating with both the genomic instability and survival in myeloma patients. We used two different myeloma patient datasets (gse26863, n=246 and IFM 170 patient dataset) which had both the gene expression and SNP/CGH array-based copy number information for each patient. Genomic instability in each patient was determined by counting the total number of amplification and/or deletion events; an event was defined as a change in ≥3 and/or 5 consecutive SNPs/probes. We identified 34 nucleases whose elevated expression correlated with increased genomic instability in gse26863 dataset. Of these, the elevated expression of 21 nucleases also correlated with increased genomic instability in 170 dataset. Elevated expression of seven of these genes also correlated with poor overall survival (p=0.00005) as well as event free survival (P=0.0003) in myeloma patients (n=170). We further tested one of these nucleases (APEX2) in both the loss and gain of function studies and found that its suppression significantly reduces DNA breaks and dysregulated HR, an important activity underlying ongoing genomic rearrangements and instability in myeloma. Upregulation of APEX2 was associated with excessive DNA breaks, dysregulation of HR, acquisition of new genomic changes over time in myeloma cells. We also investigated the prognostic significance of nucleolytic activity in cell lines and patient samples using a plasmid degradation assay in which supercoiled DNA is converted to open circular and linear forms by the MM cell lystae prepared from purified CD138+ patient MM cells or MM cell lines. The ratio of supercoiled to total DNA per lane was graphed across successive time points (0, 3, 6, 12, 24 minutes) and analyzed via nonlinear regression using PRISM (statistical software) to calculate a half-life and k-constant. The longer half life suggests lower nuclease activity in MM cells. This assay was able to differentiate MGUS and smoldering myeloma (SMM) patients with long half-life of plasmid (9499 minutes) versus newly diagnosed MM (9 minutes) in which there was variability with some patients with plasmid degradation pattern closer to SMM versus some with significantly higher activity. A large number of (N = 410) clinically annotated sample patients are currently being evaluted for both functional and clinical correlation of nuclease activity. In summary, we show correlation between nucleases activity and genomic instability with impact on survival in MM. The genes in this signature not only provide novel markers to predict clinical outcome but also potential targets for prevention/reduction of genomic evolution. Investigation of the role of each of the seven genes, separately and in combination, in the overall nucleolytic activity, genomic instability, and pathways involved in the regulation of cell cycle, DNA repair/maintenance checkpoints, apoptosis and survival is currently ongoing. Disclosures Avet-Loiseau: jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.
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Mahon, Francois-Xavier, Stéphanie Dulucq, Francois Guilhot, Laurence Legros, Philippe Rousselot, Coralie Belanger, Gabriel Etienne, Josy Reiffers, and Delphine Rea. "A More Sensitive RQ-PCR to Assess Complete Molecular Remission Does Not Allow the Prediction of Relapse After Discontinuation of Imatinib In Chronic Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 2298. http://dx.doi.org/10.1182/blood.v116.21.2298.2298.
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Abstract Abstract 2298 We previously reported that imatinib treatment could be safely discontinued in chronic myeloid leukemia (CML) patients who had achieved a sustained complete molecular remission (CMR) lasting for at least two years in succession. However, in the multicenter French STIM study, 60 % of patients loose CMR mainly within the first 6 months after discontinuation and the probability to remain in CMR 2 years after imatinib discontinuation is around 40 %. The molecular relapse originates in persisting leukemic cells that are undetectable by current RQ-PCR techniques. The sensitivity of these routinely used techniques is below a detection threshold corresponding to a 5-log reduction in the leukemic burden. We thus hypothesized that improving the sensitivity of leukemic cell detection would be of valuable help to better select those patients who can possibly be cured after imatinib treatment. In the present work, we have increased the sensitivity of the current RQ-PCR, based on repeated PCR and on an increased number of normal ABL copies that were analyzed, in order to augment the probability to amplify BCR-ABL. For each sample, 5 microg of RNA were used to synthesize 5 cDNA and for each cDNA, 2 PCR for BCR-ABL and one for ABL were carried out, i.e 10 BCR-ABL PCR points were performed per sample. For each microg of RNA the number of ABL copies was over 20 000, so the number of ABL copies analyzed was over 100 000. The detection threshold of BCR-ABL was calculated and considered at 40 cycles of PCR (theshold Ct). The control plasmid (pME-2) (Kindly given by Martin Müller and Andreas Hochhaus from European LeukemiaNet) used as standard curve dilution was also included to test the sensitivity and for instance 4 copies were detected in 19/20 cases, 2 copies 7/10 cases,1 copy in 2/10, 0.4 copy in 1/10. Thirty one samples from healthy donors or non CML patients (BCR-ABL negative) served as controls. Among the 310 (10 × 21) PCR for BCR-ABL we found only one positive well. 65 patients enrolled in the STIM study were analyzed at the time of imatinib discontinuation, using this new RQPCR technique. In the STIM study relapse was defined as the positivity of Bcr-Abl transcripts using classical QRT-PCR confirmed by a second analysis point indicating the increase in relation to the first analysis point performed on 2 successive assessments. Among 650 PCR for BCR-ABL, 46 wells were found positive. 9 patients were found at least 3 /10 positive PCR and among them 6 relapsed. 22 patients were found only at least 1 /10 positive and among them 15 relapsed. In 43 patients, BCR-ABL was never detected using the more sensitive RQ-PCR technique and 22 of them relapsed. To conclude, the use of a new RQPCR technique with a sensitivity of detection of BCR-ABL close to 6-log does not allow the prediction of molecular relapse following imatinib discontinuation in patients in CMR. Our results are in agreement with what was previously reported using PCR on genomic DNA. The persistence of leukemic cells in CMR patients does not automatically lead to CML relapse. Disclosures: Belanger: Novartis Pharma: Employment.
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Xia, G. X., D. Manen, T. Goebel, P. Linder, G. Churchward, and L. Caro. "A copy-number mutant of plasmid pSC101." Molecular Microbiology 5, no. 3 (March 1991): 631–40. http://dx.doi.org/10.1111/j.1365-2958.1991.tb00734.x.
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Trivedi, Ram Narayan, Parvez Akhtar, Jonathan Meade, Patrick Bartlow, Mohammad M. Ataai, Saleem A. Khan, and Michael M. Domach. "High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacBby IntroducingincMutations." Applied and Environmental Microbiology 80, no. 23 (September 12, 2014): 7154–60. http://dx.doi.org/10.1128/aem.02445-14.
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ABSTRACTFor small-copy-number pUC-type plasmids, theinc1andinc2mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due toincmutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of theincmutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C,inc2mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use ofincmutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.
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Mann, Brandon A., and James M. Slauch. "Transduction of Low-Copy Number Plasmids by Bacteriophage P22." Genetics 146, no. 2 (June 1, 1997): 447–56. http://dx.doi.org/10.1093/genetics/146.2.447.
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The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers. Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22. This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSC101 derivatives. We show that P22HT transduces pSC101 derivatives that share homology with the chromosome by two distinct mechanisms. In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination. This chromosomal fragment is then packaged in the transducing particle. The second mechanism is a size-dependent mechanism involving a putative plasmid multimer. We propose that this multimer is formed by interplasmidic recombination. In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome. It has been proposed that the phage packages a linear concatemer created during rolling circle replication of pBR322, similar in fashion to phage genome packaging. This study investigates the role of RecA, RecD, and RecF recombination proteins in plasmid/plasmid and plasmid/chromosome interactions that form packageable substrates in the donor. We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes.
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Fong, Ryan, Zhihao Hu, C. Richard Hutchinson, Jianqiang Huang, Stanley Cohen, and Camilla Kao. "Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction." Applied and Environmental Microbiology 73, no. 4 (December 1, 2006): 1296–307. http://dx.doi.org/10.1128/aem.01888-06.
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ABSTRACT A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.
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Sano, Emiko, Sophie Maisnier-Patin, John Paul Aboubechara, Semarhy Quiñones-Soto, and John R. Roth. "Plasmid Copy Number Underlies Adaptive Mutability in Bacteria." Genetics 198, no. 3 (August 29, 2014): 919–33. http://dx.doi.org/10.1534/genetics.114.170068.
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Allen, Steven P., and Hans P. Blaschek. "Plasmid copy number and stability determination inClostridium perfringenstransformants." FEMS Microbiology Letters 72, no. 3 (November 1990): 323–27. http://dx.doi.org/10.1111/j.1574-6968.1990.tb03910.x.
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Ehrenberg, M. "Hypothesis: hypersensitive plasmid copy number control for ColE1." Biophysical Journal 70, no. 1 (January 1996): 135–45. http://dx.doi.org/10.1016/s0006-3495(96)79555-3.
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Dwidar, Mohammed, and Yohei Yokobayashi. "Riboswitch Signal Amplification by Controlling Plasmid Copy Number." ACS Synthetic Biology 8, no. 2 (January 25, 2019): 245–50. http://dx.doi.org/10.1021/acssynbio.8b00454.
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Del Solar, Gloria, and Manuel Espinosa. "Plasmid copy number control: an ever-growing story." Molecular Microbiology 37, no. 3 (January 18, 2002): 492–500. http://dx.doi.org/10.1046/j.1365-2958.2000.02005.x.
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Wadood, Abdul, Mitsuko Dohmoto, Shigeki Sugiura, and Kazuo Yamaguchi. "Characterization of copy number mutants of plasmid pSC101." Journal of General and Applied Microbiology 43, no. 6 (1997): 309–16. http://dx.doi.org/10.2323/jgam.43.309.
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Brendel, Volker, and Alan S. Perelson. "Quantitative Model of ColE1 Plasmid Copy Number Control." Journal of Molecular Biology 229, no. 4 (February 1993): 860–72. http://dx.doi.org/10.1006/jmbi.1993.1092.
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Watve, Mukta M., Neelesh Dahanukar, and Milind G. Watve. "Sociobiological Control of Plasmid Copy Number in Bacteria." PLoS ONE 5, no. 2 (February 24, 2010): e9328. http://dx.doi.org/10.1371/journal.pone.0009328.
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Cesareni, G., and D. W. Banner. "Regulation of plasmid copy number by complementary RNAs." Trends in Biochemical Sciences 10, no. 8 (August 1985): 303–6. http://dx.doi.org/10.1016/0968-0004(85)90168-9.
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Suarez, Jorge, Yong Hu, Ayako Makino, Eduardo Fricovsky, Hong Wang, and Wolfgang H. Dillmann. "Alterations in mitochondrial function and cytosolic calcium induced by hyperglycemia are restored by mitochondrial transcription factor A in cardiomyocytes." American Journal of Physiology-Cell Physiology 295, no. 6 (December 2008): C1561—C1568. http://dx.doi.org/10.1152/ajpcell.00076.2008.
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Mitochondrial transcription factor A (TFAM) is essential for mitochondrial DNA transcription and replication. TFAM transcriptional activity is decreased in diabetic cardiomyopathy; however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM overexpression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30 mM) for 48 h, and we examined whether TFAM overexpression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations toward normal. Our results shown that TFAM overexpression increased to more than twofold mitochondria copy number in cells treated either with normal (5.5 mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM overexpression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose, which was associated with diminished sarco(endo)plasmic reticulum Ca2+-ATPase 2a and cytochrome- c oxidase subunit 1 expression. These parameters were returned to control values after TFAM overexpression. High glucose-induced protein oxidation was reduced by TFAM overexpression, indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked β- N-acetylglucosamine glycosylation. In conclusion, TFAM overexpression protected cell function against the damage induced by high glucose in cardiomyocytes.