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Sample records for plasmid replication origin

  1. The replication origin of a repABC plasmid

    Directory of Open Access Journals (Sweden)

    Cevallos Miguel A

    2011-06-01

    Full Text Available Abstract Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The ori

  2. Streptomyces linear plasmids that contain a phage-like, centrally located, replication origin.

    Science.gov (United States)

    Chang, P C; Kim, E S; Cohen, S N

    1996-12-01

    Unlike previously studied linear replicons containing 5' DNA termini covalently bound to protein, pSLA2, a 17 kb linear plasmid of Streptomyces rochei, initiates replication internally rather than at the telomeres (Chang and Cohen, 1994). Here we identify and characterize the replication origin of pSLA2, showing that it contains a series of direct repeats (iterons) within a centrally located gene encoding an essential DNA-binding protein (Rep1); a second essential protein (Rep2), which resembles prokaryotic DNA helicases and has ATPase activity stimulated by single-stranded DNA, is expressed from the same transcript. A 430 bp locus separated by almost 2 kb from the iterons of the origin specifies an as yet undefined additional function required in cis for plasmid replication. pSCL, a 12 kb linear plasmid of Streptomyces clavuligerus, contains, near the centre of the plasmid, a region configured like the pSLA2 origin. The replication regions of pSLA2 and pSCL, which are capable of propagating plasmid DNA in either a circular or linear form (Shiffman and Cohen, 1992; Chang and Cohen, 1994) resemble those of temperate bacteriophages of the Enterobacteriacae and Bacillus. Our observations suggest that Streptomyces linear plasmids may occupy an evolutionarily intermediate position between circular plasmids and linear phage replicons.

  3. Molecular analysis of the replication origin of the Lactococcus lactis plasmid pCI305

    NARCIS (Netherlands)

    Foley, S; Bron, S; Venema, G; Daly, C; Fitzgerald, GF

    1996-01-01

    The replication origin region, ori, of the Lactococcus lactis subsp. lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (Rep

  4. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  5. Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1.

    Science.gov (United States)

    Berkner, Silvia; Hinojosa, Mery Pina; Prangishvili, David; Lipps, Georg

    2014-10-01

    We have determined the minimal replicon of the crenarchaeal plasmid pRN1. It consists of 3097 base pairs amounting to 58% of the genome of pRN1. The minimal replicon comprises replication operon orf56/orf904 coding for a transcriptional repressor and the replication protein of pRN1. An upstream region of 64 bp that contains the promoter of the replication operon is essential as well as 166 bp of sequence downstream of the orf904 gene. This region contains a putative transcriptional terminator and a 100 nucleotides long stem-loop structure. Only the latter structure was shown to be required for replication. In addition replication was sustained when the stem-loop was displaced to another part of the pRN1 sequence. By mutational analysis we also find that the integrity of the stem-loop structure is required to maintain the replication of pRN1-derived constructs. As similar stem-loop structures are also present in other members of the pRN family, we suggest that this conserved structural element could be the origin of replication for the pRN plasmids. Further bioinformatic analysis revealed that the domain structure of the replication protein and the presence of a similar stem-loop structure as the putative replication origin are also found in several bacteriophages.

  6. Replication of plasmids in gram-negative bacteria.

    OpenAIRE

    1989-01-01

    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  7. In vitro replication of plasmids containing human ribosomal gene sequences: origin localization and dependence on an aprotinin-binding cytosolic protein.

    Science.gov (United States)

    Coffman, F D; Georgoff, I; Fresa, K L; Sylvester, J; Gonzalez, I; Cohen, S

    1993-11-01

    We previously investigated the role of an aprotinin-binding protein (ADR) in the initiation of DNA replication in isolated quiescent nuclei. In the present study, we have used a cell-free DNA replication system to test the ability of plasmid vectors which contain sequences from the human ribosomal RNA gene to serve as replicative templates in vitro when exposed to ADR-containing preparations. Significant dTTP incorporation was seen using DNA from either a 7-kb sequence in the 5' spacer region (CHE) or a 7-kb sequence which begins near the end of the 28S coding region and extends into the 3' spacer region (ADBB), while sequences from other regions of the rRNA gene mediated little or no dTTP incorporation. The characteristics of plasmid-directed dTTP incorporation indicate that most incorporation is due to DNA replication and not repair or damage-initiated processes. To conclusively demonstrate origin-dependent replication in the plasmid system and to further map replication origins, an approach was developed using ddGTP to restrict the length of daughter strands followed by hybridization of these replication products to restriction fragments spanning the putative origin region. This approach allowed us to identify replication origin activity apart from parent strand repair or synthesis initiated at random damaged sites. One of the origins was localized to a 1375-bp fragment within the 5' spacer region, and this fragment contains sequences homologous to those found in other replication origins.

  8. Rational design of a plasmid origin that replicates efficiently in both gram-positive and gram-negative bacteria.

    Directory of Open Access Journals (Sweden)

    Anton V Bryksin

    Full Text Available BACKGROUND: Most plasmids replicate only within a particular genus or family. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an engineered high copy number expression vector, pBAV1K-T5, that produces varying quantities of active reporter proteins in Escherichia coli, Acinetobacter baylyi ADP1, Agrobacterium tumefaciens, (all gram-negative, Streptococcus pneumoniae, Leifsonia shinshuensis, Peanibacillus sp. S18-36 and Bacillus subtilis (gram-positive. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the efficiency of pBAV1K-T5 replication in different bacterial species, thereby facilitating the study of proteins that don't fold well in E. coli and pathogens not amenable to existing genetic tools.

  9. Functional amyloids as inhibitors of plasmid DNA replication

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    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-del Álamo, María; Fernández-Tresguerres, M. Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  10. Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids.

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    Gravesen, A; von Wright, A; Josephsen, J; Vogensen, F K

    1997-01-01

    Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids. Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094. The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced. Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences. Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA. In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA. A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication. This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

  11. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  12. DNA replication origins in archaea

    OpenAIRE

    Zhenfang eWu; Jingfang eLiu; Haibo eYang; Hua eXiang

    2014-01-01

    DNA replication initiation, which starts at specific chromosomal site (known as replication origins), is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s) to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB) that are located adjacent to ...

  13. Chromosomal context and replication properties of ARS plasmids in Schizosaccharomyces pombe

    Indian Academy of Sciences (India)

    Aditya S Pratihar; Vishnu P Tripathi; Mukesh P Yadav; Dharani D Dubey

    2015-12-01

    Short, specific DNA sequences called as Autonomously Replicating Sequence (ARS) elements function as plasmid as well as chromosomal replication origins in yeasts. As compared to ARSs, different chromosomal origins vary greatly in their efficiency and timing of replication probably due to their wider chromosomal context. The two Schizosaccharomyces pombe ARS elements, ars727 and ars2OO4, represent two extremities in their chromosomal origin activity - ars727 is inactive and late replicating, while ars2OO4 is a highly active, early-firing origin. To determine the effect of chromosomal context on the activity of these ARS elements, we have cloned them with their extended chromosomal context as well as in the context of each other in both orientations and analysed their replication efficiency by ARS and plasmid stability assays. We found that these ARS elements retain their origin activity in their extended/altered context. However, deletion of a 133-bp region of the previously reported ars727-associated late replication enforcing element (LRE) caused advancement in replication timing of the resulting plasmid. These results confirm the role of LRE in directing plasmid replication timing and suggest that the plasmid origin efficiency of ars2OO4 or ars727 remains unaltered by the extended chromosomal context.

  14. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  15. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

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    Yang, X; Daniell, H; McFadden, B

    1994-09-01

    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

  16. Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

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    Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

    1984-03-01

    A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed.

  17. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  18. Origin and Evolution of Rickettsial Plasmids.

    Science.gov (United States)

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as

  19. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

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    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  20. Transcription-replication collision increases recombination efficiency between plasmids.

    Science.gov (United States)

    Jialiang, Li; Feng, Chen; Zhen, Xu; Jibing, Chen; Xiang, Lv; Lingling, Zhang; Depei, Liu

    2013-11-01

    It has been proposed that the stalling of the replication forks can induce homologous recombination in several organisms, and that arrested replication forks may offer nuclease targets, thereby providing a substrate for proteins involved in double-strand repair. In this article, we constructed a plasmid with the potential for transcription-replication collision (TRC), in which DNA replication and RNA transcription occur on the same DNA template simultaneously. Theoretically, transcription will impede DNA replication and increase homologous recombination. To validate this hypothesis, another plasmid was constructed that contained a homologous sequence with the exception of some mutated sites. Co-transfection of these two plasmids into 293T cells resulted in increased recombination frequency. The ratio of these two plasmids also affected the recombination frequency. Moreover, we found high expression levels of RAD51, which indicated that the increase in the recombination rate was probably via the homologous recombination pathway. These results indicate that mutant genes in plasmids can be repaired by TRC-induced recombination.

  1. Characterization of the replicon from the lactococcal theta-replicating plasmid pJW563.

    Science.gov (United States)

    Gravesen, A; Josephsen, J; von Wright, A; Vogensen, F K

    1995-09-01

    The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kb EcoRI fragment. This DNA fragment was sequenced ans a 1155-bp open reading frame, repB563, encoding a putative protein RepB563 of 385 amino acids was found. An AT-rich noncoding region, repA563, was found upstream of repB563. This segment included several direct and inverted repeats. A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translationally coupled to repB563, RepB563 supplied in trans could support replication of a plasmid containing repA563 and a truncated repB563. This observation suggests that RepB563 is a trans-acting replication protein, and repA563 the cis-acting origin of replication, repA563, repB563, and the beginning of ORF X showed high homology to similar regions in a family of lactococcal theta-replicating plasmids. The repA DNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated. The comparison revealed highly conserved areas among this family of plasmids. In addition, variable domains emerged, presumably having a plasmid specific function, pVS40 and pC1305 were plasmids with replication proteins showing high homology to RepB563. Despite this homology, replication from repA563 could not be supported by the pVS40 or pC1305 replication protein supplied in trans. Likewise the pJW563 protein could not support replication from the pVS40 origin. pJW563 was found to be compatible with the pVS40 and pC1305 replicons. The results indicate that pJW563 belongs to the widespread family of lactococcal theta-replicating pladmids. Despite the high homology between their replicons, the interaction between the replication origin and the protein is highly specific in many cases rendering the plasmids compatible.

  2. Integration host factor is required for replication of pYGK-derived plasmids in Aggregatibacter actinomycetemcomitans.

    Science.gov (United States)

    Torres-Escobar, Ascención; Juárez-Rodríguez, María D; Demuth, Donald R

    2014-08-01

    In this study, we show that integration host factor protein (IHF) is required for replication of pYGK plasmids in Aggregatibacter actinomycetemcomitans. YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α- or β- subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα-IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF-dependent in the family Pasteurellaceae. In addition, using site-direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

  3. Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells.

    Science.gov (United States)

    Faurez, Florence; Dory, Daniel; Henry, Aurélie; Bougeard, Stéphanie; Jestin, André

    2010-04-01

    In this study, a method was developed to measure replication rates of rolling-circle replicon-based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 Rep gene. The results show that the replication rate depends on the length of the PCV2 replicon-based plasmid and not on the respective position of the Rep promoter and the promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a replication booster. This method can evaluate the replication potential of replicon-based plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine purposes.

  4. Using autonomous replication to physically and genetically define human origins of replication

    Energy Technology Data Exchange (ETDEWEB)

    Krysan, P.J.

    1993-01-01

    The author previously developed a system for studying autonomous replication in human cells involving the use of sequences from the Epstein-Barr virus (EBV) genome to provide extrachromosomal plasmids with a nuclear retention function. Using this system, it was demonstrated that large fragments of human genomic DNA could be isolated which replicate autonomously in human cells. In this study the DNA sequences which function as origins of replication in human cells are defined physically and genetically. These experiments demonstrated that replication initiates at multiple locations distributed throughout the plasmid. Another line of experiments addressed the DNA sequence requirements for autonomous replication in human cells. These experiments demonstrated that human DNA fragments have a higher replication activity than bacterial fragments do. It was also found, however, that the bacterial DNA sequence could support efficient replication if enough copies of it were present on the plasmid. These findings suggested that autonomous replication in human cells does not depend on extensive, specific DNA sequences. The autonomous replication system which the author has employed for these experiments utilizes a cis-acting sequence from the EBV origin and the trans-acting EBNA-1 protein to provide plasmids with a nuclear retention function. It was therefore relevant to verify that the autonomous replication of human DNA fragments did not depend on the replication activity associated with the EBV sequences utilized for nuclear retention. To accomplish this goal, the author demonstrated that plasmids carrying the EBV sequences and large fragments of human DNA could support long-term autonomous replication in hamster cells, which are not permissive for EBV replication.

  5. A New Shuttle Plasmid That Stably Replicates in Clostridium acetobutylicum.

    Science.gov (United States)

    Lee, Sang-Hyun; Kwon, Min-A; Choi, Sunwha; Kim, Sooah; Kim, Jungyeon; Shin, Yong-An; Kim, Kyoung Heon

    2015-10-01

    We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

  6. Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

    OpenAIRE

    Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

    1984-01-01

    A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D11...

  7. CHARACTERIZATION OF SINGLE-STRAND ORIGINS OF CRYPTIC ROLLING-CIRCLE PLASMIDS FROM BACILLUS-SUBTILIS

    NARCIS (Netherlands)

    MEIJER, WJJ; VENEMA, G; BRON, S

    1995-01-01

    In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication, The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060, The SSO

  8. Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

    Science.gov (United States)

    Tresguerres, E F; Nieto, C; Casquero, I; Cánovas, J L

    1986-01-01

    Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell. PMID:3511032

  9. Isolation of a minireplicon of the plasmid pG6303 of Lactobacillus plantarum G63 and characterization of the plasmid-encoded Rep replication protein

    Indian Academy of Sciences (India)

    Jing Fan; Xuedong Xi; Yan Huang; Zhongli Cui

    2015-06-01

    A cryptic 10.0-kb plasmid pG6303 from a multiplasmid-containing Lactobacillus plantarum G63 was studied. The analysis of replicon was facilitated by the construction of shuttle vectors and electrotransformation into L. plantarum. The pG6303 replicon included (i) an open reading frame encoding the putative Rep replication initiation protein; and (ii) the putative origin of replication. The Rep protein was expressed as a fusion with the hexa-histidine (His) at its C-terminal end and purified by Ni-affinity chromatography. The electrophoretic mobility shift assays in pG6303 showed that the purified Rep protein specifically bound from 5582 to 5945 bp, differing from the putative origin of replication of pG6303. We speculate that pG6303 replication is a new mode of plasmid replication.

  10. DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast

    OpenAIRE

    Wang, Yangzhou; Vujcic, Marija; Kowalski, David

    2001-01-01

    Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex ...

  11. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.

    OpenAIRE

    Pal, S. K.; Chattoraj, D K

    1988-01-01

    The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a ...

  12. Replication Origin Specification Gets a Push.

    Science.gov (United States)

    Plosky, Brian S

    2015-12-03

    During the gap between G1 and S phases when replication origins are licensed and fired, it is possible that DNA translocases could disrupt pre-replicative complexes (pre-RCs). In this issue of Molecular Cell, Gros et al. (2015) find that pre-RCs can be pushed along DNA and retain the ability to support replication.

  13. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages.

    Science.gov (United States)

    Willingham-Lane, Jennifer M; Berghaus, Londa J; Giguère, Steeve; Hondalus, Mary K

    2016-01-01

    The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

  14. Replication intermediate analysis confirms that chromosomal replication origin initiates from an unusual intergenic region in Caulobacter crescentus.

    Science.gov (United States)

    Brassinga, A K; Marczynski, G T

    2001-11-01

    The alpha-proteobacterium Caulobacter crescentus possesses a developmental cell cycle that restricts chromosome replication to a stalked cell type. The proposed C.crescentus chromosome replication origin (Cori) lies between hemE and RP001, an unusual intergenic region not previously associated with bacterial replication origins, although a similar genomic arrangement is also present at the putative replication origin in the related bacterium Rickettsia prowazekii. The cloned Cori supports autonomous plasmid replication selectively in the stalked cell type implying that replication of the entire chromosome also initiates between hemE and RP001. To confirm this location, we applied the 2-D (N/N) agarose gel electrophoresis technique to resolve and identify chromosome replication intermediates throughout a 30 kb region spanning Cori. Replication initiation in Cori was uniquely characterized by an 'origin bubble and Y-arc' pattern and this observation was supported by simple replication fork 'Y-arc' patterns that characterized the regions flanking Cori. These replication forks originated bi-directionally from within Cori as determined by the fork direction assay. Therefore, chromosomal replication initiates from the unusual hemE/RP001 intergenic region that we propose represents a new class of replication origins.

  15. Cloning of an origin of DNA replication of Xenopus laevis

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, S.; Taylor, J.H.

    1980-09-01

    DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the (/sup 3/H)-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated (/sup 3/H)thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment.

  16. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    Science.gov (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

  17. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter

    1992-01-01

    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  18. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    Science.gov (United States)

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  19. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

    Science.gov (United States)

    Giraldo-Suárez, R; Fernández-Tresguerres, E; Díaz-Orejas, R; Malki, A; Kohiyama, M

    1993-01-01

    Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains. Images PMID:8265367

  20. Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

    Science.gov (United States)

    Ruiz-Masó, José A.; Bordanaba-Ruiseco, Lorena; Sanz, Marta; Menéndez, Margarita; del Solar, Gloria

    2016-01-01

    Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site

  1. Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

    Science.gov (United States)

    Olsson, Jan A; Berg, Otto; Nordström, Kurt; Dasgupta, Santanu

    2012-03-01

    The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

  2. Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

    Directory of Open Access Journals (Sweden)

    Juan López-Villarejo

    2015-02-01

    Full Text Available kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

  3. In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501.

    Science.gov (United States)

    Brantl, S; Nuez, B; Behnke, D

    1992-07-01

    Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of pIP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, pI, pII and pIII within this region. A putative fourth promoter (pIV) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter pI while the repR gene is transcribed from promoter pII located just downstream of copR. The pII transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter pIII. The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR expression is proposed. Comparative analysis of the replication regions of pAM beta 1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.

  4. Regulation of replication of lambda phage and lambda plasmid DNAs at low temperature.

    Science.gov (United States)

    Gabig, M; Obuchowski, M; Srutkowska, S; Wegrzyn, G

    1998-06-01

    It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.

  5. Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

    Science.gov (United States)

    Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

    2015-09-01

    Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

  6. Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4.

    Science.gov (United States)

    Tocchetti, A; Serina, S; Oliva, I; Dehò, G; Ghisotti, D

    2001-01-15

    DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein alpha. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in alpha (alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on lambda immunity in E.coli for in vivo detection of protein-protein interactions, we found that (i) alpha protein interacts with Cnr, whereas alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr) interactions occur and the interaction domain is located within the C-terminal of alpha; (iii) Cnr-Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both alpha-alpha and alpha(cr)-alpha(cr) dimerization. Our data suggest that Cnr and alpha interact in at least two ways, which may have different functional roles in P4 replication control.

  7. The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators.

    Science.gov (United States)

    Orlova, Natalia; Gerding, Matthew; Ivashkiv, Olha; Olinares, Paul Dominic B; Chait, Brian T; Waldor, Matthew K; Jeruzalmi, David

    2016-12-27

    The conserved DnaA-oriC system is used to initiate replication of primary chromosomes throughout the bacterial kingdom; however, bacteria with multipartite genomes evolved distinct systems to initiate replication of secondary chromosomes. In the cholera pathogen, Vibrio cholerae, and in related species, secondary chromosome replication requires the RctB initiator protein. Here, we show that RctB consists of four domains. The structure of its central two domains resembles that of several plasmid replication initiators. RctB contains at least three DNA binding winged-helix-turn-helix motifs, and mutations within any of these severely compromise biological activity. In the structure, RctB adopts a head-to-head dimeric configuration that likely reflects the arrangement in solution. Therefore, major structural reorganization likely accompanies complex formation on the head-to-tail array of binding sites in oriCII Our findings support the hypothesis that the second Vibrionaceae chromosome arose from an ancestral plasmid, and that RctB may have evolved additional regulatory features.

  8. Characterization of the replication region of the Bacillus subtilis plasmid pLS20 : a novel type of replicon

    NARCIS (Netherlands)

    Meijer, WJJ; De Boer, AJ; van Tongeren, S; Venema, G; Bron, S

    1995-01-01

    A 3.1 kb fragment of the large (~55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficie

  9. DNA replication origin activation in space and time.

    Science.gov (United States)

    Fragkos, Michalis; Ganier, Olivier; Coulombe, Philippe; Méchali, Marcel

    2015-06-01

    DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

  10. Cis-acting elements in the lytic origin of DNA replication of Marek's disease virus type 1.

    Science.gov (United States)

    Katsumata, A; Iwata, A; Ueda, S

    1998-12-01

    The replication origin of Marek's disease virus (MDV) type 1 was analysed by using a transient replication assay with plasmids containing various fragments of MDV strain Md5 genomic DNA. Plasmid pMBH, containing the BamHI-H fragment, showed replication activity in MDV-infected chicken embryonic fibroblasts (CEF). By deletion analysis of pMBH, two regions, the promoter-enhancer region of the MDV pp38 gene and the 132 bp tandem direct repeat, were shown to be required for replication activity. Replication of pMBH was not observed in uninfected CEF, suggesting that a trans-acting factor(s) encoded by the MDV genome was necessary for replication.

  11. Decreased replication origin activity in temporal transition regions.

    Science.gov (United States)

    Guan, Zeqiang; Hughes, Christina M; Kosiyatrakul, Settapong; Norio, Paolo; Sen, Ranjan; Fiering, Steven; Allis, C David; Bouhassira, Eric E; Schildkraut, Carl L

    2009-11-30

    In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication. Introduction of a transcription unit into the Igh TTR, activation of gene transcription, and enhancement of local histone modifications characteristic of active chromatin did not lead to origin activation. Moreover, very few replication initiation events were observed when two ectopic replication origin sequences were inserted into the TTR. These findings indicate that the Igh TTR represents a repressive compartment that inhibits replication initiation, thus maintaining the boundaries between early and late replication domains.

  12. Curing of plasmid pBMB28 from Bacillus thuringiensis YBT-020 using an unstable replication region.

    Science.gov (United States)

    Wang, Pengxia; Zhu, Qian; Shang, Hui; Zhu, Yiguang; Sun, Ming

    2016-02-01

    Bacillus thuringiensis serovar finitimus strain YBT-020 is the well-studied spore-crystal association (SCA) phenotypic strain, whose parasporal crystals adhere to spore after lysis of the mother cell. Its endogenous plasmids pBMB26 and pBMB28 were proved essential for this SCA phenotype. In our previous study, using conventional methods, pBMB26 cured derivative and both pBMB26 and pBMB28 cured derivative of YBT-020 were obtained. However, YBT-020 solely cured of pBMB28 could not be obtained. In this study, an unstable replication region of pBMB28 was identified and was used to construct an incompatible plasmid pRep28B. This incompatible plasmid was successfully used to cure plasmid pBMB28 and was easily eliminated through segregational instability under the optimum growth temperature of YBT-020. Therefore, an endogenous plasmid was cured from the B. thuringiensis strain utilizing plasmid incompatibility. Moreover, using an unstable replication region instead of a temperature sensitive (Ts) replication region is better to cure the incompatible plasmid because it can avoid culturing at higher temperature. This method provides an efficient method for plasmid curing in B. thuringiensis and other bacteria.

  13. Universal Temporal Profile of Replication Origin Activation in Eukaryotes

    Science.gov (United States)

    Goldar, Arach

    2011-03-01

    The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

  14. Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1.

    Science.gov (United States)

    Joshua, Chijioke J; Perez, Luis D; Keasling, Jay D

    2013-01-01

    Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.

  15. Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1.

    Directory of Open Access Journals (Sweden)

    Chijioke J Joshua

    Full Text Available Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.

  16. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    Science.gov (United States)

    Mercer, A A; Morelli, G; Heuzenroeder, M; Kamke, M; Achtman, M

    1984-12-01

    Escherichia coli K1 isolates of various O types were previously assigned to different clonal groups. Members of the two clones defined by membrane pattern 9 (MP9) and serotypes O18:K1 and O1:K1 had been found to be very similar to each other. The plasmid contents of these bacteria confirmed this conclusion. Both groups carried a self-transmissible plasmid of the FI incompatibility group that coded for colicin production and a major outer membrane protein called the plasmid-coded protein (PCP). The size of this plasmid varied from 76 to 96 megadaltons, but restriction endonuclease digestion and DNA heteroduplex analysis revealed that these plasmids were highly related. O18:K1 bacteria of MP6 had previously been determined to represent a subclone, related to but different from O18:K1 MP9 bacteria. These MP6 bacteria carried a different, smaller IncFI plasmid which did not code for colicin production or the PCP protein. This smaller plasmid was primarily related to the larger plasmid within the regions of DNA encoding incompatibility, replication, and conjugation. O1:K1 bacteria of MP5 contained other unrelated plasmids in agreement with the previous conclusion that they are unrelated to O1:K1 bacteria of MP9. The bacteria examined had been isolated from two continents over a time span of 38 years, and the results attest to conservative inheritance of plasmids within bacteria of common descent.

  17. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    Science.gov (United States)

    Mercer, A A; Morelli, G; Heuzenroeder, M; Kamke, M; Achtman, M

    1984-01-01

    Escherichia coli K1 isolates of various O types were previously assigned to different clonal groups. Members of the two clones defined by membrane pattern 9 (MP9) and serotypes O18:K1 and O1:K1 had been found to be very similar to each other. The plasmid contents of these bacteria confirmed this conclusion. Both groups carried a self-transmissible plasmid of the FI incompatibility group that coded for colicin production and a major outer membrane protein called the plasmid-coded protein (PCP). The size of this plasmid varied from 76 to 96 megadaltons, but restriction endonuclease digestion and DNA heteroduplex analysis revealed that these plasmids were highly related. O18:K1 bacteria of MP6 had previously been determined to represent a subclone, related to but different from O18:K1 MP9 bacteria. These MP6 bacteria carried a different, smaller IncFI plasmid which did not code for colicin production or the PCP protein. This smaller plasmid was primarily related to the larger plasmid within the regions of DNA encoding incompatibility, replication, and conjugation. O1:K1 bacteria of MP5 contained other unrelated plasmids in agreement with the previous conclusion that they are unrelated to O1:K1 bacteria of MP9. The bacteria examined had been isolated from two continents over a time span of 38 years, and the results attest to conservative inheritance of plasmids within bacteria of common descent. Images PMID:6094355

  18. Functional interactions of DNA topoisomerases with a human replication origin.

    Science.gov (United States)

    Abdurashidova, Gulnara; Radulescu, Sorina; Sandoval, Oscar; Zahariev, Sotir; Danailov, Miltcho B; Demidovich, Alexander; Santamaria, Laura; Biamonti, Giuseppe; Riva, Silvano; Falaschi, Arturo

    2007-02-21

    The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

  19. Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

    Science.gov (United States)

    Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A; Agu, Chukwuma A; Wang, Xindan; Bernal, Juan A; Sherratt, David J; de la Cueva-Méndez, Guillermo

    2014-02-18

    Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

  20. Causation and the origin of life. Metabolism or replication first?

    Science.gov (United States)

    Pross, Addy

    2004-06-01

    The conceptual gulf that separates the 'metabolism first' and 'replication first' mechanisms for the emergence of life continues to cloud the origin of life debate. In the present paper we analyze this aspect of the origin of life problem and offer arguments in favor of the 'replication first' school. Utilizing Wicken's two-tier approach to causation we argue that a causal connection between replication and metabolism can only be demonstrated if replication would have preceded metabolism. In conjunction with existing empirical evidence and theoretical reasoning, our analysis concludes that there is no substantive evidence for a 'metabolism first' mechanism for life's emergence, while a coherent case can be made for the 'replication first' group of mechanisms. The analysis reaffirms our conviction that life is an extreme expression of kinetic control, and that the emergence of metabolic pathways can be understood by considering life as a manifestation of 'replicative chemistry'.

  1. NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

    Science.gov (United States)

    Bellelli, Roberto; Castellone, Maria Domenica; Guida, Teresa; Limongello, Roberto; Dathan, Nina Alayne; Merolla, Francesco; Cirafici, Anna Maria; Affuso, Andrea; Masai, Hisao; Costanzo, Vincenzo; Grieco, Domenico; Fusco, Alfredo; Santoro, Massimo; Carlomagno, Francesca

    2014-07-01

    NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.

  2. Trapping DNA replication origins from the human genome.

    Science.gov (United States)

    Eki, Toshihiko; Murakami, Yasufumi; Hanaoka, Fumio

    2013-04-17

    Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins' structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5-3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks.

  3. Sequence analysis and characterization of rolling-circle replicating plasmid pVCM01 from Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Penido, A. F. B.

    2013-12-01

    Full Text Available Aims: Characterization of cryptic plasmid pVCM01 (accession number JX133088 isolated from Salmonella enterica Enteritidis. Methodology and results: The complete sequence of pVCM01 was obtained. This plasmid possesses 1981 bp, with G+C content of 57% in agreement of the range of Salmonella genomic DNA. pVCM01 has a high degree of similarity to pB and pJ plasmids. It possesses six main open reading frames, only one have a very high degree of amino acid identity with protein involved in the rolling-circle-like replication (RCR. Based on the sequence similarities, pVCM01 plasmid belonged to the pC194/pUB110 rolling-circle replicating plasmid family. The Rep pVCM01 possesses the motifs: FLTLTVRN, HPHFHTL, SGDGYVKHERW, which were present in all Rep proteins. Conclusion, significance and impact of study: The small size of pVCM01 plasmid and its stability in E. coli cells, make it an attractive candidate to develop new vectors, such as cloning and/or expression vector.

  4. Opening the strands of replication origins – still an open question

    Directory of Open Access Journals (Sweden)

    Dhruba Chattoraj

    2016-09-01

    Full Text Available The local separation of duplex DNA strands (strand opening is necessary for initiating basic transactions on DNA such as transcription, replication and homologous recombination. Strand opening is commonly a stage at which these processes are regulated. Many different mechanisms are used to open the DNA duplex, the details of which are of great current interest. In this review, we focus on a few well-studied cases of DNA replication origin opening in bacteria. In particular, we discuss the opening of origins that support the theta ( mode of replication, which is used by all chromosomal origins and many extra-chromosomal elements such as plasmids and phages. Although the details of opening can vary among different origins, a common theme is binding of the initiator to multiple sites at the origin, causing stress that opens an adjacent and intrinsically unstable A+T rich region. The initiator stabilizes the opening by capturing one of the open strands. How the initiator binding energy is harnessed for strand opening remains to be understood.

  5. The hunt for origins of DNA replication in multicellular eukaryotes

    DEFF Research Database (Denmark)

    Urban, J. M.; Foulk, M. S.; Casella, Cinzia;

    2015-01-01

    Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope...... that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed....

  6. A replicator was not involved in the origin of life.

    Science.gov (United States)

    Shapiro, R

    2000-03-01

    Many scientific theories of the origin of life suggest that life began with the spontaneous formation of a replicator (a self-copying organic polymer) within an unorganized chemical mixture, or "soup." A profound difficulty exists, however, with the idea of RNA, or any other replicator, at the start of life. Existing replicators can serve as templates for the synthesis of additional copies of themselves, but this device cannot be used for the preparation of the very first such molecule, which must arise spontaneously from an unorganized mixture. The formation of an information-bearing homopolymer through undirected chemical synthesis appears very improbable. The difficulties involved in such a synthesis are illustrated by considering the prospects for the assembly of a polypeptide of L-alpha-amino acids, based on the contents of the Murchison meteorite as an example of a mixture of abiotic origin. In that mixture, potential replicator components would be accompanied by a host of interfering substances, which include chain terminators (simple carboxylic acids and amines), branch-formers, D-amino acids, and many classes of substances for which incorporation would disrupt the necessary structural regularity of the replicator. Laboratory experiments dealing with the nonenzymatic synthesis of biopolymers have not addressed the specificity problem. The possibility that formation of the first replicator took place through a very improbable event cannot be excluded, but greater attention should be given to metabolism-first theories, which avoid this difficulty.

  7. Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins

    DEFF Research Database (Denmark)

    Lozano, C.; Garcia-Migura, L.; Aspiroz, C.

    2012-01-01

    An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybrid...

  8. Characterization of the replication, transfer, and plasmid/lytic phage cycle of the Streptomyces plasmid-phage pZL12.

    Science.gov (United States)

    Zhong, Li; Cheng, Qiuxiang; Tian, Xinli; Zhao, Liqian; Qin, Zhongjun

    2010-07-01

    We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

  9. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  10. High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

    OpenAIRE

    Sakai, Y.; Goh, T K; Tani, Y.

    1993-01-01

    We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as...

  11. Confidently estimating the number of DNA replication origins.

    Science.gov (United States)

    Bhaskar, Anand; Keich, Uri

    2010-01-01

    We present a method for estimating and providing a confidence interval for the number of DNA replication origins in the genome of the yeast Kluyveromyces lactis. The method requires an initial set of verified sites from which a position specific frequency matrix (PSFM) can be constructed. We further assume that we have access to a sparingly used experimental procedure which can verify the functionality of a few, but not all, computationally predicted sites. While our motivation comes from estimating the number of autonomously replicating sequences (ARSs), our method can also be applied to estimating the genome-wide number of "functional" transcription factor binding sites, where functionality is determined by experimental verification of the transcription factor binding event using, for example, ChIP data. The reliability of our method is demonstrated by correctly predicting the known number of Saccharomyces cerevisiae ARSs as well as the number of S. cerevisiae probes that bind to the transcription factor ABF1.

  12. A personal reflection on the replicon theory: from R1 plasmid to replication timing regulation in human cells.

    Science.gov (United States)

    Masai, Hisao

    2013-11-29

    Fifty years after the Replicon Theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here, I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases.

  13. Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

    Science.gov (United States)

    Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Chen, Chun-Long; Arneodo, Alain; Goldar, Arach; d'Aubenton-Carafa, Yves; Thermes, Claude; Audit, Benjamin; Hyrien, Olivier

    2011-12-01

    Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general

  14. Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20

    NARCIS (Netherlands)

    Meijer, WJJ; Smith, M; Wake, RG; deBoer, AL; Venema, G; Bron, S

    1996-01-01

    We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions, Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B. subtilis chr

  15. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice.

    Science.gov (United States)

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S; Pushko, Peter

    2014-11-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF.

  16. Diversity and homogeneity among small plasmids of Aeromonas salmonicida subsp. salmonicida linked with geographical origin

    Directory of Open Access Journals (Sweden)

    Sabrina A Attéré

    2015-11-01

    Full Text Available Furunculosis, which is caused by Aeromonas salmonicida subsp. salmonicida, is a major salmonid disease in fish farms worldwide. Several plasmids found in this bacterium confer phenotypes such drug resistance and virulence. Small plasmids (pAsa1, pAsa2, pAsa3, and pAsal1 related to ColE1- and ColE2-type replicons are usually present in its normal plasmidome. In the present study, with the objective to investigate if these plasmids display particularities related to the origin of the isolates bearing them, a total of 153 isolates, including 78 new and 75 previously described, were analyzed for the presence of small plasmids by PCR and DNA restriction fragment profiling. A geographical dichotomy between Canadian and European isolates for their propensity to do not have pAsa3 or pAsal1 was found. In addition, the genotyping analysis led to the identification of two European isolates harboring an unusual pAsal1. An investigation by next-generation sequencing (NGS of these two isolates shed light on two pAsal1 variants (pAsal1C and pAsal1D. As with pAsal1B, another pAsal1 variant previously described, these two new variants bore a second insertion sequence (ISAS5 in addition to the usual ISAS11. The characterization of these variants suggested that they could predominate over the wild-type pAsal1 in stressful conditions such as growth at temperatures of 25°C and above. To obtain a comprehensive portrait of the mutational pressure on small plasmids, 26 isolates whose DNA had been sequenced by NGS were investigated. pAsa3 and pAsal1 were more prone to mutations than pAsa1 and pAsa2, especially in the mobA gene, which encodes a relaxase and a primase. Lastly, the average copy number of each plasmid per cell was assessed using raw sequencing data. A clear trend with respect to the relative proportion per cell of each plasmid was identified. Our large-scale study revealed a geographical dichotomy in small plasmid repertoire in addition to a clear trend

  17. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Jokinen, Jenny; Lukashevich, Igor S. [Department of Pharmacology and Toxicology, School of Medicine, Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2014-11-15

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

  18. Rif1 Regulates Initiation Timing of Late Replication Origins throughout the S. cerevisiae Genome

    OpenAIRE

    Peace, Jared M.; Anna Ter-Zakarian; Aparicio, Oscar M

    2014-01-01

    Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identi...

  19. Role for RNA: DNA hybrids in origin-independent replication priming in a eukaryotic system

    OpenAIRE

    Stuckey, Ruth; García Rodriguez, Néstor; Aguilera López, Andrés; Wellinger, Ralf Erik

    2015-01-01

    DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops)...

  20. Fine-tuning synthesis of Yersinia pestis LcrV from runaway-like replication balanced-lethal plasmid in a Salmonella enterica serovar typhimurium vaccine induces protection against a lethal Y. pestis challenge in mice.

    Science.gov (United States)

    Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M; Branger, Christine G; Tinge, Steven A; Curtiss, Roy

    2010-06-01

    A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

  1. Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range.

    Science.gov (United States)

    Fernández-Tresguerres, M E; Martín, M; García de Viedma, D; Giraldo, R; Díaz-Orejas, R

    1995-01-01

    pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon. PMID:7635822

  2. Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

    Science.gov (United States)

    Li, Ruichao; Xie, Miaomiao; Lv, Jingzhang; Wai-Chi Chan, Edward; Chen, Sheng

    2017-03-01

    To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

  3. Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

    Science.gov (United States)

    Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

    2015-07-01

    The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication.

  4. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif.

    Science.gov (United States)

    García de Viedma, D; Serrano-López, A; Díaz-Orejas, R

    1995-01-01

    The initiator protein of the plasmid pPS10, RepA, has a putative helix-turn-helix (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. [D. García de Viedma, et al. (1996) EMBO J. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations in RepA, associated with defficiencies in autoregulation, map either at the putative HTH motif or in its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and oriV regions in vitro. Revertants of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same. Images PMID:8559664

  5. Genome-wide studies highlight indirect links between human replication origins and gene regulation.

    Science.gov (United States)

    Cadoret, Jean-Charles; Meisch, Françoise; Hassan-Zadeh, Vahideh; Luyten, Isabelle; Guillet, Claire; Duret, Laurent; Quesneville, Hadi; Prioleau, Marie-Noëlle

    2008-10-14

    To get insights into the regulation of replication initiation, we systematically mapped replication origins along 1% of the human genome in HeLa cells. We identified 283 origins, 10 times more than previously known. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Origin sequences are evolutionarily conserved, and half of them map within or near CpG islands. Most of the origins overlap transcriptional regulatory elements, providing further evidence of a connection with gene regulation. Moreover, we identify c-JUN and c-FOS as important regulators of origin selection. Half of the identified replication initiation sites do not have an open chromatin configuration, showing the absence of a direct link with gene regulation. Replication timing analyses coupled with our origin mapping suggest that a relatively strict origin-timing program regulates the replication of the human genome.

  6. Initiation of DNA replication from non-canonical sites on an origin-depleted chromosome.

    Directory of Open Access Journals (Sweden)

    Naomi L Bogenschutz

    Full Text Available Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.

  7. Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.

    Science.gov (United States)

    Akiyama, Masahiro Tatsumi; Oshima, Taku; Chumsakul, Onuma; Ishikawa, Shu; Maki, Hisaji

    2016-08-01

    Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.

  8. Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

    Science.gov (United States)

    O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

    2015-01-01

    Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

  9. Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

    Science.gov (United States)

    Gibbs, Mark J; Smeianov, Vladimir V; Steele, James L; Upcroft, Peter; Efimov, Boris A

    2006-06-01

    Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.

  10. Choosing a suitable method for the identification of replication origins in microbial genomes

    Directory of Open Access Journals (Sweden)

    Chengcheng eSong

    2015-09-01

    Full Text Available As the replication of genomic DNA is arguably the most important task performed by a cell and given that it is controlled at the initiation stage, the events that occur at the replication origin play a central role in the cell cycle. Making sense of DNA replication origins is important for improving our capacity to study cellular processes and functions in the regulation of gene expression, genome integrity in much finer detail. Thus, clearly comprehending the positions and sequences of replication origins which are fundamental to chromosome organization and duplication is the first priority of all. In view of such important roles of replication origins, tremendous work has been aimed at identifying and testing the specificity of replication origins. A number of computational tools based on various skew types have been developed to predict replication origins. Using various in silico approaches such as Ori-Finder 2, and databases such as DoriC and oriDB, researchers have predicted the locations of replication origins sites for thousands bacterial chromosomes and archaeal genomes. Based on the predicted results, we should choose an effective method for identifying and confirming the interactions at origins of replication. Here we describe the main existing experimental methods that aimed to determine the replication origin regions and list some of the many the practical applications of these methods.

  11. Diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Nicolas M Berbenetz

    2010-09-01

    Full Text Available Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers positions nucleosomes adjacent to the origin to promote replication origin function.

  12. Modular evolution of TnGBSs, a new family of integrative and conjugative elements associating insertion sequence transposition, plasmid replication, and conjugation for their spreading.

    Science.gov (United States)

    Guérillot, Romain; Da Cunha, Violette; Sauvage, Elisabeth; Bouchier, Christiane; Glaser, Philippe

    2013-05-01

    Integrative and conjugative elements (ICEs) have a major impact on gene flow and genome dynamics in bacteria. The ICEs TnGBS1 and TnGBS2, first identified in Streptococcus agalactiae, use a DDE transposase, unlike most characterized ICEs, which depend on a phage-like integrase for their mobility. Here we identified 56 additional TnGBS-related ICEs by systematic genome analysis. Interestingly, all except one are inserted in streptococcal genomes. Sequence comparison of the proteins conserved among these ICEs defined two subtypes related to TnGBS1 or TnGBS2. We showed that both types encode different conjugation modules: a type IV secretion system, a VirD4 coupling protein, and a relaxase and its cognate oriT site, shared with distinct lineages of conjugative elements of Firmicutes. Phylogenetic analysis suggested that TnGBSs evolved from two conjugative elements of different origins by the successive recruitment of a transposition module derived from insertion sequences (ISs). Furthermore, TnGBSs share replication modules with different plasmids. Mutational analyses and conjugation experiments showed that TnGBS1 and TnGBS2 combine replication and transposition upstream promoters for their transfer and stabilization. Despite an evolutionarily successful horizontal dissemination within the genus Streptococcus, these ICEs have a restricted host range. However, we reveal that for TnGBS1 and TnGBS2, this host restriction is not due to a transfer incompatibility linked to the conjugation machineries but most likely to their ability for transient maintenance through replication after their transfer.

  13. CtrA response regulator binding to the Caulobacter chromosome replication origin is required during nutrient and antibiotic stress as well as during cell cycle progression.

    Science.gov (United States)

    Bastedo, D Patrick; Marczynski, Gregory T

    2009-04-01

    The Caulobacter crescentus chromosome replication origin (Cori) has five binding sites for CtrA, an OmpR/PhoB family 'response regulator'. CtrA is degraded in replicating 'stalked' cells but is abundant in the non-replicating 'swarmer' cells, where it was proposed to repress replication by binding to Cori. We systematically mutated all Cori CtrA binding sites, and examined their consequences in the contexts of autonomous Cori-plasmid replication and in the natural chromosome locus. Remarkably, the C. crescentus chromosome tolerates severe mutations in all five CtrA binding sites, demonstrating that CtrA is not essential for replication. Further physiological and cell cycle experiments more rigorously supported the original hypothesis that CtrA represses replication. However, our experiments argued against another hypothesis that residual and/or replenished CtrA protein in stalked cells might prevent extra or unscheduled chromosome replication before cell division. Surprisingly, we also demonstrated that Cori CtrA binding sites are very advantageous and can become essential when cells encounter nutrients and antibiotics. Therefore, the CtrA cell cycle regulator co-ordinates replication with viable cell growth in stressful and rapidly changing environments. We argue that this new role for CtrA provided the primary selective pressure for evolving control by CtrA.

  14. Nucleosome occupancy as a novel chromatin parameter for replication origin functions

    Science.gov (United States)

    Rodriguez, Jairo; Lee, Laura; Lynch, Bryony; Tsukiyama, Toshio

    2017-01-01

    Eukaryotic DNA replication initiates from multiple discrete sites in the genome, termed origins of replication (origins). Prior to S phase, multiple origins are poised to initiate replication by recruitment of the pre-replicative complex (pre-RC). For proper replication to occur, origin activation must be tightly regulated. At the population level, each origin has a distinct firing time and frequency of activation within S phase. Many studies have shown that chromatin can strongly influence initiation of DNA replication. However, the chromatin parameters that affect properties of origins have not been thoroughly established. We found that nucleosome occupancy in G1 varies greatly around origins across the S. cerevisiae genome, and nucleosome occupancy around origins significantly correlates with the activation time and efficiency of origins, as well as pre-RC formation. We further demonstrate that nucleosome occupancy around origins in G1 is established during transition from G2/M to G1 in a pre-RC-dependent manner. Importantly, the diminished cell-cycle changes in nucleosome occupancy around origins in the orc1-161 mutant are associated with an abnormal global origin usage profile, suggesting that proper establishment of nucleosome occupancy around origins is a critical step for regulation of global origin activities. Our work thus establishes nucleosome occupancy as a novel and key chromatin parameter for proper origin regulation. PMID:27895110

  15. A subset of replication proteins enhances origin recognition and lytic replication by the Epstein-Barr virus ZEBRA protein.

    Directory of Open Access Journals (Sweden)

    Ayman El-Guindy

    Full Text Available ZEBRA is a site-specific DNA binding protein that functions as a transcriptional activator and as an origin binding protein. Both activities require that ZEBRA recognizes DNA motifs that are scattered along the viral genome. The mechanism by which ZEBRA discriminates between the origin of lytic replication and promoters of EBV early genes is not well understood. We explored the hypothesis that activation of replication requires stronger association between ZEBRA and DNA than does transcription. A ZEBRA mutant, Z(S173A, at a phosphorylation site and three point mutants in the DNA recognition domain of ZEBRA, namely Z(Y180E, Z(R187K and Z(K188A, were similarly deficient at activating lytic DNA replication and expression of late gene expression but were competent to activate transcription of viral early lytic genes. These mutants all exhibited reduced capacity to interact with DNA as assessed by EMSA, ChIP and an in vivo biotinylated DNA pull-down assay. Over-expression of three virally encoded replication proteins, namely the primase (BSLF1, the single-stranded DNA-binding protein (BALF2 and the DNA polymerase processivity factor (BMRF1, partially rescued the replication defect in these mutants and enhanced ZEBRA's interaction with oriLyt. The findings demonstrate a functional role of replication proteins in stabilizing the association of ZEBRA with viral DNA. Enhanced binding of ZEBRA to oriLyt is crucial for lytic viral DNA replication.

  16. Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

    OpenAIRE

    Smith, Owen K.; Aladjem, Mirit I.

    2014-01-01

    The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes ...

  17. Open chromatin encoded in DNA sequence is the signature of 'master' replication origins in human cells.

    Science.gov (United States)

    Audit, Benjamin; Zaghloul, Lamia; Vaillant, Cédric; Chevereau, Guillaume; d'Aubenton-Carafa, Yves; Thermes, Claude; Arneodo, Alain

    2009-10-01

    For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions approximately 300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as 'master' replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these 'master' origins are likely to play a key role in genome dynamics during evolution and in pathological situations.

  18. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K.; Pearson, G D

    1985-01-01

    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

  19. Control of bacterial chromosome replication by non-coding regions outside the origin

    DEFF Research Database (Denmark)

    Frimodt-Møller, Jakob; Charbon, Godefroid; Løbner-Olesen, Anders

    2017-01-01

    Chromosome replication in Eubacteria is initiated by initiator protein(s) binding to specific sites within the replication origin, oriC. Recently, initiator protein binding to chromosomal regions outside the origin has attracted renewed attention; as such binding sites contribute to control the f...

  20. Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

    Science.gov (United States)

    Ge, Xin Quan

    2010-01-01

    Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3–related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome. PMID:21173116

  1. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts

    Science.gov (United States)

    Newman, Timothy J.; Mamun, Mohammed A.; Nieduszynski, Conrad A.; Blow, J. Julian

    2013-01-01

    During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts—Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe—also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10−8, which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms. PMID:23963700

  2. High-throughput mapping of origins of replication in human cells.

    Science.gov (United States)

    Lucas, Isabelle; Palakodeti, Aparna; Jiang, Yanwen; Young, David J; Jiang, Nan; Fernald, Anthony A; Le Beau, Michelle M

    2007-08-01

    Mapping origins of replication has been challenging in higher eukaryotes. We have developed a rapid, genome-wide method to map origins of replication in asynchronous human cells by combining the nascent strand abundance assay with a highly tiled microarray platform, and we validated the technique by two independent assays. We applied this method to analyse the enrichment of nascent DNA in three 50-kb regions containing known origins of replication in the MYC, lamin B2 (LMNB2) and haemoglobin beta (HBB) genes, a 200-kb region containing the rare fragile site, FRAXA, and a 1,075-kb region on chromosome 22; we detected most of the known origins and also 28 new origins. Surprisingly, the 28 new origins were small in size and located predominantly within genes. Our study also showed a strong correlation between origin replication timing and chromatin acetylation.

  3. Acquisition through horizontal gene transfer of plasmid pSMA198 by Streptococcus macedonicus ACA-DC 198 points towards the dairy origin of the species.

    Directory of Open Access Journals (Sweden)

    Konstantinos Papadimitriou

    Full Text Available Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex.We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland showing that pSMA198's acquisition is not a recent event.Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species.

  4. High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sakai, Y; Goh, T K; Tani, Y

    1993-06-01

    We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.

  5. Origin of the membrane compartment for cowpea mosaic virus replication

    NARCIS (Netherlands)

    Carette, J.E.

    2002-01-01

    Replication of many positive-stranded RNA viruses takes place in association with intracellular membranes. Often these membranes are induced upon infection by vesiculation or rearrangement of membranes from different organelles including the early and late endomembrane system. Upon infection of cowp

  6. Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome.

    Directory of Open Access Journals (Sweden)

    Jared M Peace

    Full Text Available Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1's role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4Δ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1Δ cells, and checkpoint-defective mec1Δ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres.

  7. Evolutionary dynamics of RNA-like replicator systems: A bioinformatic approach to the origin of life.

    Science.gov (United States)

    Takeuchi, Nobuto; Hogeweg, Paulien

    2012-09-01

    We review computational studies on prebiotic evolution, focusing on informatic processes in RNA-like replicator systems. In particular, we consider the following processes: the maintenance of information by replicators with and without interactions, the acquisition of information by replicators having a complex genotype-phenotype map, the generation of information by replicators having a complex genotype-phenotype-interaction map, and the storage of information by replicators serving as dedicated templates. Focusing on these informatic aspects, we review studies on quasi-species, error threshold, RNA-folding genotype-phenotype map, hypercycle, multilevel selection (including spatial self-organization, classical group selection, and compartmentalization), and the origin of DNA-like replicators. In conclusion, we pose a future question for theoretical studies on the origin of life.

  8. BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

    DEFF Research Database (Denmark)

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline

    2016-01-01

    During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators...... recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby...

  9. Recent advances in the genome-wide study of DNA replication origins in yeast

    Directory of Open Access Journals (Sweden)

    Chong ePeng

    2015-02-01

    Full Text Available DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs. Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genome. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some nonconventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve the replication origins prediction.

  10. Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon.

    Science.gov (United States)

    Conti, Chiara; Herrick, John; Bensimon, Aaron

    2007-08-01

    Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV-18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene.

  11. Translation initiation of the replication initiator repB gene of promiscuous plasmid pMV158 is led by an extended non-SD sequence.

    Science.gov (United States)

    López-Aguilar, Celeste; Ruiz-Masó, José A; Rubio-Lepe, Tania Samir; Sanz, Marta; del Solar, Gloria

    2013-07-01

    RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence.

  12. The origin recognition complex interacts with a subset of metabolic genes tightly linked to origins of replication.

    Directory of Open Access Journals (Sweden)

    Erika Shor

    2009-12-01

    Full Text Available The origin recognition complex (ORC marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant only sites that bind ORC tightly (such as HMR-E remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC "affinity" genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.

  13. Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria.

    Science.gov (United States)

    Giusti, María de los Ángeles; Pistorio, Mariano; Lozano, Mauricio J; Tejerizo, Gonzalo A Torres; Salas, María Eugenia; Martini, María Carla; López, José Luis; Draghi, Walter O; Del Papa, María Florencia; Pérez-Mendoza, Daniel; Sanjuán, Juan; Lagares, Antonio

    2012-05-01

    Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own

  14. ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication.

    Science.gov (United States)

    Wegrzyn, A; Czyz, A; Gabig, M; Wegrzyn, G

    2000-01-01

    The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

  15. Interaction of RECQ4 and MCM10 is important for efficient DNA replication origin firing in human cells

    DEFF Research Database (Denmark)

    Kliszczak, Maciej; Sedlackova, Hana; Pitchai, Ganesha P

    2015-01-01

    DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two esse...

  16. A role for the weak DnaA binding sites in bacterial replication origins

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility...

  17. Characterization of the Complete Nucleotide Sequences of IncA/C2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin.

    Science.gov (United States)

    Papagiannitsis, Costas C; Kutilova, Iva; Medvecky, Matej; Hrabak, Jaroslav; Dolejska, Monika

    2017-09-01

    A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C2 plasmids. pEc158 carried none of the blaCMY-2-like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn6317-like module or a Tn21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C2 plasmid lineages. Copyright © 2017 American Society for Microbiology.

  18. Comparative investigations of Klebsiella species of clinical origin: plasmid patterns, biochemical reactions, antibiotic resistances and serotypes.

    Science.gov (United States)

    Podschun, R; Heineken, P; Ullmann, U; Sonntag, H G

    1986-09-01

    A total of 124 K. pneumoniae and 52 K. oxytoca isolates obtained from clinical specimens was investigated for plasmid patterns, biochemical reactions, antibiotic resistances and serotypes regarding to the distribution and relationships of these characters. A great diversity of plasmid patterns, bio/serotypes and resistance patterns was revealed. About 90% of strains contained plasmid DNA and up to seven plasmid bands per isolate could be shown. For K. pneumoniae, serotype 7 and for K. oxytoca, type 55 were most common. In general, little difference between both species was found and characters were similarly distributed. With respect to the site of isolation, serotype 7 was predominating in K. pneumoniae strains from the respiratory tract. Highly multiple-resistant organism were found in the largest number in specimens from the urogenital tract, in the lowest in specimens from wounds. Extensive statistical analyses did not detect any relationship among the characters investigated.

  19. Development of a vector and host system and characterization of replication of plasmid pSQ10 in moderately halophilic Nocardiopsis

    Institute of Scientific and Technical Information of China (English)

    Ana Zeng; Tao Wang; Haiyang Xia; Shiyuan Peng; Weihua Chen; Chenglin Jiang; Lihua Xu

    2011-01-01

    The genus of Nocardiopsis is a new source of antibiotics,chemicals,and enzymes.Here we reported the development of a vector and host system in moderately halophilic Nocardiopsis via an oriT-mediated conjugation.By screening about 80 Nocardiopsis strains,6 of them harbored 8 plasmids (18-80 kb).The complete nucleotide sequence of pSQ10 consisted of 18,219 bp,with 71.9% G + Ccontent,encoding 17 open reading frames,5 of them resembled those of Streptomyces plasmids.A rep locus (iteron within the gene) was identified for replication in Nocardiopsis sp.YIM 90083,and rep protein bound to its iteron sequence.This system may be useful for gene cloning and expression in Nocardiopsis.

  20. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Directory of Open Access Journals (Sweden)

    Anoushka Davé

    2014-04-01

    Full Text Available The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.

  1. The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites.

    Science.gov (United States)

    Taylor, James A; Ouimet, Marie-Claude; Wargachuk, Richard; Marczynski, Gregory T

    2011-10-01

    The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control. © 2011 Blackwell Publishing Ltd.

  2. Acquisition through Horizontal Gene Transfer of Plasmid pSMA198 by Streptococcus macedonicus ACA-DC 198 Points towards the Dairy Origin of the Species

    Science.gov (United States)

    Papadimitriou, Konstantinos; Anastasiou, Rania; Maistrou, Eleni; Plakas, Thomas; Papandreou, Nikos C.; Hamodrakas, Stavros J.; Ferreira, Stéphanie; Supply, Philip; Renault, Pierre; Pot, Bruno; Tsakalidou, Effie

    2015-01-01

    Background Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex. Methodology/Principal Findings We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198’s acquisition is not a recent event. Conclusions/Significance Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species. PMID:25584532

  3. Plasmid DNA Initiates Replication of Yellow Fever Vaccine In Vitro and Elicits Virus-Specific Immune Response in Mice

    OpenAIRE

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

    2014-01-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10ng of iDNA plasmid was sufficient to s...

  4. Transfer of drug-resistance plasmids by conjugation from nosocomial strains of Serratia marcescens to Escherichia coli in biological fluids of human origin.

    Science.gov (United States)

    Mendez, F J; Mendoza, M C; Llaneza, J J; Hardisson, C

    1982-09-01

    Six independent isolates of multi-resistant Serratia marcescens associated with nosocomial infections were examined for their ability to transfer drug-resistance plasmids by conjugation to Escherichia coli in biological fluids of human origin, such as normal and pathological urine, faeces, blood plasma and ascitic fluid. Luria broth was used as a control. Positive transfer was found in all media assayed. The different patterns of linked transferable resistance found in the transconjugants corresponded to the phenotypic expression of five plasmids. The frequencies of transfer varied with plasmid types and media employed. The culture media did not affect the phenotypic expression of the plasmids.

  5. The latent origin of replication of Epstein-Barr virus directs viral genomes to active regions of the nucleus.

    Science.gov (United States)

    Deutsch, Manuel J; Ott, Elisabeth; Papior, Peer; Schepers, Aloys

    2010-03-01

    The Epstein-Barr virus efficiently infects human B cells. The EBV genome is maintained extrachromosomally and replicates synchronously with the host's chromosomes. The latent origin of replication (oriP) guarantees plasmid stability by mediating two basic functions: replication and segregation of the viral genome. While the segregation process of EBV genomes is well understood, little is known about its chromatin association and nuclear distribution during interphase. Here, we analyzed the nuclear localization of EBV genomes and the role of functional oriP domains FR and DS for basic functions such as the transformation of primary cells, their role in targeting EBV genomes to distinct nuclear regions, and their association with epigenetic domains. Fluorescence in situ hybridization visualized the localization of extrachromosomal EBV genomes in the regions adjacent to chromatin-dense territories called the perichromatin. Further, immunofluorescence experiments demonstrated a preference of the viral genome for histone 3 lysine 4-trimethylated (H3K4me3) and histone 3 lysine 9-acetylated (H3K9ac) nuclear regions. To determine the role of FR and DS for establishment and subnuclear localization of EBV genomes, we transformed primary human B lymphocytes with recombinant mini-EBV genomes containing different oriP mutants. The loss of DS results in a slightly increased association in H3K27me3 domains. This study demonstrates that EBV genomes or oriP-based extrachromosomal vector systems are integrated into the higher order nuclear organization. We found that viral genomes are not randomly distributed in the nucleus. FR but not DS is crucial for the localization of EBV in perichromatic regions that are enriched for H3K4me3 and H3K9ac, which are hallmarks of transcriptionally active regions.

  6. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  7. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  8. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    Science.gov (United States)

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  9. Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues.

    Science.gov (United States)

    Kawakami, Hironori; Ohashi, Eiji; Kanamoto, Shota; Tsurimoto, Toshiki; Katayama, Tsutomu

    2015-10-12

    In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC.

  10. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio

    2015-05-01

    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  11. High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins.

    Science.gov (United States)

    Comoglio, Federico; Schlumpf, Tommy; Schmid, Virginia; Rohs, Remo; Beisel, Christian; Paro, Renato

    2015-05-05

    At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  12. The effect of the intra-S-phase checkpoint on origins of replication in human cells.

    Science.gov (United States)

    Karnani, Neerja; Dutta, Anindya

    2011-03-15

    Although many chemotherapy drugs activate the intra-S-phase checkpoint pathway to block S-phase progression, not much is known about how and where the intra-S-phase checkpoint regulates origins of replication in human chromosomes. A genomic analysis of replication in human cells in the presence of hydroxyurea (HU) revealed that only the earliest origins fire, but the forks stall within 2 kb and neighboring clusters of dormant origins are activated. The initiation events are located near expressed genes with a preference for transcription start and end sites, and when they are located in intergenic regions they are located near regulatory factor-binding regions (RFBR). The activation of clustered neo-origins by HU suggests that there are many potential replication initiation sites in permissive parts of the genome, most of which are not used in a normal S phase. Consistent with this redundancy, we see multiple sites bound to MCM3 (representative of the helicase) in the region flanking three out of three origins studied in detail. Bypass of the intra-S-phase checkpoint by caffeine activates many new origins in mid- and late-replicating parts of the genome. The intra-S-phase checkpoint suppresses origin firing after the loading of Mcm10, but before the recruitment of Cdc45 and AND-1/CTF4; i.e., after helicase loading but before helicase activation and polymerase loading. Interestingly, Cdc45 recruitment upon checkpoint bypass was accompanied by the restoration of global Cdk2 kinase activity and decrease in both global and origin-bound histone H3 Lys 4 trimethylation (H3K4me3), consistent with the suggestion that both of these factors are important for Cdc45 recruitment.

  13. New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria

    DEFF Research Database (Denmark)

    Ioannidis, Panagiotis; Dunning Hotopp, Julie C; Sapountzis, Panagiotis

    2007-01-01

    , the origin of DNA replication (ori) regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori...

  14. A structural framework for replication origin opening by AAA plus initiation factors

    NARCIS (Netherlands)

    Duderstadt, Karl E.; Berger, James M.

    2013-01-01

    ATP-dependent initiation factors help process replication origins and coordinate replisome assembly to control the onset of DNA synthesis. Although the specific properties and regulatory mechanisms of initiator proteins can vary greatly between different organisms, certain nucleotide-binding element

  15. Susceptibility to superhelically driven DNA duplex destabilization: a highly conserved property of yeast replication origins.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available Strand separation is obligatory for several DNA functions, including replication. However, local DNA properties such as A+T content or thermodynamic stability alone do not determine the susceptibility to this transition in vivo. Rather, superhelical stresses provide long-range coupling among the transition behaviors of all base pairs within a topologically constrained domain. We have developed methods to analyze superhelically induced duplex destabilization (SIDD in genomic DNA that take into account both this long-range stress-induced coupling and sequence-dependent local thermodynamic stability. Here we apply this approach to examine the SIDD properties of 39 experimentally well-characterized autonomously replicating DNA sequences (ARS elements, which function as replication origins in the yeast Saccharomyces cerevisiae. We find that these ARS elements have a strikingly increased susceptibility to SIDD relative to their surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do their immediately surrounding sequences, making them more than 2,000 times easier to open. Statistical analysis shows that the probability of this strong an association between SIDD sites and ARS elements arising by chance is approximately 4 x 10. This local enhancement of the propensity to separate to single strands under superhelical stress has obvious implications for origin function. SIDD properties also could be used, in conjunction with other known origin attributes, to identify putative replication origins in yeast, and possibly in other metazoan genomes.

  16. Isolation of restriction fragments containing origins of replication from complex genomes.

    Science.gov (United States)

    Mesner, Larry D; Hamlin, Joyce L

    2015-01-01

    The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of replication from asynchronous cell cultures. In one of these, nascent strands are melted from the long template DNA, and the small, origin-centered strands are isolated on sucrose gradients. The second method involves the selective entrapment of bubble-containing fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled "Purification of restriction fragments containing replication intermediates from mammalian cells for 2-D gel analysis" (Chapter 16 ).

  17. The origin recognition complex links replication, sister chromatid cohesion and transcriptional silencing in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Suter, Bernhard; Tong, Amy; Chang, Michael; Yu, Lisa; Brown, Grant W; Boone, Charles; Rine, Jasper

    2004-01-01

    Mutations in genes encoding the origin recognition complex (ORC) of Saccharomyces cerevisiae affect initiation of DNA replication and transcriptional repression at the silent mating-type loci. To explore the function of ORC in more detail, a screen for genetic interactions was undertaken using large

  18. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    Science.gov (United States)

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

  19. Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing.

    Science.gov (United States)

    Dellino, Gaetano Ivan; Cittaro, Davide; Piccioni, Rossana; Luzi, Lucilla; Banfi, Stefania; Segalla, Simona; Cesaroni, Matteo; Mendoza-Maldonado, Ramiro; Giacca, Mauro; Pelicci, Pier Giuseppe

    2013-01-01

    We report the genome-wide mapping of ORC1 binding sites in mammals, by chromatin immunoprecipitation and parallel sequencing (ChIP-seq). ORC1 binding sites in HeLa cells were validated as active DNA replication origins (ORIs) using Repli-seq, a method that allows identification of ORI-containing regions by parallel sequencing of temporally ordered replicating DNA. ORC1 sites were universally associated with transcription start sites (TSSs) of coding or noncoding RNAs (ncRNAs). Transcription levels at the ORC1 sites directly correlated with replication timing, suggesting the existence of two classes of ORIs: those associated with moderate/high transcription levels (≥1 RNA copy/cell), firing in early S and mapping to the TSSs of coding RNAs; and those associated with low transcription levels (<1 RNA copy/cell), firing throughout the entire S and mapping to TSSs of ncRNAs. These findings are compatible with a scenario whereby TSS expression levels influence the efficiency of ORC1 recruitment at G(1) and the probability of firing during S.

  20. Origin and Evolution of Nitrogen Fixation Genes on Symbiosis Islands and Plasmid in Bradyrhizobium

    Science.gov (United States)

    Okubo, Takashi; Piromyou, Pongdet; Tittabutr, Panlada; Teaumroong, Neung; Minamisawa, Kiwamu

    2016-01-01

    The nitrogen fixation (nif) genes of nodule-forming Bradyrhizobium strains are generally located on symbiosis islands or symbiosis plasmids, suggesting that these genes have been transferred laterally. The nif genes of rhizobial and non-rhizobial Bradyrhizobium strains were compared in order to infer the evolutionary histories of nif genes. Based on all codon positions, the phylogenetic tree of concatenated nifD and nifK sequences showed that nifDK on symbiosis islands formed a different clade from nifDK on non-symbiotic loci (located outside of symbiosis islands and plasmids) with elongated branches; however, these genes were located in close proximity, when only the 1st and 2nd codon positions were analyzed. The guanine (G) and cytosine (C) content of the 3rd codon position of nifDK on symbiosis islands was lower than that on non-symbiotic loci. These results suggest that nif genes on symbiosis islands were derived from the non-symbiotic loci of Bradyrhizobium or closely related strains and have evolved toward a lower GC content with a higher substitution rate than the ancestral state. Meanwhile, nifDK on symbiosis plasmids clustered with nifDK on non-symbiotic loci in the tree representing all codon positions, and the GC content of symbiotic and non-symbiotic loci were similar. These results suggest that nif genes on symbiosis plasmids were derived from the non-symbiotic loci of Bradyrhizobium and have evolved with a similar evolutionary pattern and rate as the ancestral state. PMID:27431195

  1. Origin and Evolution of Nitrogen Fixation Genes on Symbiosis Islands and Plasmid in Bradyrhizobium.

    Science.gov (United States)

    Okubo, Takashi; Piromyou, Pongdet; Tittabutr, Panlada; Teaumroong, Neung; Minamisawa, Kiwamu

    2016-09-29

    The nitrogen fixation (nif) genes of nodule-forming Bradyrhizobium strains are generally located on symbiosis islands or symbiosis plasmids, suggesting that these genes have been transferred laterally. The nif genes of rhizobial and non-rhizobial Bradyrhizobium strains were compared in order to infer the evolutionary histories of nif genes. Based on all codon positions, the phylogenetic tree of concatenated nifD and nifK sequences showed that nifDK on symbiosis islands formed a different clade from nifDK on non-symbiotic loci (located outside of symbiosis islands and plasmids) with elongated branches; however, these genes were located in close proximity, when only the 1st and 2nd codon positions were analyzed. The guanine (G) and cytosine (C) content of the 3rd codon position of nifDK on symbiosis islands was lower than that on non-symbiotic loci. These results suggest that nif genes on symbiosis islands were derived from the non-symbiotic loci of Bradyrhizobium or closely related strains and have evolved toward a lower GC content with a higher substitution rate than the ancestral state. Meanwhile, nifDK on symbiosis plasmids clustered with nifDK on non-symbiotic loci in the tree representing all codon positions, and the GC content of symbiotic and non-symbiotic loci were similar. These results suggest that nif genes on symbiosis plasmids were derived from the non-symbiotic loci of Bradyrhizobium and have evolved with a similar evolutionary pattern and rate as the ancestral state.

  2. The rolling-circle melting-pot model for porcine circovirus DNA replication

    Science.gov (United States)

    A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

  3. Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Shirley Lam

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these

  4. The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.

    Science.gov (United States)

    Kogoma, T; von Meyenburg, K

    1983-01-01

    The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.

  5. Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production

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    Marta Irla

    2016-09-01

    Full Text Available Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 g/L to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

  6. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress.

    Science.gov (United States)

    Ge, Xin Quan; Jackson, Dean A; Blow, J Julian

    2007-12-15

    In late mitosis and early G1, Mcm2-7 complexes are loaded onto DNA to license replication origins for use in the upcoming S phase. However, the amount of Mcm2-7 loaded is in significant excess over the number of origins normally used. We show here that in human cells, excess chromatin-bound Mcm2-7 license dormant replication origins that do not fire during normal DNA replication, in part due to checkpoint activity. Dormant origins were activated within active replicon clusters if replication fork progression was inhibited, despite the activation of S-phase checkpoints. After lowering levels of chromatin-bound Mcm2-7 in human cells by RNA interference (RNAi), the use of dormant origins was suppressed in response to replicative stress. Although cells with lowered chromatin-bound Mcm2-7 replicated at normal rates, when challenged with replication inhibitors they had dramatically reduced rates of DNA synthesis and reduced viability. These results suggest that the use of dormant origins licensed by excess Mcm2-7 is a new and physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. We propose that checkpoint kinase activity can preferentially suppress initiation within inactive replicon clusters, thereby directing new initiation events toward active clusters that are experiencing replication problems.

  7. A dual binding site for integration host factor and the response regulator CtrA inside the Caulobacter crescentus replication origin.

    Science.gov (United States)

    Siam, Rania; Brassinga, Ann Karen C; Marczynski, Gregory T

    2003-09-01

    The response regulator CtrA controls chromosome replication by binding to five sites, a, b, c, d, and e, inside the Caulobacter crescentus replication origin (Cori). In this study, we demonstrate that integration host factor (IHF) binds Cori over the central CtrA binding site c. Surprisingly, IHF and CtrA share DNA recognition sequences. Rather than promoting cooperative binding, IHF binding hinders CtrA binding to site c and nearby site d. Unlike other CtrA binding sites, DNA mutations in the CtrA c/IHF site uniquely impair autonomous Cori plasmid replication. These mutations also alter transcription from distant promoters more than 100 bp away. When the CtrA c/IHF site was deleted from the chromosome, these cells grew slowly and became selectively intolerant to a CtrA phosphor-mimic allele (D51E). Since CtrA protein concentration decreases during the cell cycle as IHF protein concentration increases, we propose a model in which IHF displaces CtrA in order to bend Cori and promote efficient chromosome replication.

  8. Ori-Finder 2, an integrated tool to predict replication origins in the archaeal genomes

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    Hao eLuo

    2014-09-01

    Full Text Available DNA replication is one of the most basic processes in all three domains of cellular life. With the advent of the post-genomic era, the increasing number of complete archaeal genomes has created an opportunity for exploration of the molecular mechanisms for initiating cellular DNA replication by in vivo experiments as well as in silico analysis. However, the location of replication origins (oriCs in many sequenced archaeal genomes remains unknown. We present a web-based tool Ori-Finder 2 to predict oriCs in the archaeal genomes automatically, based on the integrated method comprising the analysis of base composition asymmetry using the Z-curve method, the distribution of Origin Recognition Boxes (ORBs identified by FIMO tool, and the occurrence of genes frequently close to oriCs. The web server is also able to analyze the unannotated genome sequences by integrating with gene prediction pipelines and BLAST software for gene identification and function annotation. The result of the predicted oriCs is displayed as an HTML table, which offers an intuitive way to browse the result in graphical and tabular form. The software presented here is accurate for the genomes with single oriC, but it does not necessarily find all the origins of replication for the genomes with multiple oriCs. Ori-Finder 2 aims to become a useful platform for the identification and analysis of oriCs in the archaeal genomes, which would provide insight into the replication mechanisms in archaea. The web server is freely available at http://tubic.tju.edu.cn/Ori-Finder2/.

  9. Self-organizing proto-replicators and the origin of life.

    Science.gov (United States)

    Sayer, Richard M P

    2007-01-01

    Standard theories suggest that the first informational replicator involved RNA molecules (or a more primitive analogue) and that a preliminary step for the development of such replicating systems may have been the emergence of subunits capable of forming chains and interchain pairings. Following these hypotheses, this discussion describes various abstract simulations designed to investigate the structures resulting from such interactions for generalised subunits. Three classes of pairing strategy were considered for a range of subunit concentrations. The resulting dynamic self-organization of the systems produced high levels of structural complexity (some at low subunit concentrations and in the presence of disruptive subunits) and a significantly increased percentage of complementary base pairing (particularly in the more substantial structures). These properties of the systems, which did not require pre-existing replicators, templates or functional catalysts, were shown to be sensitive to the form of pairing strategy, subunit concentrations and various conditions that could theoretically be altered by products of the systems. Though no systems behaved as a replicator, some possessed collections of properties from which a replicating system might theoretically be constructed without requiring the introduction of additional classes of properties. The implications of such systems were considered with respect to the origin of life.

  10. Origins of replication and the nuclear matrix: the DHFR domain as a paradigm.

    Science.gov (United States)

    Dijkwel, P A; Hamlin, J L

    1995-01-01

    The eukaryotic genome appears to be organized in a loopwise fashion by periodic attachment to the nuclear matrix. The proposal that a chromatin loop corresponds to a functional domain has stirred interest in the properties of the DNA sequences at the bases of these loops, the matrix-attached regions (MARs). Evidence has been presented suggesting that certain MARs act as boundary elements isolating domains from their chromosomal context. MARs have also been found in the vicinity of promoters and enhancers and they could act by displacing these cis-regulatory elements into the proper nuclear subcompartment. Attachment to the matrix might also play a role in DNA replication. A large body of evidence indicates that replication occurs on the nuclear matrix. This implies that any DNA sequence will be attached to the matrix at a certain time during the cell cycle. This transient mode of attachment contrasts with the proposed permanent attachment of origins of DNA replication with the nuclear matrix. While some data exist that support this suggestion, the current lack of understanding of the mammalian replication origin precludes definitive conclusions regarding the role of MARs in the initiation process.

  11. Origins of DNA Replication and Amplification in the Breast Cancer Genome

    Science.gov (United States)

    2011-09-01

    identified 53,914 origins in the MCF-7 genome, with a median width of 1.5 kb using the methodology as follows: We used BEDTools ( Quinlan et al...are collaborating with David Gilbert (University of Florida – Tallahassee) to determine the replication foci higher order structure in the nucleus...Cancer Cell 10: 515-527. Quinlan AR, Hall IM. (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 26: 841-2

  12. Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue

    NARCIS (Netherlands)

    Rommens, Caius M.T.; Rudenko, George N.; Dijkwel, Paul P.; Haaren, Mark J.J. van; Ouwerkerk, Pieter B.F.; Blok, Karin M.; Nijkamp, H. John J.; Hille, Jacques

    1992-01-01

    We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within

  13. New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria

    Directory of Open Access Journals (Sweden)

    Baldo Laura

    2007-06-01

    Full Text Available Abstract Background The annotated genomes of two closely related strains of the intracellular bacterium Wolbachia pipientis have been reported without the identifications of the putative origin of replication (ori. Identifying the ori of these bacteria and related alpha-Proteobacteria as well as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria. Results Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication (ori regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE and COG1253 protein (CBS domain protein. Comparisons of the putative ori region among related Wolbachia strains showed higher conservation of bases within binding sites. Conclusion The sequences of the ori regions described here are only similar among closely related bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes, suggest that several of these obligate intracellular bacteria may have altered replication mechanisms. Based on these analyses, criteria are set forth for identifying the ori region in genome sequencing projects.

  14. Pseudo-replication of [GADV]-proteins and origin of life.

    Science.gov (United States)

    Ikehara, Kenji

    2009-04-02

    The RNA world hypothesis on the origin of life is generally considered as the key to solve the "chicken and egg dilemma" concerning the evolution of genes and proteins as observed in the modern organisms. This hypothesis, however, contains several serious weak points. We have a counterproposal called [GADV]-protein world hypothesis, abbreviated as GADV hypothesis, in which we have suggested that life originated from a [GADV]-protein world, which comprised proteins composed of four amino acids: Gly [G], Ala [A], Asp [D], and Val [V]. A new concept "pseudo-replication" is crucial for the description of the emergence of life. The new hypothesis not only plausibly explains how life originated from the initial chaotic protein world, but also how genes, genetic code, and proteins co-evolved.

  15. Ku80 binds to human replication origins prior to the assembly of the ORC complex.

    Science.gov (United States)

    Sibani, Sahar; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2005-05-31

    The Ku heterodimer, an abundant nuclear protein, binds DNA replication origins in a sequence-specific manner and promotes initiation. In this study, using HCT116 Ku80+/- haplo-insufficient and Orc2(delta/-) hypomorphic cells, the order of binding of Ku and the human origin recognition complex (HsORC) was determined. The nuclear expression of Ku80 was found to be decreased by 60% in Ku80+/- cells, while its general association with chromatin was decreased by 33%. Coimmunoprecipitation studies indicated that the Ku heterodimer associates specifically with the human HsOrc-2, -3, -4, and -6 subunits. Chromatin immunoprecipitation (ChIP) experiments, using cells synchronized to late G1, showed that the association of Ku80 with the lamin B2, beta-globin, and c-myc origins in vivo was decreased by 1.5-, 2.3-, and 2.5-fold, respectively, in Ku80+/- cells. The association of HsOrc-3, -4, and -6 was consistently decreased in all three origins examined in Ku80+/- cells, while that of HsOrc-2 showed no significant variation, indicating that the HsOrc-3, -4, and -6 subunits bind to the origins after Ku80. In Orc2(delta/-) cells, the association of HsOrc-2 with the lamin B2, beta-globin, and c-myc origins was decreased by 2.8-, 4.9-, and 2.8-fold, respectively, relative to wild-type HCT116 cells. Furthermore, nascent strand abundance at these three origins was decreased by 4.5-, 2.3-, and 2.6-fold in Orc2(delta/-) relative to HCT116 cells, respectively. Interestingly, the association of Ku80 with these origins was not affected in this hypomorphic cell line, indicating that Ku and HsOrc-2 bind to origins independently of each other.

  16. Mathematical modelling of DNA replication reveals a trade-off between coherence of origin activation and robustness against rereplication.

    Directory of Open Access Journals (Sweden)

    Anneke Brümmer

    2010-05-01

    Full Text Available Eukaryotic genomes are duplicated from multiple replication origins exactly once per cell cycle. In Saccharomyces cerevisiae, a complex molecular network has been identified that governs the assembly of the replication machinery. Here we develop a mathematical model that links the dynamics of this network to its performance in terms of rate and coherence of origin activation events, number of activated origins, the resulting distribution of replicon sizes and robustness against DNA rereplication. To parameterize the model, we use measured protein expression data and systematically generate kinetic parameter sets by optimizing the coherence of origin firing. While randomly parameterized networks yield unrealistically slow kinetics of replication initiation, networks with optimized parameters account for the experimentally observed distribution of origin firing times. Efficient inhibition of DNA rereplication emerges as a constraint that limits the rate at which replication can be initiated. In addition to the separation between origin licensing and firing, a time delay between the activation of S phase cyclin-dependent kinase (S-Cdk and the initiation of DNA replication is required for preventing rereplication. Our analysis suggests that distributive multisite phosphorylation of the S-Cdk targets Sld2 and Sld3 can generate both a robust time delay and contribute to switch-like, coherent activation of replication origins. The proposed catalytic function of the complex formed by Dpb11, Sld3 and Sld2 strongly enhances coherence and robustness of origin firing. The model rationalizes how experimentally observed inefficient replication from fewer origins is caused by premature activation of S-Cdk, while premature activity of the S-Cdk targets Sld2 and Sld3 results in DNA rereplication. Thus the model demonstrates how kinetic deregulation of the molecular network governing DNA replication may result in genomic instability.

  17. Boundaries of the origin of replication: creation of a pET-28a-derived vector with p15A copy control allowing compatible coexistence with pET vectors.

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    Sarmitha Sathiamoorthy

    Full Text Available During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1 to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+, and 2 to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+-derived plasmid with the p15A origin of replication (ori; pET-28a(+ contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+ replicon-comprising the ori, RNAI, RNAII, and Rom-with the p15A replicon, we generated pSAM, which contains the pET-28a(+ multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of

  18. Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

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    Olson Daniel G

    2012-05-01

    Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

  19. Constructive Approaches for Understanding the Origin of Self-Replication and Evolution

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    Norikazu Ichihashi

    2016-07-01

    Full Text Available The mystery of the origin of life can be divided into two parts. The first part is the origin of biomolecules: under what physicochemical conditions did biomolecules such as amino acids, nucleotides, and their polymers arise? The second part of the mystery is the origin of life-specific functions such as the replication of genetic information, the reproduction of cellular structures, metabolism, and evolution. These functions require the coordination of many different kinds of biological molecules. A direct strategy to approach the second part of the mystery is the constructive approach, in which life-specific functions are recreated in a test tube from specific biological molecules. Using this approach, we are able to employ design principles to reproduce life-specific functions, and the knowledge gained through the reproduction process provides clues as to their origins. In this mini-review, we introduce recent insights gained using this approach, and propose important future directions for advancing our understanding of the origins of life.

  20. Constructive Approaches for Understanding the Origin of Self-Replication and Evolution.

    Science.gov (United States)

    Ichihashi, Norikazu; Yomo, Tetsuya

    2016-07-13

    The mystery of the origin of life can be divided into two parts. The first part is the origin of biomolecules: under what physicochemical conditions did biomolecules such as amino acids, nucleotides, and their polymers arise? The second part of the mystery is the origin of life-specific functions such as the replication of genetic information, the reproduction of cellular structures, metabolism, and evolution. These functions require the coordination of many different kinds of biological molecules. A direct strategy to approach the second part of the mystery is the constructive approach, in which life-specific functions are recreated in a test tube from specific biological molecules. Using this approach, we are able to employ design principles to reproduce life-specific functions, and the knowledge gained through the reproduction process provides clues as to their origins. In this mini-review, we introduce recent insights gained using this approach, and propose important future directions for advancing our understanding of the origins of life.

  1. c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.

    OpenAIRE

    Iguchi-Ariga, Sanae M. M.; Itani, Teru; Yamaguchi, Masamitsu; Ariga, Hiroyoshi

    1987-01-01

    Replicating activity of SV40 origin-containing plasmid was tested in human cells as well as in monkey CosI cells. All the plasmids possessing SV40 ori sequences could replicate, even in the absence of SV40 T antigen, in human HL-60 and Raji cells which are expressing c-myc gene at high level. The copy numbers of the replicated plasmids in these human cells were 1/100 as high as in monkey CosI cells which express SV40 T antigen constitutively. Exactly the same plasmids as the transfected origi...

  2. A replication-time-controlling sequence element in Schizosaccharomyces pombe.

    Science.gov (United States)

    Tripathi, Vishnu P; Dubey, Dharani D

    2017-08-01

    Eukaryotic replication origins are highly variable in their activity and replication timing. The nature and role of cis-acting regulatory sequences that control chromosomal replication timing is not well defined. In the fission yeast, Schizosaccharomyces pombe, a 200-bp late-replication-enforcing element (LRE), has been shown to enforce late replication of ARS elements in plasmids. Here, we show that a short (133-bp) fragment of the LRE (shLRE) is required for causing late replication of adjoining origins in its native as well as in an ectopic early-replicating chromosomal location. Active from both sides of an early-replicating origin, the shLRE is a bona fide cis-acting regulatory element that imposes late replication timing in the chromosome.

  3. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    Science.gov (United States)

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  4. Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation

    OpenAIRE

    Strätz, Michael; Sauer, Uwe; Kuhn, Anita; Dürre, Peter

    1994-01-01

    Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 103 transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene int...

  5. Regulatory mechanisms that prevent re-initiation of DNA replication can be locally modulated at origins by nearby sequence elements.

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    Christopher D Richardson

    2014-06-01

    Full Text Available Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs. We have mapped the two RIPs to ∼ 60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some

  6. Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo.

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    Christine Sattler

    2016-03-01

    Full Text Available An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt. Using murine gammaherpesvirus 68 (MHV-68 as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.

  7. Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

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    Katarzyna Ewa Wegrzyn

    2016-08-01

    Full Text Available The proper initiation and occurrence of DNA synthesis depends on the formation and rearrangements of nucleoprotein complexes within the origin of DNA replication. In this review article, we present the current knowledge on the molecular mechanism of replication complex assembly at the origin of bacterial chromosome and plasmid replicon containing direct repeats (iterons within the origin sequence. We describe recent findings on chromosomal and plasmid replication initiators, DnaA and Rep proteins, respectively, and their sequence-specific interactions with double and single stranded DNA. Also, we discuss the current understanding of the activities of DnaA and Rep proteins required for replisome assembly that is fundamental to the duplication and stability of genetic information in bacterial cells.

  8. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  9. Residue profile in predivergence sequences as a guide to the origin of DNA replication

    CERN Document Server

    Davis, B K

    1999-01-01

    DNA dependent RNA polymerase core subunits abb' conserved residues at frequencies most closely matched with codons at stage 10.4-11.1 in code evolution. An excess of acidic residues (stage 2 additions) lowered this estimate, but not by more than 2.3 stages. With 1 tryptophan (stage 14 addition) in 529 conserved residues, abb' significantly under-represented this amino acid, consistent with a cut-off in its residue profile before completion of the genetic code. Residue profiles in FEN-1 homologs and DNA topoisomerase I-5' placed their origin at stage 11-13. Prokaryote septation protein, FtsZ, arose earlier, between stage 8-11. Proteolipid in an ATP-driven proton pump served as a marker for cell membrane formation. It indicated this event took place near stage 7. Cell division, DNA replication and transcription were inferred to have originated in a protocell antecedent of the last common ancestor. Late-forming residue profiles characterized RNA dependent RNA replicase, DNA polymerase, reverse transcriptase and ...

  10. DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner.

    Science.gov (United States)

    Wiedemann, Eva-Maria; Peycheva, Mihaela; Pavri, Rushad

    2016-12-13

    Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division.

  11. Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

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    Ramiro Mendoza-Maldonado

    Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

  12. c-ETS transcription factors play an essential role in the licensing of human MCM4 origin of replication.

    Science.gov (United States)

    Sidhu, Kaveri; Kumar, Vijay

    2015-11-01

    In metazoans, DNA replication is a highly regulated and ordered process that occurs during the S phase of cell cycle. It begins with the licensing of origins of replication usually found in close proximity of actively transcribing genes owing perhaps to a profound influence of transcription factors on the epigenetic signatures and architecture of chromatin. Here we show that ETS transcription factors are novel regulators of MCM4 origin, whose binding sites are localized between two divergently transcribing MCM4 and PRKDC genes. c-ETS1 and c-ETS2 were recruited to the MCM4 origin respectively during the S and G1 phases of cell cycle. c-ETS2 binding was facilitated by an active chromatin distinguished by acetylated histone H3 orchestrated by histone acetyl transferase GCN5 and followed by HBO1 mediated histone H4 acetylation. Interestingly, c-ETS2 overexpression led to increased BrdU incorporation in the S phase cells while its down-regulation by RNA interference compromised the loading of pre-replicative complex at the origin. Conversely, the recruitment of c-ETS1 at the origin coincided with histone H3 methylation signature characteristic of closed chromatin conformation. As expected, enforced expression of c-ETS1 severely compromised DNA replication whereas its down-regulation enhanced DNA replication as evident from increased BrdU incorporation. Thus, c-ETS transcription factors appear to be key regulators of MCM4 origin where c-ETS2 seems to promote DNA replication whereas c-ETS1 functions as a negative regulator. © 2015 Elsevier B.V. All rights reserved.

  13. The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain

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    Brom Susana

    2011-06-01

    Full Text Available Abstract Background Bean-nodulating Rhizobium etli originated in Mesoamerica, while soybean-nodulating Sinorhizobium fredii evolved in East Asia. S. fredii strains, such as GR64, have been isolated from bean nodules in Spain, suggesting the occurrence of conjugative transfer events between introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d requires cointegration with the endogenous self-transmissible plasmid pRet42a. Aiming at further understanding the generation of diversity among bean nodulating strains, we analyzed the plasmids of S. fredii GR64: pSfr64a and pSfr64b (symbiotic plasmid. Results The conjugative transfer of the plasmids of strain GR64 was analyzed. Plasmid pSfr64a was self-transmissible, and required for transfer of the symbiotic plasmid. We sequenced pSfr64a, finding 166 ORFs. pSfr64a showed three large segments of different evolutionary origins; the first one presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; the second one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes. Accordingly, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, but did not contribute to symbiosis. The third segment contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Plasmid pRet42a was unable to substitute pSfr64a for induction of pSym transfer, and its own transfer was significantly diminished in GR64 background. The symbiotic plasmid pSfr64b was found to differ from typical R. etli symbiotic plasmids. Conclusions S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We infer that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid

  14. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    Science.gov (United States)

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.

  15. Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells

    NARCIS (Netherlands)

    Pijlman, G.P.; Vermeesch, A.M.G.; Vlak, J.M.

    2003-01-01

    Successive Viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did

  16. Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells

    NARCIS (Netherlands)

    Pijlman, G.P.; Vermeesch, A.M.G.; Vlak, J.M.

    2003-01-01

    Successive Viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did

  17. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  18. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    Science.gov (United States)

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  19. New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

    Science.gov (United States)

    Ercilla, Amaia; Llopis, Alba; Feu, Sonia; Aranda, Sergi; Ernfors, Patrik; Freire, Raimundo; Agell, Neus

    2016-06-01

    Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.

  20. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins.

    Science.gov (United States)

    Foulk, Michael S; Urban, John M; Casella, Cinzia; Gerbi, Susan A

    2015-05-01

    Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-exo digestion of nonreplicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand-independent λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo-controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na(+) instead of K(+) in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

  1. RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells.

    Science.gov (United States)

    Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

    2015-01-01

    Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.

  2. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  3. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Directory of Open Access Journals (Sweden)

    Gaëlle Demarre

    2010-05-01

    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  4. Who or What? Self-Replication and Function-Reproduction in the Origin of Life

    Science.gov (United States)

    New, Michael H.; Stassinopoulos, Dimitris; Monaco, Regina; Pohorille, Andrew; DeVincenzi, Donald (Technical Monitor)

    2002-01-01

    In this presentation, we will present results on the fundamental properties of two classes of replicating systems: autocatalytic replicators that reproduce exact copies of a template molecule, and function reproducers that maintain a set of essential functions without replicating the identities of the functional moieties. We will describe the stability and behavior in-the-large of autocatalytic replicators. Most importantly, we have found no sharp distinction between an autocatalytic and a non-autocatalytic domain. We will also present a new derivation of von Kiedrowski's square-root rate law. Function - reproducers are proposed as an important component of protocells and we will present theoretical results on a simple model system that incorporates known peptide biophysics. For a wide range of parameters, we have shown that this type of system can improve its overall performance, even in the absence of any method for information storage. This type of system improvement is defined to be non-genomic evolution.

  5. Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin.

    Science.gov (United States)

    Wu, J R; Keezer, S M; Gilbert, D M

    1998-03-16

    The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen-independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2-AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2-AP during the ODP transition also disrupted origin choice, whereas non-transformed cells arrested in G1-phase and then passed through a delayed ODP after removal of 2-AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G1-phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S-phase-promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.

  6. Origins of DNA Replication and Amplification in the Breast Cancer Genome

    Science.gov (United States)

    2013-09-01

    Fischer G (2008). Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication based mechanisms. PLoS...genomic features. Bioinformatics. 26: 841-2. Raveendranathan M., Chattopadhyay S., Bolon Y.T., Haworth J., Clarke D.J. and Bielinsky A.K. (2006

  7. Minimal region necessary for autonomous replication of pTAR.

    Science.gov (United States)

    Gallie, D R; Kado, C I

    1988-07-01

    The native 44-kilobase-pair plasmid pTAR, discovered in a grapevine strain of Agrobacterium tumefaciens, contains a single origin of DNA replication confined to a 1.0-kilobase-pair region of the macromolecule. This region (ori) confers functions sufficient for replication in Agrobacterium and Rhizobium species but not in Pseudomonas solanacearum, Pseudomonas glumae, Pseudomonas syringae pv. savastanoi, Xanthomonas campestris pv. campestris, and Escherichia coli. ori contains a repA gene that encodes a 28,000-dalton protein required for replication. Nucleotide sequencing of repA and its promoter region revealed four 8-base-pair palindromic repeats upstream of the repA coding region. Deletion of these repeats alters repA expression and plasmid copy number. Downstream of repA are three additional repeats in a region essential for replication. A locus responsible for plasmid partitioning (parA) and a putative second locus regulating plasmid copy number are part of the origin region and are required for stable plasmid maintenance.

  8. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome...

  9. The role of APC/C(Cdh1) in replication stress and origin of genomic instability.

    Science.gov (United States)

    Greil, C; Krohs, J; Schnerch, D; Follo, M; Felthaus, J; Engelhardt, M; Wäsch, R

    2016-06-01

    It has been proposed that the APC/C(Cdh1) functions as a tumor suppressor by maintaining genomic stability. However, the exact nature of genomic instability following loss of Cdh1 is unclear. Using biochemistry and live cell imaging of single cells we found that Cdh1 knockdown (kd) leads to strong nuclear stabilization of the substrates cyclin A and B and deregulated kinetics of DNA replication. Restoration of the Cdh1-dependent G2 DNA damage checkpoint did not result in G2 arrest but blocked cells in prometaphase, suggesting that these cells enter mitosis despite incomplete replication. This results in DNA double-strand breaks, anaphase bridges, cytokinesis defects and tetraploidization. Tetraploid cells are the source of supernumerary centrosomes following Cdh1-kd, leading to multipolar mitosis or centrosome clustering, in turn resulting in merotelic attachment and lagging chromosomes. Whereas some of these events cause apoptosis during mitosis, surviving cells may accumulate chromosomal aberrations.

  10. Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

    Science.gov (United States)

    Cayrou, Christelle; Coulombe, Philippe; Vigneron, Alice; Stanojcic, Slavica; Ganier, Olivier; Peiffer, Isabelle; Rivals, Eric; Puy, Aurore; Laurent-Chabalier, Sabine; Desprat, Romain; Méchali, Marcel

    2011-01-01

    In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5′ and 3′ of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates. PMID:21750104

  11. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  12. Selection and mapping of replication origins from a 500-kb region of the human X chromosome and their relationship to gene expression.

    Science.gov (United States)

    Rivella, S; Palermo, B; Pelizon, C; Sala, C; Arrigo, G; Toniolo, D

    1999-11-15

    In higher eukaryotes the mechanism controlling initiation of DNA replication remains largely unknown. New technologies are needed to shed light on how DNA replication initiates along the genome in specific regions. To identify the human DNA sequence requirements for initiation of replication, we developed a new method that allows selection of replication origins starting from large genomic regions of human DNA. We repeatedly isolated 15 new putative replication origins (PROs) from a human DNA region of 500 kb in which 17 genes have previously been characterized. Fine-mapping of these PROs showed that DNA replication can initiate at many specific points along actively transcribed DNA in the cell lines used for our selection. In conclusion, in this paper we describe a new method to identify PROs that suggests that the availability of initiation sites is dependent on the transcriptional state of the DNA.

  13. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2006-11-01

    Full Text Available Abstract Background Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Results Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin or plasmid pOri2 (with double ColE1 replication origins, were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA cycle and the pentose phosphate (PP pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG, the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. Conclusion The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

  14. New insights into HCV replication in original cells from Aedes mosquitoes.

    Science.gov (United States)

    Fallecker, Catherine; Caporossi, Alban; Rechoum, Yassine; Garzoni, Frederic; Larrat, Sylvie; François, Olivier; Fender, Pascal; Morand, Patrice; Berger, Imre; Petit, Marie-Anne; Drouet, Emmanuel

    2017-08-22

    The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines. First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments. Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism). These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

  15. Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

    Science.gov (United States)

    Sherman, J A; Matson, S W

    1994-10-21

    Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

  16. Analysis of stress-induced duplex destabilization (SIDD properties of replication origins, genes and intergenes in the fission yeast, Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Yadav Mukesh P

    2012-11-01

    Full Text Available Abstract Background Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe. Results We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD’s role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region. Conclusion SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.

  17. Absence of RNase H allows replication of pBR322 in Escherichia coli mutants lacking DNA polymerase I.

    Science.gov (United States)

    Kogoma, T

    1984-12-01

    rnh (formerly termed sdrA) mutants of Escherichia coli K-12, capable of continuous DNA replication in the absence of protein synthesis (stable DNA replication), are devoid of ribonuclease H (RNase H, EC 3.1.26.4) activity. Plasmid pBR322 was found to replicate in rnh mutants in the absence of DNA polymerase I, the polA gene product, which is normally required for replication of this plasmid. The plasmid copy number in polA rnh double mutants was as high as in the wild-type strains. When a chimeric construct between pBR322 and pSC101 was introduced into a polA rnh double mutant, the replication of the plasmid via the pBR322 replicon was inhibited if the plasmid also carried an rnh+ gene or if the host harbored an F' plasmid carrying an rnh+ gene. Thus, DNA polymerase I-independent replication of pBR322 requires the absence of RNase H activity. This alternative mechanism requiring neither DNA polymerase I nor RNase H appears to involve a transcriptional event in the region of the normal origin of replication.

  18. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  19. Evolutionary dynamics of RNA-like replicators : A bioinformatic approach to the origin of life

    NARCIS (Netherlands)

    Takeuchi, N.

    2010-01-01

    The origin of life has always attracted scientific inquiries. The RNA world hypothesis suggests that, before the evolution of DNAs and proteins, primordial life was based on RNAs both for information storage and chemical catalysis. In its simplest form, an RNA world consists of RNA molecules that ca

  20. The avian-origin H3N2 canine influenza virus has limited replication in swine

    Science.gov (United States)

    A genetically and antigenically distinct H3N2 canine influenza of avian-origin was detected in March of 2015 in Chicago, Illinois. A subsequent outbreak was reported with over 1,000 dogs in the Midwest affected. The potential for canine-to-swine transmission was unknown. Experimental infection in pi...

  1. H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing.

    Science.gov (United States)

    Rondinelli, Beatrice; Schwerer, Hélène; Antonini, Elena; Gaviraghi, Marco; Lupi, Alessio; Frenquelli, Michela; Cittaro, Davide; Segalla, Simona; Lemaitre, Jean-Marc; Tonon, Giovanni

    2015-03-11

    DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.

  2. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below

  3. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  4. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  5. Cooperative binding mode of the inhibitors of R6K replication, pi dimers.

    Science.gov (United States)

    Bowers, Lisa M; Filutowicz, Marcin

    2008-03-28

    The replication initiator protein, pi, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of pi bind to iterons in the gamma origin of plasmid R6K, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. Consequently, pi monomers activate replication, while pi dimers inhibit replication. Recently, it was shown that the monomeric form of pi binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers outcompete dimers for replication initiation when plasmid copy number and pi supply are low. Here, we examine cooperative binding of pi dimers and explore the role that these interactions may have in the inactivation of gamma origin. To examine pi dimer/iteron interactions in the absence of competing pi monomer/iteron interactions using wild-type pi, constructs were made with key base changes to each iteron that eliminate pi monomer binding yet have no impact on pi dimer binding. Our results indicate that, in the absence of pi monomers, pi dimers bind with greater cooperativity to alternate iterons than to adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by pi dimers.

  6. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

    OpenAIRE

    1989-01-01

    P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomp...

  7. Plasmids and rickettsial evolution: insight from Rickettsia felis.

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    Full Text Available BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG or spotted fever group (SFG rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives also occur in AG (but not SFG rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in

  8. Unique and universal features of Epsilonproteobacterial origins of chromosome replication and DnaA-DnaA box interactions

    Directory of Open Access Journals (Sweden)

    Pawel Jaworski

    2016-09-01

    Full Text Available In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC. While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e. they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterised Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterised. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1 and downstream (oriC2 of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5

  9. Unique and Universal Features of Epsilonproteobacterial Origins of Chromosome Replication and DnaA-DnaA Box Interactions

    Science.gov (United States)

    Jaworski, Pawel; Donczew, Rafal; Mielke, Thorsten; Thiel, Marcel; Oldziej, Stanislaw; Weigel, Christoph; Zawilak-Pawlik, Anna

    2016-01-01

    In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC). While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e., they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterized Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterized. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or whether they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes, and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1) and downstream (oriC2) of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5′-TTCAC-3

  10. Chasing the Origin of Viruses: Capsid-Forming Genes as a Life-Saving Preadaptation within a Community of Early Replicators.

    Science.gov (United States)

    Jalasvuori, Matti; Mattila, Sari; Hoikkala, Ville

    2015-01-01

    Virus capsids mediate the transfer of viral genetic information from one cell to another, thus the origin of the first viruses arguably coincides with the origin of the viral capsid. Capsid genes are evolutionarily ancient and their emergence potentially predated even the origin of first free-living cells. But does the origin of the capsid coincide with the origin of viruses, or is it possible that capsid-like functionalities emerged before the appearance of true viral entities? We set to investigate this question by using a computational simulator comprising primitive replicators and replication parasites within a compartment matrix. We observe that systems with no horizontal gene transfer between compartments collapse due to the rapidly emerging replication parasites. However, introduction of capsid-like genes that induce the movement of randomly selected genes from one compartment to another rescues life by providing the non-parasitic replicators a mean to escape their current compartments before the emergence of replication parasites. Capsid-forming genes can mediate the establishment of a stable meta-population where parasites cause only local tragedies but cannot overtake the whole community. The long-term survival of replicators is dependent on the frequency of horizontal transfer events, as systems with either too much or too little genetic exchange are doomed to succumb to replication-parasites. This study provides a possible scenario for explaining the origin of viral capsids before the emergence of genuine viruses: in the absence of other means of horizontal gene transfer between compartments, evolution of capsid-like functionalities may have been necessary for early life to prevail.

  11. Lipopolysaccharide induces immune activation and SIV replication in rhesus macaques of Chinese origin.

    Directory of Open Access Journals (Sweden)

    Rong Bao

    Full Text Available BACKGROUND: Chronic immune activation is a hallmark of progressive HIV infection and a key determinant of immunodeficiency in HIV-infected individuals. Bacterial lipopolysaccharide (LPS in the circulation has been implicated as a key factor in HIV infection-related systemic immune activation. We thus investigate the impact of LPS on systemic immune activation in simian immunodeficiency virus (SIV-infected rhesus macaques of Chinese origin. METHODS: The animals were inoculated intravenously with SIVmac239. The levels of plasma viral load and host inflammatory cytokines in PBMC were measured by real-time RT-PCR. CD4/CD8 ratio and systemic immune activation markers were examined by flow cytometric analysis of PBMCs. White blood cell and neutrophil counts and C Reactive Protein levels were determined using biochemistry analyzer. The plasma levels of LPS were determined by Tachypleus Amebocyte Lysate (TAL test. RESULTS: The animals inoculated with SIVmac239 became infected as evidenced by the increased plasma levels of SIV RNA and decreased CD4/CD8 ratio. LPS administration of SIV-infected animals induced a transient increase of plasma SIV RNA and immune activation, which was indicated by the elevated expression of the inflammatory cytokines and CD4+HLA-DR+ T cells in PBMCs. CONCLUSIONS: These data support the concept that LPS is a driving factor in systemic immune activation of HIV disease.

  12. The sporulation protein SirA inhibits the binding of DnaA to the origin of replication by contacting a patch of clustered amino acids.

    Science.gov (United States)

    Rahn-Lee, Lilah; Merrikh, Houra; Grossman, Alan D; Losick, Richard

    2011-03-01

    Bacteria regulate the frequency and timing of DNA replication initiation by controlling the activity of the replication initiator protein DnaA. SirA is a recently discovered regulator of DnaA in Bacillus subtilis whose synthesis is turned on at the start of sporulation. Here, we demonstrate that SirA contacts DnaA at a patch of 3 residues located on the surface of domain I of the replication initiator protein, corresponding to the binding site used by two unrelated regulators of DnaA found in other bacteria. We show that the interaction of SirA with domain I inhibits the ability of DnaA to bind to the origin of replication. DnaA mutants containing amino acid substitutions of the 3 residues are functional in replication initiation but are immune to inhibition by SirA.

  13. Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation.

    Science.gov (United States)

    Georgescu, Roxana; Yuan, Zuanning; Bai, Lin; de Luna Almeida Santos, Ruda; Sun, Jingchuan; Zhang, Dan; Yurieva, Olga; Li, Huilin; O'Donnell, Michael E

    2017-01-31

    The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.

  14. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Guss, Adam M [ORNL; Olson, Daniel G. [Thayer School of Engineering at Dartmouth; Caiazza, Nicky [Mascoma Corporation; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2012-01-01

    BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  15. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Guss Adam M

    2012-05-01

    Full Text Available Abstract Background Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. Results We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. Conclusions E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  16. Cloned origin of DNA replication in c-myc gene can function and be transmitted in transgenic mice in an episomal state.

    OpenAIRE

    Sudo, Katsuko; Ogata, Masaaki; Sato, Yoshinari; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    1990-01-01

    The c-myc protein has recently been shown to interact with a region possessing putative origin of DNA replication and enhancer activities located 2 kb upstream of the c-myc gene itself. Transgenic mice were obtained by injecting constructs containing this region, termed pmyc(H-P), into fertilized mouse eggs. The transgenic elements were capable of efficient replication in all mouse tissues examined and were maintained in an episomal state even in highly differentiated cells. Moreover, pmyc(H-...

  17. Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate.

    Directory of Open Access Journals (Sweden)

    Fay E Dawes

    Full Text Available This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-. DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

  18. Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

    Indian Academy of Sciences (India)

    Vinay Kumar Srivastava; Dharani Dhar Dubey

    2007-08-01

    Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.

  19. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu

    2006-01-01

    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  20. Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus.

    Science.gov (United States)

    Park, Miey; Kim, Min-Soo; Lee, Kyung-Min; Hwang, Sue-Yun; Ahn, Tae In

    2009-01-01

    A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.

  1. Condensin- and Replication-Mediated Bacterial Chromosome Folding and Origin Condensation Revealed by Hi-C and Super-resolution Imaging.

    Science.gov (United States)

    Marbouty, Martial; Le Gall, Antoine; Cattoni, Diego I; Cournac, Axel; Koh, Alan; Fiche, Jean-Bernard; Mozziconacci, Julien; Murray, Heath; Koszul, Romain; Nollmann, Marcelo

    2015-08-20

    Chromosomes of a broad range of species, from bacteria to mammals, are structured by large topological domains whose precise functional roles and regulatory mechanisms remain elusive. Here, we combine super-resolution microscopies and chromosome-capture technologies to unravel the higher-order organization of the Bacillus subtilis chromosome and its dynamic rearrangements during the cell cycle. We decipher the fine 3D architecture of the origin domain, revealing folding motifs regulated by condensin-like complexes. This organization, along with global folding throughout the genome, is present before replication, disrupted by active DNA replication, and re-established thereafter. Single-cell analysis revealed a strict correspondence between sub-cellular localization of origin domains and their condensation state. Our results suggest that the precise 3D folding pattern of the origin domain plays a role in the regulation of replication initiation, chromosome organization, and DNA segregation.

  2. RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

    Directory of Open Access Journals (Sweden)

    Alexis L. Santana

    2017-02-01

    Full Text Available RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS/toll-like receptor (TLR4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB signaling during murine gammaherpesvirus 68 (MHV68 infection: a latent B cell line (HE-RIT inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.

  3. RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency

    Science.gov (United States)

    Santana, Alexis L.; Oldenburg, Darby G.; Kirillov, Varvara; Malik, Laraib; Dong, Qiwen; Sinayev, Roman; Marcu, Kenneth B.; White, Douglas W.; Krug, Laurie T.

    2017-01-01

    RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA. PMID:28212352

  4. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  5. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    Science.gov (United States)

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

  6. Dissemination of plasmid-encoded AmpC β-lactamases in antimicrobial resistant Salmonella serotypes originating from humans, pigs and the swine environment.

    Science.gov (United States)

    Keelara, Shivaramu; Thakur, Siddhartha

    2014-09-17

    The aim of this study was to characterize and determine the inter-serovar exchange of AmpC β-lactamase conferring plasmids isolated from humans, pigs and the swine environment. Plasmids isolated from a total of 21 antimicrobial resistant (AMR) Salmonella isolates representing human clinical cases (n=6), pigs (n=6) and the swine farm environment (n=9) were characterized by replicon typing and restriction digestion, inter-serovar transferability by conjugation, and presence of AmpC β-lactamase enzyme encoding gene blaCMY-2 by southern hybridization. Based on replicon typing, the majority (17/21, 81%) of the plasmids belonged to the I1-Iγ Inc group and were between 70 and 103kb. The potential for inter-serovar plasmid transfer was further confirmed by the PCR detection of AMR genes on the plasmids isolated from trans-conjugants. Plasmids from Salmonella serovars Anatum, Ouakam, Johannesburg and Typhimurium isolated from the same cohort of pigs and their environment and S. Heidelberg from a single human clinical isolate had identical plasmids based on digestion with multiple restriction enzymes (EcoRI, HindIII and PstI) and southern blotting. We demonstrated likely horizontal inter-serovar exchange of plasmid-encoding AmpC β-lactamases resistance among MDR Salmonella serotypes isolated from pigs, swine farm environment and clinical human cases. This study provides valuable information on the role of the swine farm environment and by extension other livestock farm environments, as a potential reservoir of resistant bacterial strains that potentially transmit resistance determinants to livestock, in this case, swine, humans and possibly other hosts by horizontal exchange of plasmids.

  7. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  8. Full-Length Nucleotide Sequences of mcr-1-Harboring Plasmids Isolated from Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates of Different Origins.

    Science.gov (United States)

    Zurfluh, Katrin; Klumpp, Jochen; Nüesch-Inderbinen, Magdalena; Stephan, Roger

    2016-09-01

    Here, we present the full sequences of three mcr-1-carrying plasmids isolated from extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli The plasmids belong to three different replicon types and are 34,640 bp, 209,401 bp, and 247,885 bp in size. We describe for the first time a composite transposon containing mcr-1 localized on a multidrug-resistant (MDR) IncHI2 plasmid harboring additional determinants of resistance to six different classes of antibiotics, including the ESBL gene blaCTX-M-1, and heavy metal resistance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage

    Directory of Open Access Journals (Sweden)

    Hervé Técher

    2016-02-01

    Full Text Available Mammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates.

  10. The Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion.

    Directory of Open Access Journals (Sweden)

    Martina Stevanoni

    2016-07-01

    Full Text Available It is well known that DNA replication affects the stability of several trinucleotide repeats, but whether replication profiles of human loci carrying an expanded repeat differ from those of normal alleles is poorly understood in the endogenous context. We investigated this issue using cell lines from Friedreich's ataxia patients, homozygous for a GAA-repeat expansion in intron 1 of the Frataxin gene. By interphase, FISH we found that in comparison to the normal Frataxin sequence the replication of expanded alleles is slowed or delayed. According to molecular combing, origins never fired within the normal Frataxin allele. In contrast, in mutant alleles dormant origins are recruited within the gene, causing a switch of the prevalent fork direction through the expanded repeat. Furthermore, a global modification of the replication profile, involving origin choice and a differential distribution of unidirectional forks, was observed in the surrounding 850 kb region. These data provide a wide-view of the interplay of events occurring during replication of genes carrying an expanded repeat.

  11. Multiple origin usage for DNA replication in sdrA(rnh) mutants of Escherichia coli K-12. Initiation in the absence of oriC.

    Science.gov (United States)

    de Massy, B; Fayet, O; Kogoma, T

    1984-09-15

    In stable DNA replication (sdrA/rnh) mutants of Escherichia coli, initiation of rounds of DNA replication occurs in the absence of the normal origin of replication, oriC. To determine whether or not the initiation occurs at a fixed site(s) on the chromosome in sdrA mutants, the DNA from exponentially growing sdrA mutant cells with or without the oriC site (delta oriC) was analyzed for the relative copy numbers of various genes along the chromosome. The results suggest that there are at least four fixed sites or regions of the sdrA delta oriC chromosome from which DNA replication can be initiated in the absence of the oriC sequence.

  12. BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression

    Directory of Open Access Journals (Sweden)

    Shwu-Yuan Wu

    2016-08-01

    Full Text Available Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4, a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV to viral early gene and cellular matrix metalloproteinase-9 (MMP-9 promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment.

  13. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication.

    Science.gov (United States)

    Gong, Shiaoching; Yang, Xiangdong William; Li, Chenjian; Heintz, Nathaniel

    2002-12-01

    Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

  14. Replicated origin of female-biased adult sex ratio in introduced populations of the trinidadian guppy (Poecilia reticulata).

    Science.gov (United States)

    Arendt, Jeffrey D; Reznick, David N; López-Sepulcre, Andres

    2014-08-01

    There are many theoretical and empirical studies explaining variation in offspring sex ratio but relatively few that explain variation in adult sex ratio. Adult sex ratios are important because biased sex ratios can be a driver of sexual selection and will reduce effective population size, affecting population persistence and shapes how populations respond to natural selection. Previous work on guppies (Poecilia reticulata) gives mixed results, usually showing a female-biased adult sex ratio. However, a detailed analysis showed that this bias varied dramatically throughout a year and with no consistent sex bias. We used a mark-recapture approach to examine the origin and consistency of female-biased sex ratio in four replicated introductions. We show that female-biased sex ratio arises predictably and is a consequence of higher male mortality and longer female life spans with little effect of offspring sex ratio. Inconsistencies with previous studies are likely due to sampling methods and sampling design, which should be less of an issue with mark-recapture techniques. Together with other long-term mark-recapture studies, our study suggests that bias in offspring sex ratio rarely contributes to adult sex ratio in vertebrates. Rather, sex differences in adult survival rates and longevity determine vertebrate adult sex ratio.

  15. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

    2005-01-01

    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. Th

  16. Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr.

    Science.gov (United States)

    Zhang, Wan-Jiang; Xu, Xing-Ran; Schwarz, Stefan; Wang, Xiu-Mei; Dai, Lei; Zheng, Hua-Jun; Liu, Siguo

    2014-02-01

    To determine the complete nucleotide sequence of the multidrug resistance plasmid pSCEC2, isolated from a porcine Escherichia coli strain, and to analyse it with particular reference to the cfr gene region. Plasmid pSCEC2 was purified from its E. coli J53 transconjugant and then sequenced using the 454 GS-FLX System. After draft assembly, predicted gaps were closed by PCR with subsequent sequencing of the amplicons. Plasmid pSCEC2 is 135 615 bp in size and contains 200 open reading frames for proteins of ≥100 amino acids. Analysis of the sequence of pSCEC2 revealed two resistance gene segments. The 4.4 kb cfr-containing segment is flanked by two IS256 elements in the same orientation, which are believed to be involved in the dissemination of the rRNA methylase gene cfr. The other segment harbours the resistance genes floR, tet(A)-tetR, strA/strB and sul2, which have previously been found on other IncA/C plasmids. Except for these two resistance gene regions, the pSCEC2 backbone displayed >99% nucleotide sequence identity to that of other IncA/C family plasmids isolated in France, Chile and the USA. The cfr gene was identified on an IncA/C plasmid, which is well known for its broad host range and transfer and maintenance properties. The location on such a plasmid will further accelerate the dissemination of cfr and co-located resistance genes among different Gram-negative bacteria. The genetic context of cfr on plasmid pSCEC2 underlines the complexity of cfr transfer events and confirms the role that insertion sequences play in the spread of cfr.

  17. An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.

    Directory of Open Access Journals (Sweden)

    Reza Djavadian

    2016-06-01

    Full Text Available Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "βγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.

  18. RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro.

    Science.gov (United States)

    Ogawa, T; Pickett, G G; Kogoma, T; Kornberg, A

    1984-02-01

    Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium. Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro. Furthermore, a 760-base-pair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+. In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system.

  19. Analysis of a novel 8.9kb cryptic plasmid from Bacteroides uniformis, its long-term stability and spread within human microbiota.

    Science.gov (United States)

    Shkoporov, Andrei N; Khokhlova, Ekaterina V; Kulagina, Elena V; Smeianov, Vladimir V; Kuchmiy, Anna A; Kafarskaya, Lyudmila I; Efimov, Boris A

    2013-03-01

    The analysis of plasmid content in dominant Bacteroidales order intestinal strains isolated from the same child at a 5 year interval identified a 8.9 kb plasmid in Bacteroides uniformis BUN24 strain isolated at age 6 and indistinguishably sized plasmids in the isolates of B. uniformis, B. vulgatus, B. intesinalis, and Parabacteroides distasonis at age 11. We sequenced a B. uniformis BUN24 plasmid, designated pBUN24, and using molecular surveys of diverse species we established that this 8944bp molecule (G+C content 43.5%) represents a novel family of small cryptic Bacteroidales plasmids. The replication region of pBUN24 was experimentally localized to a 1707-bp fragment that includes a putative repA gene, coding for a protein of Rep_3 superfamily of replication proteins of theta-type plasmids preceded by a putative iteron-containing origin of replication. The other open reading frames (ORFs) identified in pBUN24 sequence include a putative tad-ata-type toxin-antitoxin and mobA-mobB mobilization modules, as well as seven additional cryptic ORFs. The interaction of Tad and Ada components demonstrated by a pull-down assay and the toxicity of Tad in Escherichia coli host suggests the functionality of the plasmid addiction module. Re-sequencing of plasmids in two Bacteroides strains isolated at the age of 11 showed 100% nucleotide identity to pBUN24. This data supports the notion that this plasmid is transmissible to other Bacteroidales strains in the natural ecosystem. The possible roles of toxin-antitoxin system and other proteins encoded by pBUN24 in providing an apparent ecological advantage to the plasmid-harbouring strains of a bacterial symbiont in the human gut deserve further investigation. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Frequency and diversity of small cryptic plasmids in the genus Rahnella

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    Summers David K

    2010-02-01

    Full Text Available Abstract Background Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids. Results In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified. Conclusions For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to diffent groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the

  1. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins

    DEFF Research Database (Denmark)

    Foulk, M. S.; Urban, J. M.; Casella, Cinzia;

    2015-01-01

    Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (lambda-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent...... are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K...

  2. Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication.

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    Subhash C Verma

    2011-11-01

    Full Text Available Kaposi's sarcoma associated herpesvirus (KSHV, an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA. Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD. Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA

  3. Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

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    Angel Angelov

    2011-01-01

    Full Text Available Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

  4. Construction of pTM series plasmids for gene expression in Brucella species.

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

    2016-04-01

    Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp.

  5. Disclosing the Firing Protocol of Athenian Pottery Production: A Raman and Colorimetric Study of Replicates and Original Samples

    Science.gov (United States)

    Cianchetta, I.; Trentelman, K.; Maish, J.; Walton, M.

    2014-06-01

    The work presented here examines the technological foundations of Athenian pottery production through the replication of the firing technology. Raman spectroscopy and colorimetry were used to investigate composition and color of ceramics slips.

  6. High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements.

    Science.gov (United States)

    Chang, Fujung; May, Caitlin D; Hoggard, Timothy; Miller, Jeremy; Fox, Catherine A; Weinreich, Michael

    2011-08-01

    In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.

  7. CFE-1, a novel plasmid-encoded AmpC beta-lactamase with an ampR gene originating from Citrobacter freundii.

    Science.gov (United States)

    Nakano, Ryuichi; Okamoto, Ryoichi; Nakano, Yumiko; Kaneko, Kenichi; Okitsu, Naohiro; Hosaka, Yoshio; Inoue, Matsuhisa

    2004-04-01

    A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded beta-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a beta-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC beta-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a beta-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC beta-lactamase, CFE-1, with an ampR gene derived from C. freundii.

  8. CFE-1, a Novel Plasmid-Encoded AmpC β-Lactamase with an ampR Gene Originating from Citrobacter freundii

    Science.gov (United States)

    Nakano, Ryuichi; Okamoto, Ryoichi; Nakano, Yumiko; Kaneko, Kenichi; Okitsu, Naohiro; Hosaka, Yoshio; Inoue, Matsuhisa

    2004-01-01

    A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded β-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a β-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC β-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a β-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC β-lactamase, CFE-1, with an ampR gene derived from C. freundii. PMID:15047515

  9. The evolution of strand preference in simulated RNA replicators with strand displacement: implications for the origin of transcription.

    Science.gov (United States)

    Takeuchi, Nobuto; Salazar, Laura; Poole, Anthony M; Hogeweg, Paulien

    2008-08-11

    The simplest conceivable example of evolving systems is RNA molecules that can replicate themselves. Since replication produces a new RNA strand complementary to a template, all templates would eventually become double-stranded and, hence, become unavailable for replication. Thus the problem of how to separate the two strands is considered a major issue for the early evolution of self-replicating RNA. One biologically plausible way to copy a double-stranded RNA is to displace a preexisting strand by a newly synthesized strand. Such copying can in principle be initiated from either the (+) or (-) strand of a double-stranded RNA. Assuming that only one of them, say (+), can act as replicase when single-stranded, strand displacement produces a new replicase if the (-) strand is the template. If, however, the (+) strand is the template, it produces a new template (but no replicase). Modern transcription exhibits extreme strand preference wherein anti-sense strands are always the template. Likewise, replication by strand displacement seems optimal if it also exhibits extreme strand preference wherein (-) strands are always the template, favoring replicase production. Here we investigate whether such strand preference can evolve in a simple RNA replicator system with strand displacement. We first studied a simple mathematical model of the replicator dynamics. Our results indicated that if the system is well-mixed, there is no selective force acting upon strand preference per se. Next, we studied an individual-based simulation model to investigate the evolution of strand preference under finite diffusion. Interestingly, the results showed that selective forces "emerge" because of finite diffusion. Strikingly, the direction of the strand preference that evolves [i.e. (+) or (-) strand excess] is a complex non-monotonic function of the diffusion intensity. The mechanism underlying this behavior is elucidated. Furthermore, a speciation-like phenomenon is observed under

  10. The evolution of strand preference in simulated RNA replicators with strand displacement: Implications for the origin of transcription

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2008-08-01

    Full Text Available Abstract Background The simplest conceivable example of evolving systems is RNA molecules that can replicate themselves. Since replication produces a new RNA strand complementary to a template, all templates would eventually become double-stranded and, hence, become unavailable for replication. Thus the problem of how to separate the two strands is considered a major issue for the early evolution of self-replicating RNA. One biologically plausible way to copy a double-stranded RNA is to displace a preexisting strand by a newly synthesized strand. Such copying can in principle be initiated from either the (+ or (- strand of a double-stranded RNA. Assuming that only one of them, say (+, can act as replicase when single-stranded, strand displacement produces a new replicase if the (- strand is the template. If, however, the (+ strand is the template, it produces a new template (but no replicase. Modern transcription exhibits extreme strand preference wherein anti-sense strands are always the template. Likewise, replication by strand displacement seems optimal if it also exhibits extreme strand preference wherein (- strands are always the template, favoring replicase production. Here we investigate whether such strand preference can evolve in a simple RNA replicator system with strand displacement. Results We first studied a simple mathematical model of the replicator dynamics. Our results indicated that if the system is well-mixed, there is no selective force acting upon strand preference per se. Next, we studied an individual-based simulation model to investigate the evolution of strand preference under finite diffusion. Interestingly, the results showed that selective forces "emerge" because of finite diffusion. Strikingly, the direction of the strand preference that evolves [i.e. (+ or (- strand excess] is a complex non-monotonic function of the diffusion intensity. The mechanism underlying this behavior is elucidated. Furthermore, a speciation

  11. Insights into the initiation of JC virus DNA replication derived from the crystal structure of the T-antigen origin binding domain.

    Directory of Open Access Journals (Sweden)

    Gretchen Meinke

    2014-02-01

    Full Text Available JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML. PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel "pocket"; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.

  12. Complete sequencing of the bla(NDM-1-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Sekizuka

    Full Text Available The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1 was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38 and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb and Salmonella enterica serovar Newport pSN254 (176.4 kb. The bla(NDM-1 gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1 gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1 gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1 gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.

  13. Complete sequencing of the bla(NDM-1)-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens.

    Science.gov (United States)

    Sekizuka, Tsuyoshi; Matsui, Mari; Yamane, Kunikazu; Takeuchi, Fumihiko; Ohnishi, Makoto; Hishinuma, Akira; Arakawa, Yoshichika; Kuroda, Makoto

    2011-01-01

    The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2)-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla(NDM-1) gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1) gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1)-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1) gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1) gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.

  14. Complete mitochondrial genome of the urohaze-goby Glossogobius olivaceus (Perciformes, Gobiidae) and structure comparison of light strand replication origin in Gobioidei.

    Science.gov (United States)

    Sun, Yuena; Wei, Tao; Jin, Xiaoxiao; Qin, Yu; Xu, Tianjun

    2015-01-01

    The genome of Glossogobius olivaceus (G. olivaceus) was 16,568 bp in length with a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and 2 non-coding region (the control region and origin of the light strand replication). The conserved motif 5'-GCCGG-3' was determined in the origin of light strand replication of G. olivaceus. The G. olivaceus mitogenome base composition was: T 25.65%, C 29.82% A 27.17% and G 17.36%, with a slight A+T bias of 52.82%, which was similar to most of the vertebrate mitogenomes. The mitochondrial genome of G. olivaceus had common features regarding gene arrangement and tRNA structures compared with those of other bony fishes.

  15. 我国D2-43病毒株PrM-E基因的复制型载体质粒DNA的免疫原性%The Immunogenicity of Replicative Virus Plasmid DNA Containing PrM-E Gene of Chinese D2-43 Virus Strain

    Institute of Scientific and Technical Information of China (English)

    陈水平; 秦鄂德; 于曼; 胡志君; 赵卫; 范宝昌; 王鹏程; 杨佩英

    2002-01-01

    观察含我国登革2型病毒株(D2-43)的PrM-E基因的复制型SFV(semliki forest virus)重组质粒DNA的免疫原性,为登革新型疫苗的研制提供依据.将PrM-E基因自T载体上切下,插入复制型SFV病毒载体质粒DNA中.将此重组质粒DNA以电穿孔法导入BHK21细胞,用间接免疫荧光法在感染细胞内可检测到登革2型病毒特异蛋白的表达.采用去除内毒素的质粒提取试剂盒制备重组质粒DNA,然后以不同剂量通过肌肉多点注射途径免疫Balb/c鼠,获得的鼠血清可与登革D2-43感染的C6/36抗原片起特异的抗原抗体反应.结果表明,含登革2型病毒PrM-E基因的复制型SFV病毒载体质粒DNA在Balb/c鼠中可诱导登革2型病毒特异抗体的产生,但抗体水平较低.%To study the immunogenicity of replicative plasmid DNA containing PrM-E gene of Chinese D2-43virus strain and lay a foundation for new vaccine of dengue, the PrM-E gene was cut from pGEM-T-ME with Apa I and Cla I and inserted into replicative semliki forest virus (SFV) vector. The recombinant plasmid DNA was electroporated into BHK21 cells, then expression products specific to dengue virus type 2 could be detected by IFA (indirect immunofluorescence assay) in transfected cells. Endotoxin-free plasmid DNA was prepared with EndoFree plasmid Maxi kit. Mice were immunized with different doses of the recombinant SFV plasmid DNA intramuscularly at multiple sites. The titres of antibody to dengue virus type 2 was measured by IFA. And sera with recombinant plasmid, could react with antigen of dengue 2 virus on microscope slides. The results showed that recombinant SFV plasmid DNA containing the PrM-E gene of D2-43 virus strain, could produce specific antibody to dengue 2 virus in mice, but the titre was low.

  16. Broader utilization of origins of DNA replication in cancer cell lines along a 78 kb region of human chromosome 2q34.

    Science.gov (United States)

    Valenzuela, Manuel S; Hu, Lan; Lueders, John; Walker, Robert; Meltzer, Paul S

    2012-01-01

    Human DNA replication depends on the activation of thousands of origins distributed within the genome. The actual distribution of origins is not known, nor whether this distribution is unique to a cell type, or if it changes with the proliferative state of the cell. In this study, we have employed a real-time PCR-based nascent strand DNA abundance assay, to determine the location of origins along a 78 kb region on Chr2q34. Preliminary studies using nascent DNA strands isolated from either HeLa and normal skin fibroblast cells showed that in both cell lines peaks of high origin activity mapped in similar locations. However, the overall origin profile in HeLa cells corresponded to broad origin activation zones, whereas in fibroblasts a more punctuated profile of origin activation was observed. To investigate the relevance of this differential origin profile, we compared the origin distribution profiles in breast cancer cell lines MDA-MB-231, BT-474, and MCF-7, to their normal counterpart MCF-10A. In addition, the CRL7250 cell line was also used as a normal control. Our results validated our earlier observation and showed that the origin profile in normal cell lines exhibited a punctuated pattern, in contrast to broader zone profiles observed in the cancer cell lines. A quantitative analysis of origin peaks revealed that the number of activated origins in cancer cells is statistically larger than that obtained in normal cells, suggesting that the flexibility of origin usage is significantly increased in cancer cells compared to their normal counterparts.

  17. Origin of life. Primordial genetics: Information transfer in a pre-RNA world based on self-replicating beta-sheet amyloid conformers.

    Science.gov (United States)

    Maury, Carl Peter J

    2015-10-07

    The question of the origin of life on Earth can largely be reduced to the question of what was the first molecular replicator system that was able to replicate and evolve under the presumably very harsh conditions on the early Earth. It is unlikely that a functional RNA could have existed under such conditions and it is generally assumed that some other kind of information system preceded the RNA world. Here, I present an informational molecular system that is stable, self-replicative, environmentally responsive, and evolvable under conditions characterized by high temperatures, ultraviolet and cosmic radiation. This postulated pregenetic system is based on the amyloid fold, a functionally unique polypeptide fold characterized by a cross beta-sheet structure in which the beta strands are arranged perpendicular to the fiber axis. Beside an extraordinary structural robustness, the amyloid fold possesses a unique ability to transmit information by a three-dimensional templating mechanism. In amyloidogenesis short peptide monomers are added one by one to the growing end of the fiber. From the same monomeric subunits several structural variants of amyloid may be formed. Then, in a self-replicative mode, a specific amyloid conformer can act as a template and confer its spatially encoded information to daughter molecular entities in a repetitive way. In this process, the specific conformational information, the spatially changed organization, is transmitted; the coding element is the steric zipper structure, and recognition occurs by amino acid side chain complementarity. The amyloid information system fulfills several basic requirements of a primordial evolvable replicator system: (i) it is stable under the presumed primitive Earth conditions, (ii) the monomeric building blocks of the informational polymer can be formed from available prebiotic compounds, (iii) the system is self-assembling and self-replicative and (iv) it is adaptive to changes in the environment and

  18. DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

    2012-01-01

    In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

  19. Distribution of small native plasmids in Streptococcus pyogenes in India.

    Science.gov (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  20. A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

    Directory of Open Access Journals (Sweden)

    Kvitko Brian H

    2012-03-01

    Full Text Available Abstract Background The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (oriR6Kγ is dependent on the pir gene-encoded π protein. Originally encoded by R6K, this protein is usually provided in trans in hosts engineered to support replication of plasmids harboring oriR6Kγ. In Escherichia coli this is commonly achieved by chromosomal integration of pir either via lysogenization with a λpir phage or homologous recombination at a pre-determined locus. Findings Current methods for construction of host strains for oriR6Kγ-containing plasmids involve procedures that do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn7-based method for rapid and reliable construction of pir+ host strains. Using a curable mini-Tn7 delivery plasmid, pir expressing derivatives of several commonly used E. coli cloning and mobilizer strains were isolated using both the wild-type pir+ gene as well as the copy-up pir-116 allele. In addition, we isolated pir+ and pir-116 expressing derivatives of a clinical isolate of Salmonella enterica serovar Typhimurium. In both E. coli and S. enterica serovar Typhimurium, the presence of the pir+ wild-type or pir-116 alleles allowed the replication of oriR6Kγ-containing plasmids. Conclusions A mini-Tn7 system was employed for rapid and reliable engineering of E. coli and S. enterica serovar Typhimurium host strains for plasmids containing oriR6Kγ. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with oriR6Kγ.

  1. A Novel Mechanism for Activator-Controlled Initiation of DNA Replication that Resolves the Auto-regulation Sequestration Paradox

    Science.gov (United States)

    Nilsson, K.; Ehrenberg, M.

    For bacterial genes to be inherited to the next bacterial generation, the gene containing DNA sequences must be duplicated before cell division so that each daughter cell contains a complete set of genes. The duplication process is called DNA replication and it starts at one defined site on the DNA molecule called the origin of replication (oriC) [1]. In addition to chromosomal DNA, bacteria often also contain plasmid DNA. Plasmids are extra-chromosomal DNA molecules carrying genes that increase the fitness of their host in certain environments, with genes encoding antibiotic resistance as a notorious example [2]. The chromosome is found at a low per cell copy number and initiation of replication takes place synchronously once every cell generation [3,4], while many plasmids exist at a high copy number and replication initiates asynchronously, throughout the cell generation [5]. In this chapter we present a novel mechanism for the control of initiation of replication, where one type of molecule may activate a round of replication by binding to the origin of replication and also regulate its own synthesis accurately. This mechanism of regulating the initiation of replication also offers a novel solution to the so-called auto-regulation sequestration paradox, i.e. how a molecule sequestered by binding to DNA may at the same time accurately regulate its own synthesis [6]. The novel regulatory mechanism is inspired by the molecular set-up of the replication control of the chromosome in the bacterium Escherichia coli and is here transferred into a plasmid model. This allows us to illustrate principles of replication control in a simple way and to put the novel mechanism into the context of a previous analysis of plasmids regulated by inhibitor-dilution copy number control [7]. We analyze factors important for a sensitive response of the replication initiation rate to changes in plasmid concentration in an asynchronous model and discover a novel mechanism for creating a

  2. Identification of the replication origins from Cyanothece ATCC 51142 and their interactions with the DnaA protein: from in silico to in vitro studies

    Directory of Open Access Journals (Sweden)

    He eHuang

    2015-12-01

    Full Text Available Based on the complete genome of Cyanothece ATCC 51142, the oriCs of both the circular and linear chromosomes in Cyanothece ATCC 51142 have been predicted by utilizing a web-based system Ori-Finder. Here, we provide the experimental supports for the results of Ori-Finder to identify the origins of replication of Cyanothece ATCC 51142 and their interactions with initiator protein DnaA . The two replication origins are both composed of three characteristically arranged DnaA boxes, and an AT-rich stretch, and the oriC in circular chromosome is followed by the dnaN gene. The dnaA gene is located downstream of the origin of circular chromosome and expresses a typical DnaA protein that follows the division into four domains (I, II, III, IV, as with other members of the DnaA protein family. Here, we report the purification of DnaA (IV and characterize the interaction of the purified protein with the replication origins, in order to offer experimental supports for the prediction. Combined with experimental validation, we identified the oriCs of Cyanothece ATCC 51142. The results of EMSA and DNase I footprint assay demonstrate that the C-terminal domain of Cyanothece ATCC 51142 DnaA protein specifically binds the oriCs of both the circular and linear chromosomes, and DNase I footprint assay demonstrates that DnaA (IV has a hypersensitive affinity interaction with DnaA boxes in both oriCs. The results also confirm the results predicted by Ori-Finder.

  3. THE ENDOGENOUS BACILLUS-SUBTILIS (NATTO) PLASMIDS PTA1015 AND PTA1040 CONTAIN SIGNAL PEPTIDASE-ENCODING GENES - IDENTIFICATION OF A NEW STRUCTURAL MODULE ON CRYPTIC PLASMIDS

    NARCIS (Netherlands)

    MEIJER, WJJ; DEJONG, A; BEA, G; WISMAN, A; TJALSMA, H; VENEMA, G; BRON, S; MAARTEN, J; VANDIJL, JM

    Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis

  4. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

    2011-01-01

    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  5. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  6. Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae.

    Science.gov (United States)

    Messerschmidt, Sonja J; Kemter, Franziska S; Schindler, Daniel; Waldminghaus, Torsten

    2015-02-01

    Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, the redesign of primary chromosomes bears high risks and still requires enormous resources. An alternative approach is the addition of synthetic chromosomes to the cell. The natural secondary chromosome of Vibrio cholerae could potentially serve as template for a synthetic secondary chromosome in Escherichia coli. To test this assumption we constructed a replicon named synVicII based on the replication module of V. cholerae chromosome II (oriII). A new assay for the assessment of replicon stability was developed based on flow-cytometric analysis of unstable GFP variants. Application of this assay to cells carrying synVicII revealed an improved stability compared to a secondary replicon based on E. coli oriC. Cell cycle analysis and determination of cellular copy numbers of synVicII indicate that replication timing of the synthetic replicon in E. coli is comparable to the natural chromosome II (ChrII) in V. cholerae. The presented synthetic biology work provides the basis to use secondary chromosomes in E. coli to answer basic research questions as well as for several biotechnological applications.

  7. Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

    Directory of Open Access Journals (Sweden)

    Kazuhiro Iiyama

    2015-06-01

    Full Text Available A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

  8. Ubiquitous human 'master' origins of replication are encoded in the DNA sequence via a local enrichment in nucleosome excluding energy barriers.

    Science.gov (United States)

    Drillon, Guénola; Audit, Benjamin; Argoul, Françoise; Arneodo, Alain

    2015-02-18

    As the elementary building block of eukaryotic chromatin, the nucleosome is at the heart of the compromise between the necessity of compacting DNA in the cell nucleus and the required accessibility to regulatory proteins. The recent availability of genome-wide experimental maps of nucleosome positions for many different organisms and cell types has provided an unprecedented opportunity to elucidate to what extent the DNA sequence conditions the primary structure of chromatin and in turn participates in the chromatin-mediated regulation of nuclear functions, such as gene expression and DNA replication. In this study, we use in vivo and in vitro genome-wide nucleosome occupancy data together with the set of nucleosome-free regions (NFRs) predicted by a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix, to investigate the role of intrinsic nucleosome occupancy in the regulation of the replication spatio-temporal programme in human. We focus our analysis on the so-called replication U/N-domains that were shown to cover about half of the human genome in the germline (skew-N domains) as well as in embryonic stem cells, somatic and HeLa cells (mean replication timing U-domains). The 'master' origins of replication (MaOris) that border these megabase-sized U/N-domains were found to be specified by a few hundred kb wide regions that are hyper-sensitive to DNase I cleavage, hypomethylated, and enriched in epigenetic marks involved in transcription regulation, the hallmarks of localized open chromatin structures. Here we show that replication U/N-domain borders that are conserved in all considered cell lines have an environment highly enriched in nucleosome-excluding-energy barriers, suggesting that these ubiquitous MaOris have been selected during evolution. In contrast, MaOris that are cell-type-specific are mainly regulated epigenetically and are no longer favoured by a local abundance of intrinsic NFRs encoded in

  9. Ubiquitous human ‘master’ origins of replication are encoded in the DNA sequence via a local enrichment in nucleosome excluding energy barriers

    Science.gov (United States)

    Drillon, Guénola; Audit, Benjamin; Argoul, Françoise; Arneodo, Alain

    2015-02-01

    As the elementary building block of eukaryotic chromatin, the nucleosome is at the heart of the compromise between the necessity of compacting DNA in the cell nucleus and the required accessibility to regulatory proteins. The recent availability of genome-wide experimental maps of nucleosome positions for many different organisms and cell types has provided an unprecedented opportunity to elucidate to what extent the DNA sequence conditions the primary structure of chromatin and in turn participates in the chromatin-mediated regulation of nuclear functions, such as gene expression and DNA replication. In this study, we use in vivo and in vitro genome-wide nucleosome occupancy data together with the set of nucleosome-free regions (NFRs) predicted by a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix, to investigate the role of intrinsic nucleosome occupancy in the regulation of the replication spatio-temporal programme in human. We focus our analysis on the so-called replication U/N-domains that were shown to cover about half of the human genome in the germline (skew-N domains) as well as in embryonic stem cells, somatic and HeLa cells (mean replication timing U-domains). The ‘master’ origins of replication (MaOris) that border these megabase-sized U/N-domains were found to be specified by a few hundred kb wide regions that are hyper-sensitive to DNase I cleavage, hypomethylated, and enriched in epigenetic marks involved in transcription regulation, the hallmarks of localized open chromatin structures. Here we show that replication U/N-domain borders that are conserved in all considered cell lines have an environment highly enriched in nucleosome-excluding-energy barriers, suggesting that these ubiquitous MaOris have been selected during evolution. In contrast, MaOris that are cell-type-specific are mainly regulated epigenetically and are no longer favoured by a local abundance of intrinsic NFRs

  10. The One-Kilobase DNA Fragment Upstream of the ardC Actin Gene of Physarum polycephalum Is Both a Replicator and a Promoter

    Science.gov (United States)

    Pierron, Gérard; Pallotta, Dominick; Bénard, Marianne

    1999-01-01

    The 1-kb DNA fragment upstream of the ardC actin gene of Physarum polycephalum promotes the transcription of a reporter gene either in a transient-plasmid assay or as an integrated copy in an ectopic position, defining this region as the transcriptional promoter of the ardC gene (PardC). Since we mapped an origin of replication activated at the onset of S phase within this same fragment, we examined the pattern of replication of a cassette containing the PardC promoter and the hygromycin phosphotransferase gene, hph, integrated into two different chromosomal sites. In both cases, we show by two-dimensional agarose gel electrophoresis that an efficient, early activated origin coincides with the ectopic PardC fragment. One of the integration sites was a normally late-replicating region. The presence of the ectopic origin converted this late-replicating domain into an early-replicating domain in which replication forks propagate with kinetics indistinguishable from those of the native PardC replicon. This is the first demonstration that initiation sites for DNA replication in Physarum correspond to cis-acting replicator sequences. This work also confirms the close proximity of a replication origin and a promoter, with both functions being located within the 1-kb proximal region of the ardC actin gene. A more precise location of the replication origin with respect to the transcriptional promoter must await the development of a functional autonomously replicating sequence assay in Physarum. PMID:10207074

  11. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments ...

  12. Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA.

    Science.gov (United States)

    Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew; Ostermeier, Marc

    2011-11-01

    We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.

  13. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  14. Involvement of proliferating cell nuclear antigen (Cyclin) in DNA replication in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Zuber, M.; Tan, E.M.; Ryoji, M.

    1989-01-01

    Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase /delta/ but not the other DNA polymerases in vitro. The authors injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase /delta/ is necessary for plasmid replication in vivo, Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase /alpha/. Anti-DNA polymerase /alpha/ alone inhibited plasmid replication by 63%. Thus, DNA ploymerase /alpha/ is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase /alpha/ antibody blocked 73% of replication. They concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase /delta/. In addition, they obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymearse /alpha/, that the structure of DNA polymerase /alpha/ holoenzyme for chromosome replication is significantly different from that for plasmid replication.

  15. A leucine zipper motif determines different functions in a DNA replication protein.

    Science.gov (United States)

    Garcia de Viedma, D; Giraldo, R; Rivas, G; Fernández-Tresguerres, E; Diaz-Orejas, R

    1996-01-01

    RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors. Images PMID:8631313

  16. Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets.

    Science.gov (United States)

    Mooij, Petra; Koopman, Gerrit; Mortier, Daniëlla; van Heteren, Melanie; Oostermeijer, Herman; Fagrouch, Zahra; de Laat, Rudy; Kobinger, Gary; Li, Yan; Remarque, Edmond J; Kondova, Ivanela; Verschoor, Ernst J; Bogers, Willy M J M

    2015-01-01

    The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.

  17. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

    Science.gov (United States)

    Tobian, J A; Macrina, F L

    1982-10-01

    A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

  18. An Enterobacter plasmid as a new genetic background for the transposon Tn1331

    Directory of Open Access Journals (Sweden)

    Alavi MR

    2011-11-01

    Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into

  19. The Complete Nucleotide Sequence of the Carbapenem Resistance-Conferring Conjugative Plasmid pLD209 from a Pseudomonas putida Clinical Strain Reveals a Chimeric Design Formed by Modules Derived from Both Environmental and Clinical Bacteria

    Science.gov (United States)

    Marchiaro, Patricia M.; Brambilla, Luciano; Morán-Barrio, Jorgelina; Revale, Santiago; Pasteran, Fernando; Vila, Alejandro J.; Viale, Alejandro M.

    2014-01-01

    The complete sequence of the carbapenem-resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain is presented. pLD209 is formed by 3 well-defined regions: an adaptability module encompassing a Tn402-like class 1 integron of clinical origin containing blaVIM-2 and aacA4 gene cassettes, partitioning and transfer modules, and a replication module derived from plasmids of environmental bacteria. pLD209 is thus a mosaic of modules originating in both the clinical and environmental (nonclinical) microbiota. PMID:24395220

  20. An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942.

    Science.gov (United States)

    Liu, Y; Tsinoremas, N F

    1996-06-12

    In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC). In this study, the dnaN locus from the cyanobacterium Synechococcus sp. strain PCC 7942 was sequenced. The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria. A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp. We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor. Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus. Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

  1. Replication of a gene-environment interaction Via Multimodel inference: additive-genetic variance in adolescents' general cognitive ability increases with family-of-origin socioeconomic status.

    Science.gov (United States)

    Kirkpatrick, Robert M; McGue, Matt; Iacono, William G

    2015-03-01

    The present study of general cognitive ability attempts to replicate and extend previous investigations of a biometric moderator, family-of-origin socioeconomic status (SES), in a sample of 2,494 pairs of adolescent twins, non-twin biological siblings, and adoptive siblings assessed with individually administered IQ tests. We hypothesized that SES would covary positively with additive-genetic variance and negatively with shared-environmental variance. Important potential confounds unaddressed in some past studies, such as twin-specific effects, assortative mating, and differential heritability by trait level, were found to be negligible. In our main analysis, we compared models by their sample-size corrected AIC, and base our statistical inference on model-averaged point estimates and standard errors. Additive-genetic variance increased with SES-an effect that was statistically significant and robust to model specification. We found no evidence that SES moderated shared-environmental influence. We attempt to explain the inconsistent replication record of these effects, and provide suggestions for future research.

  2. Development of new plasmid DNA vaccine vectors with R1-based replicons

    Directory of Open Access Journals (Sweden)

    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  3. Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions.

    Science.gov (United States)

    Bollag, B; Mackeen, P C; Frisque, R J

    1996-04-01

    The human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int-) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA-binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S. I) of both viral origin regions, whereas SVT preferentially bound B.S. I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCV in vitro, and to the virus' ability to establish persistent infections in vivo.

  4. Replication Restart in Bacteria.

    Science.gov (United States)

    Michel, Bénédicte; Sandler, Steven J

    2017-07-01

    In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability. Copyright © 2017 American Society for Microbiology.

  5. iROS-gPseKNC: Predicting replication origin sites in DNA by incorporating dinucleotide position-specific propensity into general pseudo nucleotide composition

    Science.gov (United States)

    Xiao, Xuan; Ye, Han-Xiao; Liu, Zi; Jia, Jian-Hua; Chou, Kuo-Chen

    2016-01-01

    DNA replication, occurring in all living organisms and being the basis for biological inheritance, is the process of producing two identical replicas from one original DNA molecule. To in-depth understand such an important biological process and use it for developing new strategy against genetics diseases, the knowledge of duplication origin sites in DNA is indispensible. With the explosive growth of DNA sequences emerging in the postgenomic age, it is highly desired to develop high throughput tools to identify these regions purely based on the sequence information alone. In this paper, by incorporating the dinucleotide position-specific propensity information into the general pseudo nucleotide composition and using the random forest classifier, a new predictor called iROS-gPseKNC was proposed. Rigorously cross–validations have indicated that the proposed predictor is significantly better than the best existing method in sensitivity, specificity, overall accuracy, and stability. Furthermore, a user-friendly web-server for iROS-gPseKNC has been established at http://www.jci-bioinfo.cn/iROS-gPseKNC, by which users can easily get their desired results without the need to bother the complicated mathematics, which were presented just for the integrity of the methodology itself. PMID:27147572

  6. Production and characterization of chicken origin Shigella dysenteriae ghosts with double bacteriolysis plasmids%双重溶菌质粒制备鸡痢疾志贺菌菌壳的研究

    Institute of Scientific and Technical Information of China (English)

    蒋大伟; 刘军; 张瑞安; 夏力亮; 孙洋; 祝令伟; 冯书章; 许兰菊

    2013-01-01

    In order to improve lysis rate and stability of chicken origin Shigella dysenteriae ghosts,recombinant strain of Shigella with double bacteriolytic plasmids was constructed and its characteristics was studied. Two recombinant bacteriolysis plasmid pBV220: :E and pBBR IMCS-E were co-transformed into the Shigella strain isolated from chicken to produce recombinant strain Sd(pBV220::E /pBBRIMCS-E). The recombinant strain Sd(pBV220::E /pBBRIMCS-E) was grown at 28℃ with shaking until mid log-phase,followed by incubation at 42℃ to induce the expression of gene E and produce Shigella dysenteriae ghosts. The D600 value of bacterial cultures was monitored over 20 h at 30 min intervals. The morphology of chicken origin Shigella dysenteriae ghosts was observed by electron microscope. The recombinant strains Sd(pBV220 : : E/pB-BRIMCS-E) generated chicken origin Shigella dysenteriae ghosts after inducing at 42℃. The ghosts retained basic cell morphology,with the lysis rate of 99. 999 9%. The chicken origin Shigella dysenteriae ghosts were generated successfully with double bacteriolysis plasmids, which laid foundation for future development of bacterial ghost vaccine against Shigella infection in chicken.%通过将2种不同的重组溶菌质粒转化至鸡痢疾志贺菌中,构建含有双重溶菌质粒的志贺菌重组菌株,以提高该菌菌壳制备的裂解率和稳定性,并对其特性进行研究.用重组溶菌质粒pBV220::E和pBBRIMCS-E共同转化到鸡痢疾志贺菌,构建志贺菌重组菌株Sd(pBV220::E/pBBRI MCS-E),重组菌株于28 C培养至D600值为0.5时升至42℃继续培养,间隔30 min检测菌液D600值,进行重组菌株的溶菌裂解动力学比较试验,绘制裂解曲线图并计算出裂解率,制备菌壳并对其形态进行电镜观察.重组菌株Sd(pBV220::E/pBBRIMCS-E)经42℃诱导后可形成菌壳且保留了基本的细胞形态,裂解率达到了99.999 9%.本研究成功制备出鸡痢疾志贺菌菌壳,为研究利用该

  7. Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.

    Science.gov (United States)

    Krude, T; Knippers, R

    1994-08-19

    Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.

  8. Dissection of the beta-globin replication-initiation region reveals specific requirements for replicator elements during gene amplification.

    Directory of Open Access Journals (Sweden)

    Naoya Okada

    Full Text Available Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR/origin/replicator and a nuclear matrix-attachment region (MAR is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR. Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

  9. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  10. Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR.

    Science.gov (United States)

    Carapuça, Elisabete; Azzoni, Adriano R; Prazeres, Duarte M F; Monteiro, Gabriel A; Mergulhão, Filipe J M

    2007-10-01

    A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 x 104 copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.

  11. Complete nucleotide sequence and analysis of two conjugative broad host range plasmids from a marine microbial biofilm.

    Directory of Open Access Journals (Sweden)

    Peter Norberg

    Full Text Available The complete nucleotide sequence of plasmids pMCBF1 and pMCBF6 was determined and analyzed. pMCBF1 and pMCBF6 form a novel clade within the IncP-1 plasmid family designated IncP-1 ς. The plasmids were exogenously isolated earlier from a marine biofilm. pMCBF1 (62 689 base pairs; bp and pMCBF6 (66 729 bp have identical backbones, but differ in their mercury resistance transposons. pMCBF1 carries Tn5053 and pMCBF6 carries Tn5058. Both are flanked by 5 bp direct repeats, typical of replicative transposition. Both insertions are in the vicinity of a resolvase gene in the backbone, supporting the idea that both transposons are "res-site hunters" that preferably insert close to and use external resolvase functions. The similarity of the backbones indicates recent insertion of the two transposons and the ongoing dynamics of plasmid evolution in marine biofilms. Both plasmids also carry the insertion sequence ISPst1, albeit without flanking repeats. ISPs1is located in an unusual site within the control region of the plasmid. In contrast to most known IncP-1 plasmids the pMCBF1/pMCBF6 backbone has no insert between the replication initiation gene (trfA and the vegetative replication origin (oriV. One pMCBF1/pMCBF6 block of about 2.5 kilo bases (kb has no similarity with known sequences in the databases. Furthermore, insertion of three genes with similarity to the multidrug efflux pump operon mexEF and a gene from the NodT family of the tripartite multi-drug resistance-nodulation-division (RND system in Pseudomonas aeruginosa was found. They do not seem to confer antibiotic resistance to the hosts of pMCBF1/pMCBF6, but the presence of RND on promiscuous plasmids may have serious implications for the spread of antibiotic multi-resistance.

  12. Modulation of pPS10 Host Range by Plasmid-Encoded RepA Initiator Protein

    Science.gov (United States)

    Maestro, Beatriz; Sanz, Jesús M.; Díaz-Orejas, Ramón; Fernández-Tresguerres, Elena

    2003-01-01

    We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37°C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon. Four of them have broadened their host range, and one of them, unable to replicate in Pseudomonas, has therefore changed its host range. Moreover, the mutants also have increased their replication efficiency in strains other than E. coli such as Pseudomonas putida and Alcaligenes faecalis. None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography. The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein. We propose possible explanations for the host range effect of the different repA mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA. PMID:12562807

  13. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315.

    Directory of Open Access Journals (Sweden)

    Wen-Li Du

    2016-07-01

    Full Text Available Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315. It comprises an extra replicon (c2 of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1, another extra replicon(c3 of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the

  14. Aging and calorie restriction oppositely affect mitochondrial biogenesis through TFAM binding at both origins of mitochondrial DNA replication in rat liver.

    Directory of Open Access Journals (Sweden)

    Anna Picca

    Full Text Available Aging affects mitochondria in a tissue-specific manner. Calorie restriction (CR is, so far, the only intervention able to delay or prevent the onset of several age-related changes also in mitochondria. Using livers from middle age (18-month-old, 28-month-old and 32-month-old ad libitum-fed and 28-month-old calorie-restricted rats we found an age-related decrease in mitochondrial DNA (mtDNA content and mitochondrial transcription factor A (TFAM amount, fully prevented by CR. We revealed also an age-related decrease, completely prevented by CR, for the proteins PGC-1α NRF-1 and cytochrome c oxidase subunit IV, supporting the efficiency of CR to forestall the age-related decrease in mitochondrial biogenesis. Furthermore, CR counteracted the age-related increase in oxidative damage to proteins, represented by the increased amount of oxidized peroxiredoxins (PRX-SO3 in the ad libitum-fed animals. An unexpected age-related decrease in the mitochondrial proteins peroxiredoxin III (Prx III and superoxide dismutase 2 (SOD2, usually induced by increased ROS and involved in mitochondrial biogenesis, suggested a prevailing relevance of the age-reduced mitochondrial biogenesis above the induction by ROS in the regulation of expression of these genes with aging. The partial prevention of the decrease in Prx III and SOD2 proteins by CR also supported the preservation of mitochondrial biogenesis in the anti-aging action of CR. To investigate further the age- and CR-related effects on mitochondrial biogenesis we analyzed the in vivo binding of TFAM to specific mtDNA regions and demonstrated a marked increase in the TFAM-bound amounts of mtDNA at both origins of replication with aging, fully prevented by CR. A novel, positive correlation between the paired amounts of TFAM-bound mtDNA at these sub-regions was found in the joined middle age ad libitum-fed and 28-month-old calorie-restricted groups, but not in the 28-month-old ad libitum-fed counterpart suggesting

  15. Origins.

    Science.gov (United States)

    Online-Offline, 1999

    1999-01-01

    Provides an annotated list of resources dealing with the theme of origins of life, the universe, and traditions. Includes Web sites, videos, books, audio materials, and magazines with appropriate grade levels and/or subject disciplines indicated; professional resources; and learning activities. (LRW)

  16. Formation of replicating saponite from a gel in the presence of oxalate: implications for the formation of clay minerals in carbonaceous chondrites and the origin of life

    Science.gov (United States)

    Schumann, Dirk; Hartman, Hyman; Eberl, Dennis D.; Sears, S. Kelly; Hesse, Reinhard; Vali, Hojatollah

    2012-01-01

    The potential role of clay minerals in the abiotic origin of life has been the subject of ongoing debate for the past several decades. At issue are the clay minerals found in a class of meteorites known as carbonaceous chondrites. These clay minerals are the product of aqueous alteration of anhydrous mineral phases, such as olivine and orthopyroxene, that are often present in the chondrules. Moreover, there is a strong correlation in the occurrence of clay minerals and the presence of polar organic molecules. It has been shown in laboratory experiments at low temperature and ambient pressure that polar organic molecules, such as the oxalate found in meteorites, can catalyze the crystallization of clay minerals. In this study, we show that oxalate is a robust catalyst in the crystallization of saponite, an Al- and Mg-rich, trioctahedral 2:1 layer silicate, from a silicate gel at 60°C and ambient pressure. High-resolution transmission electron microscopy analysis of the saponite treated with octadecylammonium (n(C)=18) cations revealed the presence of 2:1 layer structures that have variable interlayer charge. The crystallization of these differently charged 2:1 layer silicates most likely occurred independently. The fact that 2:1 layer silicates with variable charge formed in the same gel has implications for our understanding of the origin of life, as these 2:1 clay minerals most likely replicate by a mechanism of template-catalyzed polymerization and transmit the charge distribution from layer to layer. If polar organic molecules like oxalate can catalyze the formation of clay-mineral crystals, which in turn promote clay microenvironments and provide abundant adsorption sites for other organic molecules present in solution, the interaction among these adsorbed molecules could lead to the polymerization of more complex organic molecules like RNA from nucleotides on early Earth.

  17. Formation of replicating saponite from a gel in the presence of oxalate: implications for the formation of clay minerals in carbonaceous chondrites and the origin of life.

    Science.gov (United States)

    Schumann, Dirk; Hartman, Hyman; Eberl, Dennis D; Sears, S Kelly; Hesse, Reinhard; Vali, Hojatollah

    2012-06-01

    The potential role of clay minerals in the abiotic origin of life has been the subject of ongoing debate for the past several decades. At issue are the clay minerals found in a class of meteorites known as carbonaceous chondrites. These clay minerals are the product of aqueous alteration of anhydrous mineral phases, such as olivine and orthopyroxene, that are often present in the chondrules. Moreover, there is a strong correlation in the occurrence of clay minerals and the presence of polar organic molecules. It has been shown in laboratory experiments at low temperature and ambient pressure that polar organic molecules, such as the oxalate found in meteorites, can catalyze the crystallization of clay minerals. In this study, we show that oxalate is a robust catalyst in the crystallization of saponite, an Al- and Mg-rich, trioctahedral 2:1 layer silicate, from a silicate gel at 60°C and ambient pressure. High-resolution transmission electron microscopy analysis of the saponite treated with octadecylammonium (n(C)=18) cations revealed the presence of 2:1 layer structures that have variable interlayer charge. The crystallization of these differently charged 2:1 layer silicates most likely occurred independently. The fact that 2:1 layer silicates with variable charge formed in the same gel has implications for our understanding of the origin of life, as these 2:1 clay minerals most likely replicate by a mechanism of template-catalyzed polymerization and transmit the charge distribution from layer to layer. If polar organic molecules like oxalate can catalyze the formation of clay-mineral crystals, which in turn promote clay microenvironments and provide abundant adsorption sites for other organic molecules present in solution, the interaction among these adsorbed molecules could lead to the polymerization of more complex organic molecules like RNA from nucleotides on early Earth.

  18. Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains

    Science.gov (United States)

    2017-01-01

    The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell. PMID:28486476

  19. Single-stranded annealing induced by re-initiation of replication origins provides a novel and efficient mechanism for generating copy number expansion via non-allelic homologous recombination.

    Directory of Open Access Journals (Sweden)

    Kenneth J Finn

    Full Text Available Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR. Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA-mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution.

  20. Single-stranded annealing induced by re-initiation of replication origins provides a novel and efficient mechanism for generating copy number expansion via non-allelic homologous recombination.

    Directory of Open Access Journals (Sweden)

    Kenneth J Finn

    Full Text Available Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR. Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA-mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution.

  1. Identification of two replicons in phage-plasmid P4.

    Science.gov (United States)

    Tocchetti, A; Serina, S; Terzano, S; Dehò, G; Ghisotti, D

    1998-06-05

    DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr. Replication depends on the product of the P4 alpha gene, a protein with primase and helicase activity, that binds both ori1 and crr. A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the alpha gene product, coexist in P4. The first replicon is made by the cnr and alpha genes and the ori1 and crr sites. The second is limited to the alpha and crr region. Thus, in the absence of the ori1 region, replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.

  2. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2.

    Science.gov (United States)

    Jha, Jyoti K; Li, Mi; Ghirlando, Rodolfo; Miller Jenkins, Lisa M; Wlodawer, Alexander; Chattoraj, Dhruba

    2017-04-18

    Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid-the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication of Vibrio cholerae Chr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing

  3. Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

    Science.gov (United States)

    Kulinska, Anna; Czeredys, Magdalena; Hayes, Finbarr; Jagura-Burdzy, Grazyna

    2008-07-01

    IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

  4. Plasmids in Mycoplasma species isolated from goats and sheep and their preliminary typing

    Directory of Open Access Journals (Sweden)

    Nascimento Elmiro R.

    1999-01-01

    Full Text Available One-hundred-five (105 clinical isolates of mycoplasma from caprine origin and one isolate from ovine were surveyed for plasmids, which were present in thirty-three (31% of them. These mycoplasmas originated from 13 herds. Ten of them were symptomatic for mycoplasmal disease (mastitis, polyarthritis, septicemia and three herds were asymptomatic, i.e., clinically normal. Twenty-eight isolates were Mycoplasma mycoides subspecies mycoides LC (large colony or caprine biotype, four were Mycoplasma capricolum subsp. capricolum and one was Mycoplasma cottewii. The isolated plasmids were linearized by EcoRI, EcoRV, EcoRI and EcoRV or BamHI and EcoRV, and were of five sizes (1.1, 1.6, 1.7, 1.8, and 1.9 Kbp. Based on restriction enzyme digestion and size of the linearized supercoiled extrachromosomal DNA, five plasmid types were recovered (p1II, p2III, p2V, p3I, and p4IV. The small size of these DNA elements probably exclude replicative forms of DNA virus, which are equal or larger than 8.0 Kbp.

  5. Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis

    Science.gov (United States)

    Li, Caroline M.; Miao, Yunan; Lingeman, Robert G.; Hickey, Robert J.; Malkas, Linda H.

    2016-01-01

    We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits. PMID:28036377

  6. A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.

    Science.gov (United States)

    Aakvik, Trine; Degnes, Kristin Fløgstad; Dahlsrud, Rannveig; Schmidt, Frank; Dam, Ragnar; Yu, Lihua; Völker, Uwe; Ellingsen, Trond Erling; Valla, Svein

    2009-06-01

    The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

  7. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  8. Characterisation of IncA/C2 plasmids carrying an In416-like integron with the blaVIM-19 gene from Klebsiella pneumoniae ST383 of Greek origin.

    Science.gov (United States)

    Papagiannitsis, Costas C; Dolejska, Monika; Izdebski, Radosław; Giakkoupi, Panagiota; Skálová, Anna; Chudějová, Kateřina; Dobiasova, Hana; Vatopoulos, Alkiviadis C; Derde, Lennie P G; Bonten, Marc J M; Gniadkowski, Marek; Hrabák, Jaroslav

    2016-02-01

    The complete nucleotide sequences of three multidrug resistance (MDR) IncA/C-like plasmids from Enterobacteriaceae isolates carrying the VIM-type carbapenemase-encoding integrons In4863 (blaVIM-19-aacA7-dfrA1-ΔaadA1-smr2) or In4873 (blaVIM-1-aacA7-dfrA1-ΔaadA1-smr2) were determined, which are the first In416-like elements identified in Greece. Plasmids pKP-Gr642 and pKP-Gr8143 were from Klebsiella pneumoniae ST383 isolates, whereas plasmid pEcl-Gr4873 was from an Enterobacter cloacae ST88 isolate. Sequencing showed that pKP-Gr642 (162787bp) and pKP-Gr8143 (154395bp) consisted of the type 1 IncA/C2 conserved backbone, the blaCMY-2-like gene-containing region, and the ARI-B (with the sul2 gene) and ARI-A (with a class 1 integron) resistance islands, like the plasmid pUMNK88_161 from the USA. The third plasmid, pEcl-Gr4873 (153958bp), exhibited extensive similarity with the type 2 IncA/C2 plasmid pR55 from France. pEcl-Gr4873 carried only one resistance island of a hybrid transposon structure inserted in a different location to ARI-A in type 1 A/C2 plasmids. In all three plasmids, the In416-like integrons In4863 or In4873 were identified within non-identical class II transposon structures. All three In416-like-carrying regions presented significant similarities with the MDR region of the IncA/C2 plasmid pCC416 from Italy, carrying the prototype In416 integron (blaVIM-4-aacA7-dfrA1-ΔaadA1-smr2). These findings provided the basis for speculations regarding the evolution of IncA/C2 plasmids with In416-like integrons, and confirmed the rapid evolution of some IncA/C2 plasmid lineages. Considering the broad host range of IncA/C2 molecules, it seems that pKP-Gr642, pKP-Gr8143 and pEcl-Gr4873 plasmids might support the diffusion of In416-like integrons among Enterobacteriaceae. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  9. Initiation of adenovirus DNA replication.

    OpenAIRE

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L

    1980-01-01

    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  10. A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

    Science.gov (United States)

    Hussein, Islam T.M.; Ma, Eric J.; Meixell, Brandt; Hill, Nichola J.; Lindberg, Mark S.; Albrecht , Randy A.; Bahl, Justin; Runstadler, Jonathan A.

    2016-01-01

    H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~ 12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context.

  11. Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: evidence for their involvement in regulation of the viral DNA replication.

    Science.gov (United States)

    Saribas, A Sami; Mun, Sarah; Johnson, Jaslyn; El-Hajmoussa, Mohammad; White, Martyn K; Safak, Mahmut

    2014-01-20

    JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.

  12. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains.

    Science.gov (United States)

    Kananavičiūtė, Rūta; Butaitė, Elena; Citavičius, Donaldas

    2014-01-01

    We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation. Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

  13. Characterization and comparative overview of complete sequences of the first plasmids of Pandoraea across clinical and non-clinical strains

    Directory of Open Access Journals (Sweden)

    Delicia Yong

    2016-10-01

    Full Text Available To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572 (pPF72-1, pPF72-2, Pandoraea oxalativorans DSM 23570 (pPO70-1, pPO70-2, pPO70-3, pPO70-4, Pandoraea vervacti NS15 (pPV15 and Pandoraea apista DSM 16535 (pPA35 were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful because these plasmid sequences did not match to any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified among the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens.

  14. A potential new gene (tccA) on IncP plasmid RK2 and transposon Tn1721: relationship of its product to the TrwC relaxase/helicase of IncW plasmid R388.

    Science.gov (United States)

    Jovanovic, O S; Figurski, D H

    1997-01-01

    Computational analysis of the fully sequenced 60-kb genome of broad-host-range IncP alpha plasmid RK2 revealed a previously unreported potential protein-coding sequence, an 80-codon open reading frame (tccA), located in the region between the vegetative origin of replication (oriV) and the tetR gene of the tetracycline resistance determinant. The coding region is also present in the transposon Tn1721 tet region, which is nearly identical to the tet region of RK2. Remarkably, the predicted polypeptide product of the coding region displays 56% identity and 72% similarity with the C-terminal domain of the TrwC relaxase/helicase protein of IncW plasmid R388.

  15. Modeling inhomogeneous DNA replication kinetics.

    Directory of Open Access Journals (Sweden)

    Michel G Gauthier

    Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

  16. Genomic analysis of Acidianus hospitalis W1 a host for studying crenarchaeal virus and plasmid life cycles

    DEFF Research Database (Denmark)

    You, X. Y.; Liu, Chao; Wang, S. Y.

    2011-01-01

    The Acidianus hospitalis W1 genome consists of a minimally sized chromosome of about 2.13 Mb and a conjugative plasmid pAH1 and it is a host for the model filamentous lipothrixvirus AFV1. The chromosome carries three putative replication origins in conserved genomic regions and two large regions ...... it a promising candidate for developing the first Acidianus genetic systems....... of local chromosomal regions and to minimise the impact of environmental stress. Complex and partially defective CRISPR/Cas/Cmr immune systems are present and interspersed with five vapBC gene pairs. Remnants of integrated viral genomes and plasmids are located at five intron-less tRNA genes and several...

  17. A classification system for plasmids from Enterococci and other Gram-positive bacteria

    DEFF Research Database (Denmark)

    Jensen, Lars Bogø; Garcia-Migura, Lourdes; Valenzuela, Antonio Jesus Sanchez

    2010-01-01

    A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiati...

  18. Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species

    OpenAIRE

    Brom, Susana; Girard, Lourdes; García-de los Santos, Alejandro; Sanjuan-Pinilla, Julio M.; Olivares, José; Sanjuan, Juan

    2002-01-01

    Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organi...

  19. blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

    Science.gov (United States)

    Call, Douglas R; Singer, Randall S; Meng, Da; Broschat, Shira L; Orfe, Lisa H; Anderson, Janet M; Herndon, David R; Kappmeyer, Lowell S; Daniels, Joshua B; Besser, Thomas E

    2010-02-01

    Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

  20. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology

    Science.gov (United States)

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F.; Doumith, Michel; Ellington, Matthew J.; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A. Sarah; Phan, Hang; Sheppard, Anna E.

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made. PMID:28232822

  1. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  2. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  3. Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.

    Science.gov (United States)

    Sibani, Sahar; Rampakakis, Emmanouil; Di Paola, Domenic; Zannis-Hadjopoulos, Maria

    2008-06-01

    DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.

  4. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

    Directory of Open Access Journals (Sweden)

    Susu He

    2016-12-01

    Full Text Available The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance.

  5. Database Replication

    CERN Document Server

    Kemme, Bettina

    2010-01-01

    Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

  6. Plasmid interference for curing antibiotic resistance plasmids in vivo

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  7. Both foot-and-mouth disease virus and bovine viral diarrhea virus replication are inhibited by Mx1 protein originated from porcine.

    Science.gov (United States)

    Shi, Huijun; Fu, Qiang; Ren, Yan; Wang, Dawei; Qiao, Jun; Wang, Pengyan; Zhang, Hui; Chen, Chuangfu

    2015-01-01

    Mx1 protein is I type interferons (IFNs)-induced 76-kDa guanosine triphosphatases (GTPases) that belong to the dynamin superfamily of large GTPases. Mx1 proteins have attracted attention because some display antiviral activity against pathogenic RNA and DNA viruses. Meanwhile, Mx1 gene generally exists in organisms or cells of mammalian, fish and chicken. Blocking a wide range of RNA virus replication by inhibiting nuclear viral mRNA synthesis is a unique property of Mx1 protein. In order to investigate a novel prevention measure against foot-and-mouth disease virus (FMDV) and bovine viral diarrhea virus (BVDV), which frequently break out in Xinjiang Uygur Autonomous Region of China, we investigated the effects of porcine Mx1 protein on FMDV and BVDV replication by measuring viral reverse transcriptase activity at various time intervals. In our study, Mx1 protein was overexpressed in BHK-21 and MDBK cells mediated by lentivirus prior to infect with FMDV and BVDV. FMDV and BVDV replication levels were monitored by quantitative real-Time PCR. The results showed porcine Mx1 overexpression significantly inhibited both FMDV and BVDV replication within 12 and 36 hours post-infection (pi). The finding may provide a new therapeutic approach for preventing from FDMV and BVDV infection.

  8. Abiotic self-replication.

    Science.gov (United States)

    Meyer, Adam J; Ellefson, Jared W; Ellington, Andrew D

    2012-12-18

    The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled. The ability to mutate in a discrete or quantized way, without frequent reversion, may be an additional requirement for Darwinian evolution, in which case the notion that Darwinian evolution defines life may be less of a tautology than previously thought. In this Account, we examine a variety of in vitro systems of increasing complexity, from simple chemical replicators up to complex systems based on in vitro transcription and translation. Comparing and contrasting these systems provides an interesting window onto the molecular origins of life. For nucleic acids, the story likely begins with simple chemical replication, perhaps of the form A + B → T, in which T serves as a template for the joining of A and B. Molecular variants capable of faster replication would come to dominate a population, and the development of cycles in which templates could foster one another's replication would have led to increasingly complex replicators and from thence to the initial genomes. The initial genomes may have been propagated by RNA replicases, ribozymes capable of joining oligonucleotides and eventually polymerizing mononucleotide substrates. As ribozymes were added to the genome to fill gaps in the chemistry necessary for replication, the backbone of a putative RNA world would have emerged. It is likely that such replicators would have been plagued by molecular parasites, which would have been passively replicated by the RNA world machinery without contributing to it. These molecular parasites would have been a major driver for the development of compartmentalization/cellularization, as more robust compartments could have outcompeted parasite-ridden compartments. The eventual outsourcing of metabolic

  9. Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

    DEFF Research Database (Denmark)

    Clausen, Anders; Mikkelsen, Marie Just; Schrøder, I.

    2004-01-01

    The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 (A. thermophilum), growing optimally at 70degreesC, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence...... was found, but no single stranded intermediates, characteristic of rolling circle replication, were found on Southern blots. The larger plasmid, pBAL, was found to be a 8294 bp plasmid with a GC content of 39%. It revealed 17 ORFs, of which three showed similarity at the amino acid (aa) level to known...

  10. Chromosome replication and segregation in bacteria.

    Science.gov (United States)

    Reyes-Lamothe, Rodrigo; Nicolas, Emilien; Sherratt, David J

    2012-01-01

    In dividing cells, chromosome duplication once per generation must be coordinated with faithful segregation of newly replicated chromosomes and with cell growth and division. Many of the mechanistic details of bacterial replication elongation are well established. However, an understanding of the complexities of how replication initiation is controlled and coordinated with other cellular processes is emerging only slowly. In contrast to eukaryotes, in which replication and segregation are separate in time, the segregation of most newly replicated bacterial genetic loci occurs sequentially soon after replication. We compare the strategies used by chromosomes and plasmids to ensure their accurate duplication and segregation and discuss how these processes are coordinated spatially and temporally with growth and cell division. We also describe what is known about the three conserved families of ATP-binding proteins that contribute to chromosome segregation and discuss their inter-relationships in a range of disparate bacteria.

  11. Presence and analysis of plasmids in human and animal associated arcobacter species.

    Science.gov (United States)

    Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip; Deforce, Dieter; Ingmer, Hanne; Vandenberg, Olivier; Van den Abeele, Anne-Marie; Houf, Kurt

    2014-01-01

    In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

  12. Presence and analysis of plasmids in human and animal associated arcobacter species.

    Directory of Open Access Journals (Sweden)

    Laid Douidah

    Full Text Available In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

  13. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenlokke; Riber, Leise; Kot, Witold;

    2016-01-01

    on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed...... to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired...

  14. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication.

    Science.gov (United States)

    Seco, Elena M; Ayora, Silvia

    2017-08-21

    Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA 'initiation primer' on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    Directory of Open Access Journals (Sweden)

    Dexi Bi

    Full Text Available Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.

  16. Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus.

    Directory of Open Access Journals (Sweden)

    Daehwan Chung

    Full Text Available The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105, and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

  17. The evolution of collective restraint: policing and obedience among non-conjugative plasmids.

    Science.gov (United States)

    Kentzoglanakis, Kyriakos; García López, Diana; Brown, Sam P; Goldstein, Richard A

    2013-04-01

    The repression of competition by mechanisms of policing is now recognized as a major force in the maintenance of cooperation. General models on the evolution of policing have focused on the interplay between individual competitiveness and mutual policing, demonstrating a positive relationship between within-group diversity and levels of policing. We expand this perspective by investigating what is possibly the simplest example of reproductive policing: copy number control (CNC) among non-conjugative plasmids, a class of extra-chromosomal vertically transmitted molecular symbionts of bacteria. Through the formulation and analysis of a multi-scale dynamical model, we show that the establishment of stable reproductive restraint among plasmids requires the co-evolution of two fundamental plasmid traits: policing, through the production of plasmid-coded trans-acting replication inhibitors, and obedience, expressed as the binding affinity of plasmid-specific targets to those inhibitors. We explain the intrinsic replication instabilities that arise in the absence of policing and we show how these instabilities are resolved by the evolution of copy number control. Increasing levels of policing and obedience lead to improvements in group performance due to tighter control of local population size (plasmid copy number), delivering benefits both to plasmids, by reducing the risk of segregational loss and to the plasmid-host partnership, by increasing the rate of cell reproduction, and therefore plasmid vertical transmission.

  18. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    Science.gov (United States)

    Houtteman, S W; Elder, R T

    1993-03-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

  19. The evolution of collective restraint: policing and obedience among non-conjugative plasmids.

    Directory of Open Access Journals (Sweden)

    Kyriakos Kentzoglanakis

    2013-04-01

    Full Text Available The repression of competition by mechanisms of policing is now recognized as a major force in the maintenance of cooperation. General models on the evolution of policing have focused on the interplay between individual competitiveness and mutual policing, demonstrating a positive relationship between within-group diversity and levels of policing. We expand this perspective by investigating what is possibly the simplest example of reproductive policing: copy number control (CNC among non-conjugative plasmids, a class of extra-chromosomal vertically transmitted molecular symbionts of bacteria. Through the formulation and analysis of a multi-scale dynamical model, we show that the establishment of stable reproductive restraint among plasmids requires the co-evolution of two fundamental plasmid traits: policing, through the production of plasmid-coded trans-acting replication inhibitors, and obedience, expressed as the binding affinity of plasmid-specific targets to those inhibitors. We explain the intrinsic replication instabilities that arise in the absence of policing and we show how these instabilities are resolved by the evolution of copy number control. Increasing levels of policing and obedience lead to improvements in group performance due to tighter control of local population size (plasmid copy number, delivering benefits both to plasmids, by reducing the risk of segregational loss and to the plasmid-host partnership, by increasing the rate of cell reproduction, and therefore plasmid vertical transmission.

  20. Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1

    NARCIS (Netherlands)

    Hargreaves, D.; Giraldo, R.; Santos-Sierra, S.; Boelens, R.; Rice, D.W.; Díaz Orejas, R.; Rafferty, J.B.

    2002-01-01

    DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The

  1. Geminiviruses: a tale of a plasmid becoming a virus

    Directory of Open Access Journals (Sweden)

    Krupovic Mart

    2009-05-01

    Full Text Available Abstract Background Geminiviruses (family Geminiviridae are small single-stranded (ss DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons. Results Phylogenetic and clustering analyses of various RCR Reps suggest that Rep proteins of geminiviruses share a most recent common ancestor with Reps encoded on plasmids of phytoplasmas, parasitic wall-less bacteria replicating both in plant and insect cells and therefore occupying a common ecological niche with geminiviruses. Capsid protein of Satellite tobacco necrosis virus was found to be the best template for homology-based structural modeling of the geminiviral capsid protein. Good stereochemical quality of the generated models indicates that the geminiviral capsid protein shares the same structural fold, the viral jelly-roll, with the vast majority of icosahedral plant-infecting ssRNA viruses. Conclusion We propose a plasmid-to-virus transition scenario, where a phytoplasmal plasmid acquired a capsid-coding gene from a plant RNA virus to give rise to the ancestor of geminiviruses.

  2. [Epidemiologic study of 2 S. typhimurium outbreaks using plasmid fingerprints].

    Science.gov (United States)

    Baumgartner, A; Breer, C; Schopfer, K

    1989-04-05

    An outbreak of salmonellosis in an old people's home is reported. The infectious agent, S. typhi-murium, was isolated not only from several inmates but also from sick cows of the farm belonging to the home, in animal feed, from employees of the local butcher's shop, and finally in sludge from the local sewage plant. Plasmid analysis provided evidence of a common origin for the isolated S. typhi-murium strains. The incriminated strains harboured, together with two low-molecular-weight plasmids, a plasmid of approximately 50 Mdal, which was also demonstrated in some other S. typhi-murium strains isolated from clinical cases in the area around St. Gallen.

  3. Characterisation of IncA/C2 plasmids carrying an In416-like integron with the blaVIM-19 gene from Klebsiella pneumoniae ST383 of Greek origin

    NARCIS (Netherlands)

    Papagiannitsis, Costas C; Dolejska, Monika; Izdebski, Radosław; Giakkoupi, Panagiota; Skálová, Anna; Chudějová, Kateřina; Dobiasova, Hana; Vatopoulos, Alkiviadis C; Derde, Lennie P G; Bonten, Marc J M; Gniadkowski, Marek; Hrabák, Jaroslav

    2016-01-01

    The complete nucleotide sequences of three multidrug resistance (MDR) IncA/C-like plasmids from Enterobacteriaceae isolates carrying the VIM-type carbapenemase-encoding integrons In4863 (blaVIM-19-aacA7-dfrA1-ΔaadA1-smr2) or In4873 (blaVIM-1-aacA7-dfrA1-ΔaadA1-smr2) were determined, which are the fi

  4. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    Science.gov (United States)

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  5. Plasmid copy number noise in monoclonal populations of bacteria

    Science.gov (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  6. IncA/C plasmids: An emerging threat to human and animal health?

    Science.gov (United States)

    Johnson, Timothy J; Lang, Kevin S

    2012-01-01

    Incompatibility group IncA/C plasmids are large, low copy, theta-replicating plasmids that have been described in the literature for over 40 years. However, they have only recently been intensively studied on the genomic level because of their associations with the emergence of multidrug resistance in enteric pathogens of humans and animals. These plasmids are unique among other enterobacterial plasmids in many aspects, including their modular structure and gene content. While the IncA/C plasmid genome structure has now been well defined, many questions remain pertaining to their basic biological mechanisms of dissemination and regulation. Here, we discuss the history of IncA/C plasmids in light of our recent understanding of their population distribution, genomics, and effects on host bacteria.

  7. Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

    DEFF Research Database (Denmark)

    Basta, Tamara; Smyth, John; Forterre, Patrick

    2009-01-01

    to establish a system for studying plasmid-virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter....... Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid-virus interactions. AFV1 infection and propagation leads to a loss...... of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed...

  8. Organization of Replication of Ribosomal DNA in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Linskens, Maarten H.K.; Huberman, Joel A.

    1988-01-01

    Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the

  9. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  10. Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon

    DEFF Research Database (Denmark)

    Chen, Zhengjun; Lin, Jinzhong; Ma, Chengjie

    2014-01-01

    %. These vectors were employed to express a green fluorescent protein (GFP) using the promoter of S-layer protein SlpA from Lactobacillus acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. In conclusion, these shuttle vectors provide efficient genetic tools for DNA...

  11. Evaluating replicability of laboratory experiments in economics.

    Science.gov (United States)

    Camerer, Colin F; Dreber, Anna; Forsell, Eskil; Ho, Teck-Hua; Huber, Jürgen; Johannesson, Magnus; Kirchler, Michael; Almenberg, Johan; Altmejd, Adam; Chan, Taizan; Heikensten, Emma; Holzmeister, Felix; Imai, Taisuke; Isaksson, Siri; Nave, Gideon; Pfeiffer, Thomas; Razen, Michael; Wu, Hang

    2016-03-25

    The replicability of some scientific findings has recently been called into question. To contribute data about replicability in economics, we replicated 18 studies published in the American Economic Review and the Quarterly Journal of Economics between 2011 and 2014. All of these replications followed predefined analysis plans that were made publicly available beforehand, and they all have a statistical power of at least 90% to detect the original effect size at the 5% significance level. We found a significant effect in the same direction as in the original study for 11 replications (61%); on average, the replicated effect size is 66% of the original. The replicability rate varies between 67% and 78% for four additional replicability indicators, including a prediction market measure of peer beliefs.

  12. A novel nucleoid-associated protein coordinates chromosome replication and chromosome partition.

    Science.gov (United States)

    Taylor, James A; Panis, Gaël; Viollier, Patrick H; Marczynski, Gregory T

    2017-09-06

    We searched for regulators of chromosome replication in the cell cycle model Caulobacter crescentus and found a novel DNA-binding protein (GapR) that selectively aids the initiation of chromosome replication and the initial steps of chromosome partitioning. The protein binds the chromosome origin of replication (Cori) and has higher-affinity binding to mutated Cori-DNA that increases Cori-plasmid replication in vivo. gapR gene expression is essential for normal rapid growth and sufficient GapR levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified dynamic DNA-binding distributions for GapR, with the strongest associations at the partitioning (parABS) locus near Cori. Using molecular-genetic and fluorescence microscopy experiments, we showed that GapR also promotes the first steps of chromosome partitioning, the initial separation of the duplicated parS loci following replication from Cori. This separation occurs before the parABS-dependent partitioning phase. Therefore, this early separation, whose mechanisms is not known, coincides with the poorly defined mechanism(s) that establishes chromosome asymmetry: C. crescentus chromosomes are partitioned to distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that GapR coordinates chromosome replication with asymmetry-establishing chromosome separation, noting that both roles are consistent with the phylogenetic restriction of GapR to asymmetrically dividing bacteria. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Life Origination Hydrate Theory (LOH-Theory) and Mitosis and Replication Hydrate Theory (MRH-Theory): three-dimensional PC validation

    Science.gov (United States)

    Kadyshevich, E. A.; Dzyabchenko, A. V.; Ostrovskii, V. E.

    2014-04-01

    Size compatibility of the CH4-hydrate structure II and multi-component DNA fragments is confirmed by three-dimensional simulation; it is validation of the Life Origination Hydrate Theory (LOH-Theory).

  14. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

    Energy Technology Data Exchange (ETDEWEB)

    Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo; Miller Jenkins, Lisa M.; Wlodawer, Alexander; Chattoraj, Dhruba; Dunny, Gary M.

    2017-04-18

    Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that

  15. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

    Directory of Open Access Journals (Sweden)

    Chunying Zhong

    Full Text Available Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp at the mid-exponential growth stage (OD(600 = 2.0 of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively. These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

  16. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  17. Resolution of Multimeric Forms of Circular Plasmids and Chromosomes.

    Science.gov (United States)

    Crozat, Estelle; Fournes, Florian; Cornet, François; Hallet, Bernard; Rousseau, Philippe

    2014-10-01

    One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes.

  18. First detection of AmpC β-lactamase bla(CMY-2) on a conjugative IncA/C plasmid in a Vibrio parahaemolyticus isolate of food origin.

    Science.gov (United States)

    Li, Ruichao; Lin, Dachuan; Chen, Kaichao; Wong, Marcus Ho Yin; Chen, Sheng

    2015-07-01

    Vibrio parahaemolyticus is an important causative agent of gastroenteritis, with the consumption of contaminated seafood being the major transmission route. Resistance to penicillin is common among V. parahaemolyticus strains, whereas cephalosporin resistance remains rare. In an attempt to assess the current prevalence and characteristics of antibiotic resistance of this pathogen in common food samples, a total of 54 (17% of the total samples) V. parahaemolyticus strains were isolated from 318 meat and seafood samples purchased from supermarkets and wet markets in Shenzhen, China, in 2013. These isolates exhibited high-level resistance to ampicillin, yet they were mostly susceptible to other antimicrobials, except for two that were resistant to extended-spectrum cephalosporins. The β-lactamase gene blaPER-1 was detectable in one strain, V. parahaemolyticus V43, which was resistant to both third- and fourth-generation cephalosporins. Compared to other blaPER-1-positive V. parahaemolyticus strains reported in our previous studies, strain V43 was found to harbor an ∼200-kb conjugative plasmid carrying genes that were different from the antimicrobial resistance genes reported from the previous studies. The β-lactamase gene blaCMY-2 was detectable for the first time in another V. parahaemolyticus isolate, V4, which was resistant to third-generation cephalosporins. This blaCMY-2 gene was shown to be located in an ∼150-kb IncA/C-type conjugative plasmid with a genetic structure consisting of traB-traV-traA-ISEcp1-blaCMY-2-blc-sugE-encR-orf1-orf2-orf3-orf4-dsbC-traC, which is identical to that of other IncA/C conjugative plasmids in Enterobacteriaceae, albeit with a different size. These findings indicate that the transmission of extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase genes via conjugative plasmids can mediate the development of extended-spectrum cephalosporin resistance in V. parahaemolyticus, thereby posing a potential threat to public health

  19. The complete genome sequence and analysis of a plasmid-bearing myxobacterial strain Myxococcus fulvus 124B02 (M 206081).

    Science.gov (United States)

    Chen, Xiao-Jing; Han, Kui; Feng, Jing; Zhuo, Li; Li, Ya-Jie; Li, Yue-Zhong

    2016-01-01

    Myxobacteria, phylogenetically located in the delta division of the Proteobacteria, are well known for characterized social behaviors and large genomes of more than 9 Mb in size. Myxococcus fulvus is a typical species of the genus Myxococcus in the family Myxococcaceae. M. fulvus 124B02, originally isolated from a soil sample collected in Northeast China, is the one and only presently known myxobacterial strain that harbors an endogenous autonomously replicating plasmid, named pMF1. The endogenous plasmid is of importance for understanding the genome evolution of myxobacteria, as well as for the development of genetic engineering tools in myxobacteria. Here we describe the complete genome sequence of this organism. M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. Comparative genomic analyses suggest that pMF1 has a longstanding sustention within myxobacteria, and probably contributes to the genome expansion of myxobacteria.

  20. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  1. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  2. Construction and Identification of Plasmid pTA-TUB2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TU B2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TU B2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×105) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg*mL-1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non-selective medium.

  3. Outbreak of KPC-2-producing Enterobacteriaceae caused by clonal dissemination of Klebsiella pneumoniae ST307 carrying an IncX3-type plasmid harboring a truncated Tn4401a.

    Science.gov (United States)

    Kim, Jung Ok; Song, Sae Am; Yoon, Eun-Jeong; Shin, Jeong Hwan; Lee, Hyukmin; Jeong, Seok Hoon; Lee, Kyungwon

    2017-04-01

    Over a 5-month period between the end of June and the beginning of November in 2015, a KPC-producing Enterobacteriaceae outbreak occurred in a general hospital in Busan, South Korea, being associated with a total of 50 clinical isolates from 47 patients. Multilocus sequence typing and pulsed-field gel electrophoresis were carried out for strain typing and whole-genome sequencing was performed to characterize the plasmids. A clonal spread of K. pneumoniae sequence type 307 (ST307) carrying a self-transferable IncX3-type plasmid harboring blaKPC-2 was responsible for the outbreak. Sporadic emergence of K. pneumoniae ST697 carrying an IncFII-type plasmid and a ST11 isolate harboring a small plasmid devoid of any known origin of replication were observed to be associated with blaKPC-3, but no further dissemination of these strains was identified. The results indicated a healthcare-associated infection associated with a blaKPC-harboring plasmid dissemination and a clonal spread of KPC-producing Enterobacteriaceae.

  4. Plasmids of the pRM/pRF family occur in diverse Rickettsia species.

    Science.gov (United States)

    Baldridge, Gerald D; Burkhardt, Nicole Y; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2008-02-01

    The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.

  5. Effect of excessive cadmium chloride on the plasmids of E. coli HB 101 in vivo

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl2 constantly for 24 h, these plasmids were isolated from E. coli, and the effect of excessive CdCl2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl2 did not inhibit the replication and amp+ gene's expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl2 treatment for 24 hours might cause the sequence's change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.

  6. A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae

    DEFF Research Database (Denmark)

    Val, Marie-Eve; Marbouty, Martial; Martins, Francisco de Lemos

    2016-01-01

    of the important differences between plasmids and chromosomes is that the latter replicate during a defined period of the cell cycle, ensuring a single round of replication per cell. Vibrio cholerae carries two circular chromosomes, Chr1 and Chr2, which are replicated in a well-orchestrated manner with the cell...

  7. Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant.

    Science.gov (United States)

    Schlüter, Andreas; Krause, Lutz; Szczepanowski, Rafael; Goesmann, Alexander; Pühler, Alfred

    2008-08-31

    Plasmid metagenome nucleotide sequence data were recently obtained from wastewater treatment plant (WWTP) bacteria with reduced susceptibility to selected antimicrobial drugs by applying the ultrafast 454-sequencing technology. The sequence dataset comprising 36,071,493 bases (346,427 reads with an average read length of 104 bases) was analysed for genetic diversity and composition by using a newly developed bioinformatic pipeline based on assignment of environmental gene tags (EGTs) to protein families stored in the Pfam database. Short amino acid sequences deduced from the plasmid metagenome sequence reads were compared to profile hidden Markov models underlying Pfam. Obtained matches evidenced that many reads represent genes having predicted functions in plasmid replication, stability and plasmid mobility which indicates that WWTP bacteria harbour genetically stabilised and mobile plasmids. Moreover, the data confirm a high diversity of plasmids residing in WWTP bacteria. The mobile organic peroxide resistance plasmid pMAC from Acinetobacter baumannii was identified as reference plasmid for the most abundant replication module type in the sequenced sample. Accessory plasmid modules encode different transposons, insertion sequences, integrons, resistance and virulence determinants. Most of the matches to Transposase protein families were identified for transposases similar to the one of the chromate resistance transposon Tn5719. Noticeable are hits to beta-lactamase protein families which suggests that plasmids from WWTP bacteria encode different enzymes possessing beta-lactam-hydrolysing activity. Some of the sequence reads correspond to antibiotic resistance genes that were only recently identified in clinical isolates of human pathogens. EGT analysis thus proofed to be a very valuable method to explore genetic diversity and composition of the present plasmid metagenome dataset.

  8. Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

    Science.gov (United States)

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

  9. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  10. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.

    Science.gov (United States)

    Al-Allaf, Faisal A; Tolmachov, Oleg E; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2013-02-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5'-Olig2cDNA-IRES-dsRed2-3', we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor.

  11. An Autonomously Replicating Transforming Vector for Sulfolobus solfataricus

    Science.gov (United States)

    Cannio, Raffaele; Contursi, Patrizia; Rossi, Mosè; Bartolucci, Simonetta

    1998-01-01

    A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C. PMID:9620978

  12. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  13. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    Science.gov (United States)

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  14. Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

    Science.gov (United States)

    Garcillán-Barcia, M Pilar; Ruiz del Castillo, Belén; Alvarado, Andrés; de la Cruz, Fernando; Martínez-Martínez, Luis

    2015-01-01

    Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements.

  15. Temporal order of evolution of DNA replication systems inferred by comparison of cellular and viral DNA polymerases

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2006-12-01

    Full Text Available Abstract Background The core enzymes of the DNA replication systems show striking diversity among cellular life forms and more so among viruses. In particular, and counter-intuitively, given the central role of DNA in all cells and the mechanistic uniformity of replication, the core enzymes of the replication systems of bacteria and archaea (as well as eukaryotes are unrelated or extremely distantly related. Viruses and plasmids, in addition, possess at least two unique DNA replication systems, namely, the protein-primed and rolling circle modalities of replication. This unexpected diversity makes the origin and evolution of DNA replication systems a particularly challenging and intriguing problem in evolutionary biology. Results I propose a specific succession for the emergence of different DNA replication systems, drawing argument from the differences in their representation among viruses and other selfish replicating elements. In a striking pattern, the DNA replication systems of viruses infecting bacteria and eukaryotes are dominated by the archaeal-type B-family DNA polymerase (PolB whereas the bacterial replicative DNA polymerase (PolC is present only in a handful of bacteriophage genomes. There is no apparent mechanistic impediment to the involvement of the bacterial-type replication machinery in viral DNA replication. Therefore, I hypothesize that the observed, markedly unequal distribution of the replicative DNA polymerases among the known cellular and viral replication systems has a historical explanation. I propose that, among the two types of DNA replication machineries that are found in extant life forms, the archaeal-type, PolB-based system evolved first and had already given rise to a variety of diverse viruses and other selfish elements before the advent of the bacterial, PolC-based machinery. Conceivably, at that stage of evolution, the niches for DNA-viral reproduction have been already filled with viruses replicating with the

  16. Mobilization of Bacillus thuringiensis plasmid pTX14-3.

    Science.gov (United States)

    Andrup, L; Bendixen, H H; Jensen, G B

    1995-05-01

    The Bacillus thuringiensis subsp. israelensis (Bti) plasmid pTX14-3 has been reported to contain a gene, mob14-3, with considerable homology to genes encoding mobilization proteins from other gram-positive bacteria. We have used the aggregation-mediated conjugation system recently discovered in Bti to compare the mobilization kinetics of different derivatives of plasmid pTX14-3. Plasmid pTX14-3 has been found to replicate by the rolling-circle mechanism and to contain a locus suppressing the formation of high-molecular-weight DNA. We found that deleting a DNA fragment containing this locus increased the transfer frequency about twofold. The mobilization frequency of the plasmid containing the intact mob14-3 gene did not indicate a mobilization-enhancing activity of the encoded polypeptide. However, the presence of the mob14-3 gene seemed to increase the stability of the plasmid in exponential growth.

  17. A quantitative model of DNA replication in Xenopus embryos: reliable replication despite stochasticity

    Science.gov (United States)

    Cheng-Hsin Yang, Scott; Bechhoefer, John

    2008-03-01

    DNA synthesis in Xenopus frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the replication time and may lead to cell death if replication takes longer than the cell cycle time (˜ 25 min.). Surprisingly, although the typical replication time is about 20 min., in vivo experiments show that replication fails to complete only about 1 in 250 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two mechanisms: the first uses a regular spatial distribution of origins, while the second uses randomly located origins but increases their probability of initiation as the cell cycle proceeds. Here, we show that both mechanisms yield similar end-time distributions, implying that regular origin spacing is not needed for control of replication time. Moreover, we show that the experimentally inferred time-dependent initiation rate satisfies the observed low failure probability and nearly optimizes the use of replicative proteins.

  18. The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

    Directory of Open Access Journals (Sweden)

    Jyoti K. Jha

    2017-04-01

    Full Text Available Replication of Vibrio cholerae chromosome 2 (Chr2 depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.

  19. pGIAK1, a heavy metal resistant plasmid from an obligate alkaliphilic and halotolerant bacterium isolated from the Antarctic Concordia station confined environment.

    Directory of Open Access Journals (Sweden)

    Suxia Guo

    Full Text Available pGIAK1 is a 38-kb plasmid originating from the obligate alkaliphilic and halotolerant Bacillaceae strain JMAK1. The strain was originally isolated from the confined environments of the Antarctic Concordia station. Analysis of the pGIAK1 38,362-bp sequence revealed that, in addition to its replication region, this plasmid contains the genetic determinants for cadmium and arsenic resistances, putative methyltransferase, tyrosine recombinase, spore coat protein and potassium transport protein, as well as several hypothetical proteins. Cloning the pGIAK1 cad operon in Bacillus cereus H3081.97 and its ars operon in Bacillus subtilis 1A280 conferred to these hosts cadmium and arsenic resistances, respectively, therefore confirming their bona fide activities. The pGIAK1 replicon region was also shown to be functional in Bacillus thuringiensis, Bacillus subtilis and Staphylococcus aureus, but was only stably maintained in B. subtilis. Finally, using an Escherichia coli - B. thuringiensis shuttle BAC vector, pGIAK1 was shown to display conjugative properties since it was able to transfer the BAC plasmid among B. thuringiensis strains.

  20. Spatial regulation and organization of DNA replication within the nucleus

    OpenAIRE

    2009-01-01

    Duplication of chromosomal DNA is a temporally and spatially regulated process. The timing of DNA replication initiation at various origins is highly coordinated; some origins fire early and others late during S phase. Moreover, inside the nuclei, the bulk of DNA replication is physically organized in replication factories, consisting of DNA polymerases and other replication proteins. In this review article, we discuss how DNA replication is organized and regulated spatially within the nucleu...

  1. Diversity and stability of plasmids from glycopeptide resistant Enterococcus faecium isolated from pigs in Denmark

    DEFF Research Database (Denmark)

    Hasman, H.; Villadsen, A. G.; Aarestrup, Frank Møller

    2005-01-01

    was seen at the end of the 7-year period, coinciding with the ban in 1998 of the macrolide tylosin as growth promoter for pig production. The stability of the plasmid in its original host was compared with stability of the same plasmid in BM4105RF, when both strains were maintained in liquid cultures...

  2. Diversity and stability of plasmids from glycopeptide resistant Enterococcus faecium isolated from pigs in Denmark

    DEFF Research Database (Denmark)

    Hasman, H.; Villadsen, A. G.; Aarestrup, Frank Møller

    2005-01-01

    was seen at the end of the 7-year period, coinciding with the ban in 1998 of the macrolide tylosin as growth promoter for pig production. The stability of the plasmid in its original host was compared with stability of the same plasmid in BM4105RF, when both strains were maintained in liquid cultures...

  3. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  4. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    Science.gov (United States)

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2016-01-01

    Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

  5. Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid.

    Science.gov (United States)

    Duquesne, Fabien; Hébert, Laurent; Sévin, Corinne; Breuil, Marie-France; Tapprest, Jackie; Laugier, Claire; Petry, Sandrine

    2010-10-01

    To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene. © 2010 ANSES. Journal compilation © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

  6. Spatial regulation and organization of DNA replication within the nucleus.

    Science.gov (United States)

    Natsume, Toyoaki; Tanaka, Tomoyuki U

    2010-01-01

    Duplication of chromosomal DNA is a temporally and spatially regulated process. The timing of DNA replication initiation at various origins is highly coordinated; some origins fire early and others late during S phase. Moreover, inside the nuclei, the bulk of DNA replication is physically organized in replication factories, consisting of DNA polymerases and other replication proteins. In this review article, we discuss how DNA replication is organized and regulated spatially within the nucleus and how this spatial organization is linked to temporal regulation. We focus on DNA replication in budding yeast and fission yeast and, where applicable, compare yeast DNA replication with that in bacteria and metazoans.

  7. Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple.

    Science.gov (United States)

    Huang, T C; Burr, T J

    1999-05-01

    Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.

  8. Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

    Science.gov (United States)

    Zurfluh, Katrin; Nüesch-Inderbinen, Magdalena; Klumpp, Jochen; Poirel, Laurent; Nordmann, Patrice; Stephan, Roger

    2017-01-01

    Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones. There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some "epidemic" plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

  9. Single molecule analysis of Trypanosoma brucei DNA replication dynamics.

    Science.gov (United States)

    Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco; Marques, Catarina A; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L; Elias, Maria Carolina

    2015-03-11

    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.

  10. Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication.

    Science.gov (United States)

    Luria, Neta; Lu, Liqun; Chejanovsky, Nor

    2012-05-01

    IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  11. Conserved Structural Motifs at the C-Terminus of Baculovirus Protein IE0 are Important for its Functions in Transactivation and Supporting hr5-mediated DNA Replication

    Directory of Open Access Journals (Sweden)

    Neta Luria

    2012-05-01

    Full Text Available IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV. IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  12. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    Science.gov (United States)

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-03-09

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes.

  13. Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; Habuchi, Satoshi; van Oijen, Antoine M.; Walter, Johannes C.

    2010-01-01

    The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes.

  14. Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; Habuchi, Satoshi; van Oijen, Antoine M.; Walter, Johannes C.

    2010-01-01

    The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. So

  15. DNA-binding proteins regulating pIP501 transfer and replication

    Directory of Open Access Journals (Sweden)

    Elisabeth Grohmann

    2016-08-01

    Full Text Available pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently found in clinical Enterococcus faecalis and Enterococcus faecium isolates. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual role: It acts as transcriptional repressor at the repR promoter and prevents convergent transcription of RNAIII and repR mRNA (RNAII, thereby indirectly increasing RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII. Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including filamentous streptomycetes and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter, which overlaps with the origin of transfer (oriT. The T4SS operon encodes the DNA-binding proteins TraJ (VirD4

  16. DNA-Binding Proteins Regulating pIP501 Transfer and Replication

    Science.gov (United States)

    Grohmann, Elisabeth; Goessweiner-Mohr, Nikolaus; Brantl, Sabine

    2016-01-01

    pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently detected in clinical Enterococcus faecalis and Enterococcus faecium strains. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual function: It acts as transcriptional repressor at the repR promoter and, in addition, prevents convergent transcription of RNAIII and repR mRNA (RNAII), which indirectly increases RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII). Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS) encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including Streptomyces and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter overlapping with the origin of transfer (oriT). The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling

  17. Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii.

    Science.gov (United States)

    Kent, Bethany N; Foecking, Mark F; Calcutt, Michael J

    2012-10-01

    A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.

  18. Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1.

    Science.gov (United States)

    Singh, M; Kreutzer, R; Acker, G; Klingmüller, W

    1988-01-01

    A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.

  19. High-Resolution Replication Profiles Define the Stochastic Nature of Genome Replication Initiation and Termination

    Directory of Open Access Journals (Sweden)

    Michelle Hawkins

    2013-11-01

    Full Text Available Eukaryotic genome replication is stochastic, and each cell uses a different cohort of replication origins. We demonstrate that interpreting high-resolution Saccharomyces cerevisiae genome replication data with a mathematical model allows quantification of the stochastic nature of genome replication, including the efficiency of each origin and the distribution of termination events. Single-cell measurements support the inferred values for stochastic origin activation time. A strain, in which three origins were inactivated, confirmed that the distribution of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci.

  20. ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation

    Science.gov (United States)

    Freudenau, Inga; Lutter, Petra; Baier, Ruth; Schleef, Martin; Bednarz, Hanna; Lara, Alvaro R.; Niehaus, Karsten

    2015-01-01

    Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production. PMID:26389114

  1. ColE1-plasmid production in Escherichia coli: Mathematical Simulation and Experimental Validation

    Directory of Open Access Journals (Sweden)

    Inga eFreudenau

    2015-09-01

    Full Text Available Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the last years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3 and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ by using ordinary differential equations (ODE and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN, and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ±0.7 to 34 ±7 RNAI molecules per cell and 0.44 ±0.1 to 3 ±0.9 RNAII molecules per cell. The determined plasmid copy numbers (PCN averaged between 46 ±26 to 48 ±30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ±203 to 1086 ±298 RNAI molecules per cell and 22 ±2 to 75 ±10 RNAII molecules per cell with an averaged PCN of 1514 ±1301 to 5806 ±4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the pDNA production.

  2. Archaeal DNA replication.

    Science.gov (United States)

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed.

  3. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

    Science.gov (United States)

    Gaimster, Hannah; Summers, David

    2015-03-01

    Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Modular construction of plasmids by parallel assembly of linear vector components.

    Science.gov (United States)

    Gao, XinZheng; Yan, Pu; Shen, Wentao; Li, Xiaoying; Zhou, Peng; Li, Yuenan

    2013-06-15

    Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

  5. Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake.

    Science.gov (United States)

    Vogel, R F; Lohmann, M; Weller, A N; Hugas, M; Hammes, W P

    1991-11-15

    Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.

  6. DNA replication stress: causes, resolution and disease.

    Science.gov (United States)

    Mazouzi, Abdelghani; Velimezi, Georgia; Loizou, Joanna I

    2014-11-15

    DNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Various pertubations, originating from endogenous or exogenous sources, can interfere with proper progression and completion of the replication process, thus threatening genome integrity. Coordinated regulation of replication and the DNA damage response is therefore fundamental to counteract these challenges and ensure accurate synthesis of the genetic material under conditions of replication stress. In this review, we summarize the main sources of replication stress and the DNA damage signaling pathways that are activated in order to preserve genome integrity during DNA replication. We also discuss the association of replication stress and DNA damage in human disease and future perspectives in the field. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Co-resident plasmids travel together.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  9. The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment

    Science.gov (United States)

    Flórez, Ana Belén; Mayo, Baltasar

    2015-01-01

    Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication (ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42

  10. How frog embryos replicate their DNA reliably

    Science.gov (United States)

    Bechhoefer, John; Marshall, Brandon

    2007-03-01

    Frog embryos contain three billion base pairs of DNA. In early embryos (cycles 2-12), DNA replication is extremely rapid, about 20 min., and the entire cell cycle lasts only 25 min., meaning that mitosis (cell division) takes place in about 5 min. In this stripped-down cell cycle, there are no efficient checkpoints to prevent the cell from dividing before its DNA has finished replication - a disastrous scenario. Even worse, the many origins of replication are laid down stochastically and are also initiated stochastically throughout the replication process. Despite the very tight time constraints and despite the randomness introduced by origin stochasticity, replication is extremely reliable, with cell division failing no more than once in 10,000 tries. We discuss a recent model of DNA replication that is drawn from condensed-matter theories of 1d nucleation and growth. Using our model, we discuss different strategies of replication: should one initiate all origins as early as possible, or is it better to hold back and initiate some later on? Using concepts from extreme-value statistics, we derive the distribution of replication times given a particular scenario for the initiation of origins. We show that the experimentally observed initiation strategy for frog embryos meets the reliability constraint and is close to the one that requires the fewest resources of a cell.

  11. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  12. Control of DNA replication by anomalous reaction-diffusion kinetics

    Science.gov (United States)

    Bechhoefer, John; Gauthier, Michel

    2010-03-01

    DNA replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. Experiments indicate that these origins are triggered over the whole genome at a rate I(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (S) phase, before rapidly decreasing to zero at the end of the replication process. We propose a simple model for the control of DNA replication in which the rate of initiation of replication origins is controlled by protein-DNA interactions. Analyzing recent data from Xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. Initiation of origins is suppressed when the diffusion-limited search time dominates. To fit the experimental data, we find that the interaction between DNA and the rate-limiting protein must be subdiffusive.

  13. Effect of low temperature on stability of theta-type plasmids in Carnobacterium maltaromaticum.

    Science.gov (United States)

    Bohaychuk, Valerie M; van Belkum, Marco J; Stiles, Michael E; McMullen, Lynn M

    2008-03-01

    The heterologous production of useful peptides such as bacteriocins by lactic acid bacteria (LAB) has been studied for use in the biopreservation of foods. Recombinant plasmids can suffer drawbacks such as segregational instability affecting the production of these peptides in certain environments such as absence of selective pressure or low temperature. The link between growth temperature characteristics of parental strains and stability of theta-type plasmids at a low temperature was investigated. The growth of four parental strains at 4 degrees C and stability of five derivative theta-type plasmids transformed into Carnobacterium maltaromaticum UAL26 at 25 and 4 degrees C were determined. Two plasmids (pCD11 and pCaT) derived from psychrotrophic LAB and plasmid, pHW800, from Enterococcus faecium 226 with unknown growth temperature characteristics, had excellent stability when strains were grown at 4 degrees C. Plasmids (pTRKH2 and pUCB820) derived from LAB that did not grow at refrigeration temperatures were not stable at 4 degrees C. When a DNA fragment from pCD11 containing 22-bp repeats, a putative replication initiation site, and the gene for the RepA protein was inserted into pTRKH2, the resulting derivative plasmid was 100% stable at 4 degrees C.

  14. Broad-Host-Range IncP-1 plasmids and their resistance potential

    Directory of Open Access Journals (Sweden)

    Magdalena ePopowska

    2013-03-01

    Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

  15. Development and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae.

    Directory of Open Access Journals (Sweden)

    Shukriti Sharma

    Full Text Available Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.

  16. Dynamics of Escherichia coli chromosome segregation during multifork replication.

    Science.gov (United States)

    Nielsen, Henrik J; Youngren, Brenda; Hansen, Flemming G; Austin, Stuart

    2007-12-01

    Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.

  17. Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326.

    Science.gov (United States)

    Sun, Yuhui; He, Xinyi; Liang, Jingdan; Zhou, Xiufen; Deng, Zixin

    2009-02-01

    The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85(-)) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti(-), rep*, orf85(-)) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti(+) pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.

  18. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  19. Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae strains isolated in North-East Italy.

    Science.gov (United States)

    Kocsis, B; Mazzariol, A; Kocsis, E; Koncan, R; Fontana, R; Cornaglia, G

    2013-02-01

    We investigated the prevalence of plasmid-mediated quinolone resistance genes in 756 clinical isolates of Enterobacteriaceae originating from Microbiology Diagnostic Laboratories of North-East Italy. Five point zero two percent of isolates carried a qnr determinant while the aac(6')-Ib-cr determinant was detected in 9·25% of isolates. We also investigated the association between the plasmid-mediated quinolone resistance and the beta-lactamase genes, and characterized the plasmids carrying these determinants of resistance.

  20. Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

    Science.gov (United States)

    Lim, Suk-Kyung; Tanimoto, Koichi; Tomita, Haruyoshi; Ike, Yasuyoshi

    2006-10-01

    The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information

  1. A dimeric Rep protein initiates replication of a linear archaeal virus genome: implications for the Rep mechanism and viral replication

    DEFF Research Database (Denmark)

    Oke, Muse; Kerou, Melina; Liu, Huanting

    2011-01-01

    that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between...... positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral...

  2. Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells.

    Science.gov (United States)

    Spuul, Pirjo; Balistreri, Giuseppe; Hellström, Kirsi; Golubtsov, Andrey V; Jokitalo, Eija; Ahola, Tero

    2011-05-01

    For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.

  3. Chlamydophila felis: plasmid detection in Italian isolates.

    Science.gov (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella

    2010-04-01

    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  4. Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes.

    Science.gov (United States)

    Wu, LiHong; Liu, Yang; Kong, DaoChun

    2014-05-01

    Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

  5. Characterization of plasmids in extensively drug-resistant acinetobacter strains isolated in India and Pakistan.

    Science.gov (United States)

    Jones, Lim S; Carvalho, Maria J; Toleman, Mark A; White, P Lewis; Connor, Thomas R; Mushtaq, Ammara; Weeks, Janis L; Kumarasamy, Karthikeyan K; Raven, Katherine E; Török, M Estée; Peacock, Sharon J; Howe, Robin A; Walsh, Timothy R

    2015-02-01

    The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.

  6. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

    Science.gov (United States)

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-04-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.

  7. Regulation of Unperturbed DNA Replication by Ubiquitylation

    Directory of Open Access Journals (Sweden)

    Sara Priego Moreno

    2015-06-01

    Full Text Available Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key role in coordination of the DNA damage response is well established. In this review we focus, however, on the ways ubiquitylation controls the process of unperturbed DNA replication. We summarise the accumulated knowledge showing the leading role of ubiquitin driven protein degradation in setting up conditions favourable for replication origin licensing and S-phase entry. Importantly, we also present the emerging major role of ubiquitylation in coordination of the active DNA replication process: preventing re-replication, regulating the progression of DNA replication forks, chromatin re-establishment and disassembly of the replisome at the termination of replication forks.

  8. Sequence analysis and minimal replicon determination of a new haloarchaeal plasmid pHF2 isolated from Haloferax sp. strain Q22.

    Science.gov (United States)

    Chen, Shaoxing; Wang, Chuangming; Xiang, Hua

    2016-01-01

    A new cryptic plasmid, pHF2 (2520 bp), was isolated from Haloferax sp. strain Q22 (CGMCC 1.15317), a haloarchaeal strain living in a subterranean halite deposit. Sequence analysis revealed that it is the smallest plasmid in the genus Haloferax so far, and three syntropic open reading frames (ORF1, ORF2, and ORF3) were identified on the same strand. ORF1 encodes a putative replication initiation protein (Rep). Three typical motifs (I, II, and III) were presented in the Rep proteins of rolling-circle replicating (RCR) plasmids. The amino acid sequence of the Rep protein is very similar to that of another haloarchaeal plasmid pNB101 in Natronobacterium sp. AS-7091 (coverage 97%, identity 56%). The minimal replicon (~1000 bp) of pHF2 was determined through the construction of a series of truncated plasmids. Interestingly, we also found that the incomplete rep gene still can drive plasmid replication. This plasmid has provided another valuable extra-chromosomal genetic resource, and deepened our knowledge in DNA replication.

  9. Progressive Rearrangement of Telomeric Sequences Added to Both the ITR Ends of the Yeast Linear pGKL Plasmid

    Directory of Open Access Journals (Sweden)

    Gunge Norio

    2003-01-01

    Full Text Available Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. In Saccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid which carried the host telomeric repeats TG1-3 of 300-350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat, suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG1-3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids.

  10. Comparative Genomics of the Conjugation Region of F-like Plasmids: Five Shades of F

    Science.gov (United States)

    Fernandez-Lopez, Raul; de Toro, Maria; Moncalian, Gabriel; Garcillan-Barcia, M. Pilar; de la Cruz, Fernando

    2016-01-01

    The F plasmid is the foremost representative of a large group of conjugative plasmids, prevalent in Escherichia coli, and widely distributed among the Enterobacteriaceae. These plasmids are of clinical relevance, given their frequent association with virulence determinants, colicins, and antibiotic resistance genes. Originally defined by their sensitivity to certain male-specific phages, IncF plasmids share a conserved conjugative system and regulatory circuits. In order to determine whether the genetic architecture and regulation circuits are preserved among these plasmids, we analyzed the natural diversity of F-like plasmids. Using the relaxase as a phylogenetic marker, we identified 256 plasmids belonging to the IncF/ MOBF12group, present as complete DNA sequences in the NCBI database. By comparative genomics, we identified five major groups of F-like plasmids. Each shows a particular operon structure and alternate regulatory systems. Results show that the IncF/MOBF12 conjugation gene cluster conforms a diverse and ancient group, which evolved alternative regulatory schemes in its adaptation to different environments and bacterial hosts. PMID:27891505

  11. Diversity and stability of Plasmids from glycopeptide-resistant Enterococcus faecium (GRE) isolated from pigs in Denmark

    DEFF Research Database (Denmark)

    Hasman, Henrik; Villadsen, A.G.; Aarestrup, Frank Møller

    2005-01-01

    was seen at the end of the 7-year period, coinciding with the ban in 1998 of the macrolide tylosin as growth promoter for pig production. The stability of the plasmid in its original host was compared with stability of the same plasmid in BM4105RF, when both strains were maintained in liquid cultures...

  12. Kalilo plasmids are a family of four distinct members with individual global distributions across species.

    Science.gov (United States)

    He, C; Nastasja de Groot; Bok, J W; Griffiths, A J

    2000-01-01

    Kalilo is a linear 9-kb plasmid, isolated originally from Hawaiian strains of the heterothallic fungus Neurospora intermedia. Its properties include terminal inverted repeats, two ORFs coding for a presumptive DNA and an RNA polymerase, and the ability to cause senescence in its original host and in the closely related species Neurospora crassa. We have examined natural isolates alleged to contain plasmids homologous to kalilo. Most of these isolates do in fact contain plasmids with so close an identity to kalilo as to be certain relatives. We found a new case of kalilo in Neurospora tetrasperma from Moorea-Tahiti, and a new case of LA-kalilo (previously found only in N. tetrasperma) in N. crassa from Haiti. A previously unreported, substantially shorter, kalilo variant has been found in three geographically separate isolates of the heterothallic species Neurospora discreta. Therefore, if the previously reported kalilo variant from the genus Gelasinospora is included, in all there are four members of the kalilo plasmid family. The main differences between these plasmids are in the terminal inverted repeats (TIRs). The phylogeny of the TIR sequences is largely congruent with that of nuclear DNA in the species in which they are found, suggesting that the plasmids are related by vertical descent throughout the evolution of these species. However, there are two cases of a plasmid found in a heterothallic and a pseudohomothallic species in the same global area; these cases might have arisen from more recent horizontal transmission or introgression.

  13. Control of chromosome replication in caulobacter crescentus.

    Science.gov (United States)

    Marczynski, Gregory T; Shapiro, Lucy

    2002-01-01

    Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Its chromosome replication origin (Cori) may be prototypical of the large and diverse class of alpha-proteobacteria. Cori has features that both affiliate and distinguish it from the Escherichia coli chromosome replication origin. For example, requirements for DnaA protein and RNA transcription affiliate both origins. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria.

  14. Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

    OpenAIRE

    Bröer, S; Ji, G.; Bröer, A; Silver, S

    1993-01-01

    The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic or...

  15. Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

    Directory of Open Access Journals (Sweden)

    Yanhua Cui

    2015-06-01

    Full Text Available Plasmids are widely distributed in different sources of lactic acid bacteria (LAB as self-replicating extrachromosomal genetic materials, and have received considerable attention due to their close relationship with many important functions as well as some industrially relevant characteristics of the LAB species. They are interesting with regard to the development of food-grade cloning vectors. This review summarizes new developments in the area of lactic acid bacteria plasmids and aims to provide up to date information that can be used in related future research.

  16. Geminin: a major DNA replication safeguard in higher eukaryotes

    DEFF Research Database (Denmark)

    Melixetian, Marina; Helin, Kristian

    2004-01-01

    Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...

  17. Geminin: a major DNA replication safeguard in higher eukaryotes

    DEFF Research Database (Denmark)

    Melixetian, Marina; Helin, Kristian

    2004-01-01

    Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication...

  18. Hundreds of circular novel plasmids and DNA elements identified in a rat cecum metamobilome.

    Directory of Open Access Journals (Sweden)

    Tue Sparholt Jørgensen

    Full Text Available Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences. The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database.

  19. Genomics of high molecular weight plasmids isolated from an on-farm biopurification system.

    Science.gov (United States)

    Martini, María C; Wibberg, Daniel; Lozano, Mauricio; Torres Tejerizo, Gonzalo; Albicoro, Francisco J; Jaenicke, Sebastian; van Elsas, Jan Dirk; Petroni, Alejandro; Garcillán-Barcia, M Pilar; de la Cruz, Fernando; Schlüter, Andreas; Pühler, Alfred; Pistorio, Mariano; Lagares, Antonio; Del Papa, María F

    2016-06-20

    The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.

  20. RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

    Science.gov (United States)

    Pérez-Oseguera, Angeles; Cevallos, Miguel A

    2013-11-01

    Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process.

  1. The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations.

    Science.gov (United States)

    Haugan, K; Karunakaran, P; Blatny, J M; Valla, S

    1992-01-01

    The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein. PMID:1400252

  2. Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates.

    Science.gov (United States)

    Kaplan, Ella; Sela, Noa; Doron-Faigenboim, Adi; Navon-Venezia, Shiri; Jurkevitch, Edouard; Cytryn, Eddie

    2015-01-01

    plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A) qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B) that small qnrS-harboring pGNB2-like plasmids are ubiquitous in wastewater treatment facilities and are most likely environmental in origin.

  3. Replication, falsification, and the crisis of confidence in social psychology.

    Science.gov (United States)

    Earp, Brian D; Trafimow, David

    2015-01-01

    The (latest) crisis in confidence in social psychology has generated much heated discussion about the importance of replication, including how it should be carried out as well as interpreted by scholars in the field. For example, what does it mean if a replication attempt "fails"-does it mean that the original results, or the theory that predicted them, have been falsified? And how should "failed" replications affect our belief in the validity of the original research? In this paper, we consider the replication debate from a historical and philosophical perspective, and provide a conceptual analysis of both replication and falsification as they pertain to this important discussion. Along the way, we highlight the importance of auxiliary assumptions (for both testing theories and attempting replications), and introduce a Bayesian framework for assessing "failed" replications in terms of how they should affect our confidence in original findings.

  4. Replication, falsification, and the crisis of confidence in social psychology

    Science.gov (United States)

    Earp, Brian D.; Trafimow, David

    2015-01-01

    The (latest) crisis in confidence in social psychology has generated much heated discussion about the importance of replication, including how it should be carried out as well as interpreted by scholars in the field. For example, what does it mean if a replication attempt “fails”—does it mean that the original results, or the theory that predicted them, have been falsified? And how should “failed” replications affect our belief in the validity of the original research? In this paper, we consider the replication debate from a historical and philosophical perspective, and provide a conceptual analysis of both replication and falsification as they pertain to this important discussion. Along the way, we highlight the importance of auxiliary assumptions (for both testing theories and attempting replications), and introduce a Bayesian framework for assessing “failed” replications in terms of how they should affect our confidence in original findings. PMID:26042061

  5. Persistence of Antibiotic Resistance Plasmids in Biofilms

    Science.gov (United States)

    2014-10-01

    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  6. Escalation of error catastrophe for enzymatic self-replicators

    Science.gov (United States)

    Obermayer, B.; Frey, E.

    2009-11-01

    It is a long-standing question in origin-of-life research whether the information content of replicating molecules can be maintained in the presence of replication errors. Extending standard quasispecies models of non-enzymatic replication, we analyze highly specific enzymatic self-replication mediated through an otherwise neutral recognition region, which leads to frequency-dependent replication rates. We find a significant reduction of the maximally tolerable error rate, because the replication rate of the fittest molecules decreases with the fraction of functional enzymes. Our analysis is extended to hypercyclic couplings as an example for catalytic networks.

  7. Regulation of chromosomal replication in Caulobacter crescentus.

    Science.gov (United States)

    Collier, Justine

    2012-03-01

    The alpha-proteobacterium Caulobacter crescentus is characterized by its asymmetric cell division, which gives rise to a replicating stalked cell and a non-replicating swarmer cell. Thus, the initiation of chromosomal replication is tightly regulated, temporally and spatially, to ensure that it is coordinated with cell differentiation and cell cycle progression. Waves of DnaA and CtrA activities control when and where the initiation of DNA replication will take place in C. crescentus cells. The conserved DnaA protein initiates chromosomal replication by directly binding to sites within the chromosomal origin (Cori), ensuring that DNA replication starts once and only once per cell cycle. The CtrA response regulator represses the initiation of DNA replication in swarmer cells and in the swarmer compartment of pre-divisional cells, probably by competing with DnaA for binding to Cori. CtrA and DnaA are controlled by multiple redundant regulatory pathways that include DNA methylation-dependent transcriptional regulation, temporally regulated proteolysis and the targeting of regulators to specific locations within the cell. Besides being critical regulators of chromosomal replication, CtrA and DnaA are also master transcriptional regulators that control the expression of many genes, thus connecting DNA replication with other events of the C. crescentus cell cycle. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants.

    Science.gov (United States)

    Wiesner, Magdalena; Calva, Edmundo; Fernández-Mora, Marcos; Cevallos, Miguel A; Campos, Freddy; Zaidi, Mussaret B; Silva, Claudia

    2011-01-11

    Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the

  9. Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum.

    Directory of Open Access Journals (Sweden)

    Isolde Nieszner

    Full Text Available Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriC MG. We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a "Trojan horse" plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriC MG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes.

  10. Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

    Science.gov (United States)

    Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

    1994-07-01

    We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

  11. Replication forks reverse at high frequency upon replication stress in Physarum polycephalum.

    Science.gov (United States)

    Maric, Chrystelle; Bénard, Marianne

    2014-12-01

    The addition of hydroxyurea after the onset of S phase allows replication to start and permits the successive detecting of replication-dependent joint DNA molecules and chicken foot structures in the synchronous nuclei of Physarum polycephalum. We find evidence for a very high frequency of reversed replication forks upon replication stress. The formation of these reversed forks is dependent on the presence of joint DNA molecules, the impediment of the replication fork progression by hydroxyurea, and likely on the propensity of some replication origins to reinitiate replication to counteract the action of this compound. As hydroxyurea treatment enables us to successively detect the appearance of joint DNA molecules and then of reversed replication forks, we propose that chicken foot structures are formed both from the regression of hydroxyurea-frozen joint DNA molecules and from hydroxyurea-stalled replication forks. These experiments underscore the transient nature of replication fork regression, which becomes detectable due to the hydroxyurea-induced slowing down of replication fork progression.

  12. Plasmid profiling of bacterial isolates from confined environments

    Science.gov (United States)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  13. Plasmid profiles of Moraxella bovis isolates.

    Science.gov (United States)

    McDonald, T J; Pugh, G W

    1986-04-01

    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  14. Plasmid transfer systems in the rhizobia.

    Science.gov (United States)

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  15. Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

    Directory of Open Access Journals (Sweden)

    Mónica Aguado-Urda

    Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

  16. Semiconservative replication in the quasispecies model

    Science.gov (United States)

    Tannenbaum, Emmanuel; Deeds, Eric J.; Shakhnovich, Eugene I.

    2004-06-01

    This paper extends Eigen’s quasispecies equations to account for the semiconservative nature of DNA replication. We solve the equations in the limit of infinite sequence length for the simplest case of a static, sharply peaked fitness landscape. We show that the error catastrophe occurs when μ , the product of sequence length and per base pair mismatch probability, exceeds 2 ln [2/ ( 1+1/k ) ] , where k>1 is the first-order growth rate constant of the viable “master” sequence (with all other sequences having a first-order growth rate constant of 1 ). This is in contrast to the result of ln k for conservative replication. In particular, as k→∞ , the error catastrophe is never reached for conservative replication, while for semiconservative replication the critical μ approaches 2 ln 2 . Semiconservative replication is therefore considerably less robust than conservative replication to the effect of replication errors. We also show that the mean equilibrium fitness of a semiconservatively replicating system is given by k ( 2 e-μ/2 -1 ) below the error catastrophe, in contrast to the standard result of k e-μ for conservative replication (derived by Kimura and Maruyama in 1966). From this result it is readily shown that semiconservative replication is necessary to account for the observation that, at sufficiently high mutagen concentrations, faster replicating cells will die more quickly than more slowly replicating cells. Thus, in contrast to Eigen’s original model, the semiconservative quasispecies equations are able to provide a mathematical basis for explaining the efficacy of mutagens as chemotherapeutic agents.

  17. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  18. Analysis of benzo[a]pyrene diolepoxide mutagenesis in a mammalian in vitro DNA replication system

    Energy Technology Data Exchange (ETDEWEB)

    Vasunia, K.; Cheng, L.; Carty, M. [Univ. of Cincinnati, OH (United States)] [and others

    1995-11-01

    Chemicals that interact with DNA and cause mutations, thereby activating protooncogenes or inactivating tumor suppressor genes, are thought to initiate the process of carcinogenesis. To elucidate the molecular mechanisms involved in mutagenesis of bulky adducts, we used an in vitro DNA replication system. An SV40-based shuttle vector, PZ189, was treated with anti-benzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide (BPDE) and then replicated in vitro using hypotonic extracts of human HeLa cells. The replication efficiency was monitored by the incorporation of a radiolabelled nucleotide into DNA. The products of the replication reaction were then digested with Dpn-1 to inactivate the unreplicated plasmid and the mutation frequency was evaluated by transfection into E. coli MBM7070. Our results show that BPDE-damaged plasmids undergo replication in the in vitro system. The efficiency of replication and the mutant frequency is dose-dependent, such that the replication efficiency decreases and mutation frequency increases with increasing BPDE dose to the plasmid. This study further validates the in vitro DNA replication system by demonstrating the mutagenicity of bulky adducts of BPDE.

  19. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  20. A New Replication Norm for Psychology

    Directory of Open Access Journals (Sweden)

    Etienne P LeBel

    2015-10-01

    Full Text Available In recent years, there has been a growing concern regarding the replicability of findings in psychology, including a mounting number of prominent findings that have failed to replicate via high-powered independent replication attempts. In the face of this replicability “crisis of confidence”, several initiatives have been implemented to increase the reliability of empirical findings. In the current article, I propose a new replication norm that aims to further boost the dependability of findings in psychology. Paralleling the extant social norm that researchers should peer review about three times as many articles that they themselves publish per year, the new replication norm states that researchers should aim to independently replicate important findings in their own research areas in proportion to the number of original studies they themselves publish per year (e.g., a 4:1 original-to-replication studies ratio. I argue this simple approach could significantly advance our science by increasing the reliability and cumulative nature of our empirical knowledge base, accelerating our theoretical understanding of psychological phenomena, instilling a focus on quality rather than quantity, and by facilitating our transformation toward a research culture where executing and reporting independent direct replications is viewed as an ordinary part of the research process. To help promote the new norm, I delineate (1 how each of the major constituencies of the research process (i.e., funders, journals, professional societies, departments, and individual researchers can incentivize replications and promote the new norm and (2 any obstacles each constituency faces in supporting the new norm.

  1. The 64 508 bp IncP-1beta antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1beta group.

    Science.gov (United States)

    Schlüter, A; Heuer, H; Szczepanowski, R; Forney, L J; Thomas, C M; Pühler, A; Top, E M

    2003-11-01

    The complete 64508 bp nucleotide sequence of the IncP-1beta antibiotic-resistance plasmid pB10, which was isolated from a waste-water treatment plant in Germany and mediates resistance against the antimicrobial agents amoxicillin, streptomycin, sulfonamides and tetracycline and against mercury ions, was determined and analysed. A typical class 1 integron with completely conserved 5' and 3' segments is inserted between the tra and trb regions. The two mobile gene cassettes of this integron encode a beta-lactamase of the oxacillin-hydrolysing type (Oxa-2) and a gene product of unknown function (OrfE-like), respectively. The pB10-specific gene load present between the replication module (trfA1) and the origin of vegetative replication (oriV) is composed of four class II (Tn3 family) transposable elements: (i). a Tn501-like mercury-resistance (mer) transposon downstream of the trfA1 gene, (ii). a truncated derivative of the widespread streptomycin-resistance transposon Tn5393c, (iii). the insertion sequence element IS1071 and (iv). a Tn1721-like transposon that contains the tetracycline-resistance genes tetA and tetR. A very similar Tn501-like mer transposon is present in the same target site of the IncP-1beta degradative plasmid pJP4 and the IncP-1beta resistance plasmid R906, suggesting that pB10, R906 and pJP4 are derivatives of a common ancestor. Interestingly, large parts of the predicted pB10 restriction map, except for the tetracycline-resistance determinant, are identical to that of R906. It thus appears that plasmid pB10 acquired as many as five resistance genes via three transposons and one integron, which it may rapidly spread among bacterial populations given its high promiscuity. Comparison of the pB10 backbone DNA sequences with those of other sequenced IncP-1beta plasmids reveals a mosaic structure. While the conjugative transfer modules (trb and tra regions) and the replication module are very closely related to the corresponding segments of the IncP-1

  2. Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.; Cabrera-Juarez, E.; Griffin, K.

    1984-11-01

    The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNova. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. The hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.

  3. Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice

    Institute of Scientific and Technical Information of China (English)

    WU Ying; HUANG Ai-long; TANG Ni; ZHANG Bing-qiang; LU Nian-fang

    2005-01-01

    Background RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs (siRNAs) have sufficiently inhibited hepatitis B virus (HBV) replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects.Methods A mutant RNAi vector (pSI-C mut) with two base pairs different from the original target gene sequence at the RNAi vector (pSI-C) was constructed according to the method described in this study. A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload. pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein (GFP) gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay (ELISA) assay was used to measure the concentration of HBV surface antigen (HBsAg) in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein (HBcAg) in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR (RT-PCR) analysis.Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection (p.i.), the OD values were shown to be 5.07±1.07 in infecting group and 0.62±0.59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group (P<0.01). Liver intracellular synthesis of viral core protein was sharply reduced to 0.9%±0.1%, compared with 5.4%±1.2% of positive hepatocytes in infecting group (P<0.01), and the transcriptional level of HBV C mRNA was greatly reduced by 84.7%. However, the irrelevant RNAi control plasmid

  4. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  5. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  6. [Interaction of Dystamycin Dimeric Analog with Poly(dA) x poly(dT), Poly[d(A-T)] x poly[d(A-T)] and Duplex O23 at Origin of Replication of the Herpes Simplex Virus].

    Science.gov (United States)

    Surovaya, A N; Bazhulina, N P; Lepehina, S Yu; Andronova, V L; Galegov, G A; Moiseeva, E D; Grokhovsky, S L; Gursky, G V

    2016-01-01

    The binding of distamycin dimeric analog (Pt-bis-Dst) to poly[d(A-T)] x poly[d(A-T)1, poly(dA) x poly(dT) and duplex O23 with the sequence 5'-GCCAATATATATATATTATTAGG-3' which is present at the origin of replication of herpes simplex virus OriS is investigated with the use of UV and CD spectroscopy. The distinction of the synthetic polyamide from a natural antibiotic lies in the fact that in the synthetic polyamide there are two distamycin moieties bound via a glycine cis-diamino platinum group. It was shown that the binding of Pt-bis-Dst to poly[d(A-T)] x poly[d(A-T)] and poly(dA) x poly(dT) reaches saturation if one molecule of the ligand occurs at approximately every 8 bp. With further increase in the ratio of the added ligand to the base pairs in CD spectra of complexes with poly[d(A-T)] x poly[d(A-T)], we observed that the maximum wavelength band tend to be shifted towards longer wavelengths, while in the spectral region of 290-310 nm a "shoulder", that was absent in the spectra of the complexes obtained at low polymer coverages by the ligand, appeared. At high molar concentration ratios of ligand to oligonucleotide Pt-bis-Dst can bind to poly[d(A-T)] x poly[d(A-T)] in the form of hairpins or may form associates by the interaction between the distamycin moieties of neighboring molecules of Pt-bis-Dst. The structure of the complexes is stabilized by interactions between pirrolcarboxamide moieties of two molecules of Pt-bis-Dst adsorbed on adjacent overlapping binding sites. These interactions are probably also responsible for the concentration-dependent spectral changes observed during the formation of a complex between Pt-bis-Dst and poly[d(A-T)] x poly[d(A-T)]. Spectral changes are almost absent in binding of Pt-bis-Dst to poly(dA) x poly(dT). Binding of Pt-bis-Dst to duplex O23 reaches saturation if two ligand molecules occur in a duplex that contains a cluster of 18 AT pairs. With increasing the molar concentration ratio of the ligand to the duplex CD

  7. Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

    DEFF Research Database (Denmark)

    Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

    2014-01-01

    The transfer of conjugal plasmids is the main bacterial process of horizontal gene transfer to potentially distantly related bacteria. These extrachromosomal, circular DNA molecules host genes that code for their own replication and transfer to other organisms. Because additional accessory genes ...

  8. Transfer and loss of naturally-occuring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

    NARCIS (Netherlands)

    Lakzian, A.; Murphy, P.J.; Giller, K.E.

    2007-01-01

    Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and